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Title:
METHODS OF ASSAYING FOR THE PRESENCE OF MICROORGANISMS
Document Type and Number:
WIPO Patent Application WO/2019/143812
Kind Code:
A1
Abstract:
The present disclosure provides methods, systems, and kits for processing and assaying a sample. Sample processing may involve separation of solid materials or other processes for preparing for an assay (e.g., polymerare chain reaction and immunofluorescene-based target detection). The methods, systems, and kits provided herein may be used to, for example, analyse a milk sample (e.g., from a cow) for the presence or absence of one or more microorganisms.

Inventors:
RAGLAND, Ryan, Lee (35 West Mount Airy Avenue, Philadelphia, Pennsylvania, 19119, US)
VANWESTRIENEN, Jesse, Wilson (432 Cross Street, Philadelphia, Pennsylvania, 19147, US)
Application Number:
US2019/014005
Publication Date:
July 25, 2019
Filing Date:
January 17, 2019
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
BIOMEME, INC. (1015 Chestnut Street, Suite 1401Philadelphia, Pennsylvania, 19107, US)
International Classes:
C12M1/34; G01N1/34; G01N21/76
Domestic Patent References:
WO2009036956A12009-03-26
Foreign References:
US20080145848A12008-06-19
US20160115520A12016-04-28
US5849488A1998-12-15
Attorney, Agent or Firm:
ZHANG, Mengmeng et al. (Wilson Sonsini Goodrich & Rosati, 650 Page Mill RoadPalo Alto, California, 94304, US)
Download PDF:
Claims:
CLAIMS

WHAT IS CLAIMED IS:

1. A method for assaying for a presence of one or more microorganisms in a milk sample from a cow or a collection of cows, comprising:

(a) obtaining said milk sample from said cow or said collection of cows;

(b) processing said milk sample under conditions sufficient to extract one or more nucleic acids from said one or more microorganisms, thereby yielding a processed sample;

(c) subjecting said processed sample to conditions sufficient to assay for a presence of said one or more nucleic acids, which presence of said one or more nucleic acids is indicative of said presence of said one or more microorganisms in said milk sample; and

(d) outputing a report that identifies said one or more microorganisms in said milk

sample, wherein (a)-(d) are performed in a time period of less than 4 hours.

2. The method of claim 1, wherein said time period is less than 3 hours.

3. The method of claim 2, wherein said time period is less than 2 hours.

4. The method of claim 3, wherein said time period is less than 1 hour.

5. A method for assaying for a presence of one or more microorganisms in a milk sample from a cow or a collection of cows, comprising:

(a) obtaining said milk sample from said cow or said collection of cows at a first location;

(b) processing said milk sample under conditions sufficient to extract one or more nucleic acids from said one or more microorganisms, thereby yielding a processed sample;

(c) subjecting said processed sample to conditions sufficient to assay for a presence of said one or more nucleic acids, which presence of said one or more nucleic acids is indicative of said presence of said one or more microorganisms in said milk sample; and

(d) outputting a report that identifies said one or more microorganisms in said milk

sample,

wherein (b)-(d) are performed at a second location that is within 5 miles of said first location.

6. The method of claim 5, wherein said second location is within 4 miles of said first location.

7. The method of claim 6, wherein said second location is within 3 miles of said first location.

8. The method of claim 8, wherein said second location is within 2 miles of said first location.

9. The method of claim 8, wherein said second location is within 1 mile of said first location.

10. A method for assaying for a presence of one or more microorganisms in a milk sample from a cow or a collection of cows, comprising:

(a) obtaining said milk sample from said cow or said collection of cows;

(b) processing said milk sample under conditions sufficient to extract one or more nucleic acids from said one or more microorganisms, thereby yielding a processed sample;

(c) using a mobile diagnostic device to subject said processed sample to conditions

sufficient to assay for a presence of said one or more nucleic acids, which presence of said one or more nucleic acids is indicative of said presence of said one or more microorganisms in said milk sample; and (d) outputting a report that identifies said one or more microorganisms in said milk sample.

11. The method of claim 10, wherein said mobile diagnostic device comprises a mobile electronic device.

12. The method of any one of claims 1-11, wherein said processing in (b) comprises filtering said milk sample.

13. The method of claim 12, wherein said filtering separates one or more solid components from said milk sample.

14. The method of any one of claims 1-13, wherein said processing in (b) comprises

combining said milk sample with one or more reagents capable of lysing cells of said one or more microorganisms, thereby extracting one or more nucleic acids from said one or more microorganisms.

15. The method of any one of claims 1-14, wherein said processing in (b) comprises washing said milk sample with one or more washing reagents.

16. The method of any one of claims 1-15, wherein (c) comprises subjecting said processed sample to conditions sufficient to conduct one or more nucleic acid amplification reactions on said one or more nucleic acids.

17. The method of claim 16, wherein said one or more nucleic acid amplification reactions comprise polymerase chain reactions (PCR).

18. The method of claim 16 or 17, wherein (c) comprises thermal cycling of said processed sample.

19. The method of any one of claims 16-18, wherein said one or more nucleic acid

amplification reactions are performed in the presence of one or more reagents selected from the group consisting of primers, probes, polymerases, enzymes, lyophilization reagents, deoxyribonucleotides, washes, and elution buffers.

20. The method of claim 19, wherein said one or more nucleic acid amplification reactions are performed in the presence of primers having sequence complementarity to said one or more nucleic acids.

21. The method of claim 19 or 20, wherein reagents of said one or more reagents comprise a fluorescent label or fluorophore.

22. The method of claim 21, wherein said fluorescent label or fluorophore is selected from the group consisting of TexasRed, Cy5, and FAM.

23. The method of any one of claims 1-22, wherein (d) comprises outputting said report on an electronic display of an electronic device of a user.

24. The method of claim 23, wherein said electronic device is a mobile electronic device.

25. The method of claim 23 or 24, wherein (c) is performed in a thermal cycler that is

separate from said electronic device of said user.

26. The method of any one of claims 1-25, further comprising determining an amount of said one or more microorganisms in said milk sample.

27. The method of claim 26, wherein said report identifies said amount of said one or more microorganisms.

28. The method of any one of claims 1-27, wherein said one or more microorganisms are selected from the group consisting of gram negative bacteria, gram positive bacteria, Mycoplasma, Prototheca, and yeast.

29. The method of any one of claims 1-27, wherein said one or more microorganisms are selected from the group consisting of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas aeruginosa, Citrobacter, Enterobacter, Serratia marcescens, Pasteurella, Staphylococcus spp., Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus hyicus, Staphylococcus simulans,

Streptococcous uberis, Streptococcus agalactiae, Streptococcous dysgalactiae,

Streptococcous faecalis, Streptococcous faecium, Corynebacterium bovis, Bacillus licheniformis, Bacillus cereus, Trueperella pyogenes, Enterococcus spp., Enterococcus faecalis, Enterococcus faecium, Peptoniphilus indolicus, Arcanobacterium pyogenes, Lactococcus lactis, Nocardia, Mycoplasma spp., Mycoplasma alkalescens, Mycoplasma bovis, Mycoplasma bovigenitalium, Mycoplasma califomicum, Mycoplasma canadense, Mycoplasma arginine, Mycoplasma dispar, Mycoplasma bovihimis, Prototheca spp., Prototheca zopfii, Prototheca wickerhamii, Prototheca blaschkeae, Candida albicans, and Cyrptococcus neoformans.

30. The method of any one of claims 1-27, wherein said one or more microorganisms are selected from the group consisting of Mycoplasma, Prototheca, Staphylococcus aureus, Streptococcus agalactiae, and gram positive bacteria.

31. A system for assaying for a presence of one or more microorganisms in a milk sample from a cow or a collection of cows, comprising:

a sample preparation device configured to process said milk sample from said cow or said collection of cows under conditions sufficient to extract one or more nucleic acids from said one or more microorganisms, thereby yielding a processed sample; and

an analytic device configured to subject said processed sample to conditions sufficient to (i) assay for a presence of said one or more nucleic acids, which presence of said one or more nucleic acids is indicative of said presence of said one or more microorganisms in said milk sample, and (ii) output a report that identifies said one or more

microorganisms in said milk sample, wherein said sample preparation device is configured to generate said processed sample and said analytic device is configured to output said report in a time period of less than 4 hours.

32. The system of claim 31, further comprising a sample extraction device, wherein said sample extraction device is configured to transfer said milk sample between two different containers.

33. The system of claim 32, wherein said sample extraction device comprises a housing

defining an internal fluid passage having a distal open end and a proximal open end.

34. The system of claim 33, wherein said sample extraction device further comprises a porous medium within the fluid passage between the distal open end and the proximal open end.

35. The system of claim 31, wherein said sample preparation device comprises at least one container to store or receive a reagent.

36. The system of claim 35, wherein said at least one container comprises or is configured to accept one or more conduits.

37. The system of claim 31, wherein said sample preparation device comprises one or more electrical or mechanical components to perform one or more sample processing operations.

38. The system of claim 37, wherein said one or more electrical or mechanical components comprise computers, circuits, motors, actuators, or any combination thereof.

39. The system of claim 31, wherein said analytic device is configured to accept a mobile electronic device.

40. The system of claim 31, wherein said analytic device comprises one or more excitation sources.

41. The system of claim 40, wherein said one or more excitation sources are disposed onto a carriage.

42. The system of claim 31, wherein said analytic device is configured to heat or cool said milk sample.

43. The system of claim 42, wherein said analytic device comprises a heating block to heat said milk sample.

44. The system of claim 42, wherein said analytic device comprises a fan to cool said milk sample.

45. The system of claim 39, wherein said mobile electronic device comprises a user display.

46. The system of claim 31, wherein said analytic device comprises a processor configured to control one or more processes relating to analysis of said milk sample.

47. The system of claim 31, wherein said time period is less than 3 hours.

48. The system of claim 31, wherein said time period is less than 2 hours.

49. The system of claim 31, wherein said time period is less than 1 hour.

50. A system for assaying for a presence of one or more microorganisms in a milk sample from a cow or a collection of cows, comprising:

a sample preparation device configured to process said milk sample under conditions sufficient to extract one or more nucleic acids from said one or more microorganisms, thereby yielding a processed sample; and

a mobile diagnostic device configured to subject said processed sample to conditions sufficient to (i) assay for a presence of said one or more nucleic acids, which presence of said one or more nucleic acids is indicative of said presence of said one or more microorganisms in said milk sample; and (ii) output a report that identifies said one or more microorganisms in said milk sample.

51. The system of claim 50, further comprising a sample extraction device, wherein said sample extraction device is configured to transfer said milk sample between two different containers.

52. The system of claim 51, wherein said sample extraction device comprises a housing

defining an internal fluid passage having a distal open end and a proximal open end.

53. The system of claim 52, wherein said sample extraction device further comprises a porous medium within the fluid passage between the distal open end and the proximal open end.

54. The system of claim 50, wherein said sample preparation device comprises at least one container to store or receive a reagent.

55. The system of claim 54, wherein said at least one container comprises or is configured to accept one or more conduits.

56. The system of claim 50, wherein said sample preparation device comprises one or more electrical or mechanical components to perform one or more sample processing operations.

57. The system of claim 56, wherein said one or more electrical or mechanical components comprise computers, circuits, motors, actuators, or any combination thereof.

58. The system of claim 50, wherein said mobile diagnostic device comprises one or more excitation sources.

59. The system of claim 58, wherein said one or more excitation sources are disposed onto a carriage.

60. The system of claim 50, wherein said mobile diagnostic device is configured to heat or cool said milk sample.

61. The system of claim 60, wherein said mobile diagnostic device comprises a heating block to heat said milk sample.

62. The system of claim 60, wherein said mobile diagnostic device comprises a fan to cool said milk sample.

63. The system of claim 50, wherein said mobile diagnostic device comprises a processor configured to control one or more processes relating to analysis of said milk sample.

64. The system of claim 50, wherein (i) said sample preparation device is configured to process said milk sample and (ii) said mobile diagnostic device is configured to assay for said presence of said one or more nucleic acids and output said report, within 5 miles of a location at which said milk sample is collected from said cow or said collection of cows.

65. A kit for assaying for a presence of one or more microorganisms in a milk sample from a cow or a collection of cows, comprising: (a) reagents comprising at least one nucleic acid molecule having a sequence selected from Tables 1-4, and (b) instructions that direct a user to use said reagents to subject said processed sample to conditions sufficient to (i) process said milk sample under conditions sufficient to extract one or more nucleic acids from said one or more microorganisms, thereby yielding a processed sample, and (ii) use said reagents to assay for said presence of said one or more nucleic acids to yield a report indicative of said presence of said one or more microorganisms, in a time period of less than 4 hours.

66. The kit of claim 65, wherein said at least one nucleic acid molecule is a primer.

67. The kit of claim 65, wherein said reagents comprise a plurality of nucleic acid molecules having sequences selected from Tables 1-4.

68. The kit of claim 65, wherein said reagents comprise a plurality of nucleic acid molecules having sequences of Tables 1-4.

69. The kit of claim 65, wherein said reagents further comprise polymerizing enzymes.

70. The kit of claim 65, wherein said reagents further comprise a lysis buffer, a polymerizing enzyme, dNTPs, or any combination thereof.

71. The kit of claim 65, wherein said reagents further comprise detectable labels.

72. The kit of claim 71, wherein said detectable labels are fluorophores.

Description:
PCT PATENT APPLICATION

METHODS OF ASSAYING FOR THE PRESENCE OF MICROORGANISMS

CROSS-REFERENCE

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/619,032, filed January 18, 2018, which is entirely incorporated herein by reference.

BACKGROUND

[0002] Samples may be analyzed for various purposes, including detecting or identifying the presence of a target in the sample. Analysis of a sample may involve one or more processing operations designed to prepare a sample for assaying. For example, a sample including or suspected of including nucleic acids may be processed to separate or extract the nucleic acids from a cell present in the sample (e.g., by lysing the cell) and to remove extraneous material from the sample.

[0003] Samples may derive from biological or environmental sources. For example, a sample may derive from a livestock animal (e.g., from a bodily fluid such as milk or an external portion of the animal such as an udder or teat) or its immediate environment (e.g., a feature of a housing or milking facility used with the livestock animal). Such samples may include solids and other materials that may be removed from the sample using one or more processing operations (e.g., lysis, phase separation, or another process). For example, a milk sample may include milk solids as well as cells, hair, hay, and other materials that may obscure analysis of the sample.

[0004] Analysis of a milk sample, or another sample associated with a livestock animal such as a cow, goat, or sheep, may be performed to assess the sample for the presence of a target (e.g., a nucleic acid) associated with one or more microorganisms. For example, a sample derived from a livestock animal or its environment may be analyzed for the presence of one or more microorganisms associated with mastitis.

[0005] Mastitis is an inflammatory reaction of udder tissue and is the most common disease affecting dairy cattle in the United States. Mastitis may be very costly to farms, as affected milk may need to be discarded and affected animals may require costly care or may be removed from the dairy -producing population. Mastitis may be clinical (e.g., symptomatic) or subclinical (e.g., pre-symptomatic). While milk produced by an animal suffering from clinical mastitis may be watery, thick, or ropy, milk produced by an animal suffering from subclinical mastitis may appear normal for some time before the quality degrades. By the time mastitis becomes clinical, significant economic damages may be incurred.

[0006] Mastitis may be the result of an injury to or irritation of a teat (e.g., due to a chemical, mechanical, or thermal interaction with milking machinery) or a microbial invasion of the teat. Mastitis may be detected using a cell count test, such as the California Mastitis Test that provides an indication of the number of leukocytes in a milk sample. The number of leukocytes (e.g., the somatic cell count) may be indicative of an amount of inflammation associated with the animal. However, such tests are limited in their ability to assess mastitis, as they may be highly subjective and a high somatic cell count may not be necessarily caused by microorganisms. For example, somatic cell counts may be high due to a teat injury, after calving or during a drying off period, early or late in lactation, during estrus, or due to unrelated infections. Because cell count tests may provide no insight into the presence or type of one or more microorganisms, they may not indicate whether a mastitis treatment will be effective.

[0007] An alternative to somatic cell counting is assaying a milk sample for the presence of one or more microorganisms associated with mastitis. Analysis of a sample associated with a livestock animal rapidly and/or in proximity to the site of sample collection may facilitate rapid detection of mastitis and ultimately reduce economic impacts of mastitis.

SUMMARY

[0008] Recognized herein is a need for methods, systems, and kits for processing and analyzing biological and environmental samples associated with livestock. Current methods may be limited in their ability to process and analyze a sample with sufficient ease and rapidity. The present disclosure provides methods for processing and assaying a milk sample for the presence of microorganisms associated with mastitis in proximity to the source of the sample and/or within a relatively short period of time.

[0009] In an aspect, the present disclosure provides a method for assaying for a presence or amount of one or more microorganisms in a milk sample from a cow or a collection of cows, comprising (a) obtaining the milk sample from the cow or the collection of cows; (b) processing the milk sample under conditions sufficient to extract one or more nucleic acids from the one or more microorganisms, thereby yielding a processed sample; (c) subjecting the processed sample to conditions sufficient to assay for a presence of the one or more nucleic acids, wherein a presence of the one or more nucleic acids is indicative of a presence of the one or more microorganisms in the milk sample; and (d) outputting a report that identifies the one or more microorganisms in the milk sample. In some embodiments, (a)-(d) are performed in a time period of less than 4 hours.

[0010] In some embodiments, the time period is less than 3 hours. In some embodiments, the time period is less than 2 hours. In some embodiments, the time period is less than 1 hour.

[0011] In another aspect, the present disclosure provides a method for assaying for a presence or amount of one or more microorganisms in a milk sample from a cow or a collection of cows, comprising: (a) obtaining the milk sample from the cow or the collection of cows at a first location; (b) processing the milk sample under conditions sufficient to extract one or more nucleic acids from the one or more microorganisms, thereby yielding a processed sample; (c) subjecting the processed sample to conditions sufficient to assay for a presence of the one or more nucleic acids, wherein a presence of the one or more nucleic acids is indicative of a presence of the one or more microorganisms in the milk sample; and (d) outputting a report that identifies the one or more microorganisms in the milk sample, wherein (b)-(d) are performed at a second location that is within 5 miles of the first location.

[0012] In some embodiments, the second location is within 4 miles of the first location. In some embodiments, the second location is within 3 miles of the first location. In some embodiments, the second location is within 2 miles of the first location. In some embodiments, the second location is within 1 mile of the first location.

[0013] In another aspect, the present disclosure provides a method for assaying for a presence or amount of one or more microorganisms in a milk sample from a cow or a collection of cows, comprising: (a) obtaining the milk sample from the cow or the collection of cows; (b) processing the milk sample under conditions sufficient to extract one or more nucleic acids from the one or more microorganisms, thereby yielding a processed sample; (c) using a mobile diagnostic device to subject the processed sample to conditions sufficient to assay for a presence of the one or more nucleic acids, wherein a presence of the one or more nucleic acids is indicative of a presence of the one or more microorganisms in the milk sample; and (d) outputting a report that identifies the one or more microorganisms in the milk sample.

[0014] In some embodiments, the mobile diagnostic device comprises a mobile electronic device. [0015] In some embodiments of any of the above methods, the processing in (b) comprises filtering the milk sample. In some embodiments, the filtering separates one or more solid components from the milk sample.

[0016] In some embodiments of any of the above methods, the processing in (b) comprises combining the milk sample with one or more reagents capable of lysing cells of the one or more microorganisms, thereby extracting one or more nucleic acids from the one or more

microorganisms.

[0017] In some embodiments of any of the above methods, the processing in (b) comprises washing the milk sample with one or more washing reagents.

[0018] In some embodiments of any of the above methods, (c) comprises subjecting the processed sample to conditions sufficient to conduct one or more nucleic acid amplification reactions on the one or more nucleic acids. In some embodiments, the one or more nucleic acid amplification reactions comprise polymerase chain reactions (PCR). In some embodiments, (c) comprises thermal cycling of the processed sample. In some embodiments, the one or more nucleic acid amplification reactions are performed in the presence of one or more reagents selected from the group consisting of primers, probes, polymerases, enzymes, lyophilization reagents, deoxyribonucleotides, washes, and elution buffers. In some embodiments, the one or more nucleic acid amplification reactions are performed in the presence of primers having sequence complementarity to said one or more nucleic acids. In some embodiments, reagents of the one or more reagents comprise a fluorescent label or fluorophore. In some embodiments, the fluorescent label or fluorophore is selected from the group consisting of TexasRed, Cy5, and FAM.

[0019] In some embodiments of any of the above methods, (d) comprises outputting the report on an electronic display of an electronic device of a user. In some embodiments, the electronic device is a mobile electronic device. In some embodiments, (c) is performed in a thermal cycler that is separate from the electronic device of the user.

[0020] In some embodiments of any of the above methods, the method further comprises determining an amount of the one or more microorganisms in the milk sample. In some embodiments, the report identifies the amount of the one or more microorganisms.

[0021] In some embodiments of any of the above methods, the one or more microorganisms are selected from the group consisting of gram negative bacteria, gram positive bacteria,

Mycoplasma, Prototheca, and yeast.

[0022] In some embodiments of any of the above methods, the one or more microorganisms are selected from the group consisting of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas aeruginosa, Citrobacter, Enterobacter, Serratia marcescens, Pasteurella, Staphylococcus spp., Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus hyicus, Staphylococcus simulans, Streptococcous uberis,

Streptococcus agalactiae, Streptococcous dysgalactiae, Streptococcous faecalis, Streptococcous faecium, Corynebacterium bovis, Bacillus licheniformis, Bacillus cereus, Trueperella pyogenes, Enterococcus spp., Enterococcus faecalis, Enterococcus faecium, Peptoniphilus indolicus, Arcanobacterium pyogenes, Lactococcus lactis, Nocardia, Mycoplasma spp., Mycoplasma alkalescens, Mycoplasma bovis, Mycoplasma bovigenitalium, Mycoplasma califomicum, Mycoplasma canadense, Mycoplasma arginine, Mycoplasma dispar, Mycoplasma bovihimis, Prototheca spp., Prototheca zopfii, Prototheca wickerhamii, Prototheca blaschkeae, Candida albicans, and Cyrptococcus neoformans.

[0023] In some embodiments of any of the above methods, the one or more microorganisms are selected from the group consisting of Mycoplasma, Prototheca, Staphylococcus aureus, Streptococcus agalactiae, and gram positive bacteria. [0024] Another aspect of the present disclosure provides a system for assaying for a presence of one or more microorganisms in a milk sample from a cow or a collection of cows, comprising: a sample preparation device configured to process the milk sample from the cow or the collection of cows under conditions sufficient to extract one or more nucleic acids from the one or more microorganisms, thereby yielding a processed sample; and an analytic device configured to subject the processed sample to conditions sufficient to (i) assay for a presence of the one or more nucleic acids, which presence of the one or more nucleic acids is indicative of the presence of the one or more microorganisms in the milk sample, and (ii) output a report that identifies the one or more microorganisms in the milk sample, wherein the sample preparation device is configured to generate the processed sample and the analytic device is configured to output the report in a time period of less than 4 hours. In some embodiments, the system further comprises a sample extraction device, wherein the sample extraction device is configured to transfer the milk sample between two different containers. In some embodiments, the sample extraction device comprises a housing defining an internal fluid passage having a distal open end and a proximal open end. In some embodiments, the sample extraction device further comprises a porous medium within the fluid passage between the distal open end and the proximal open end. In some embodiments, the sample preparation device comprises at least one container to store or receive a reagent. In some embodiments, the at least one container comprises or is configured to accept one or more conduits. In some embodiments, the sample preparation device comprises one or more electrical or mechanical components to perform one or more sample processing operations. In some embodiments, the one or more electrical or mechanical components comprise computers, circuits, motors, actuators, or any combination thereof. In some embodiments, the analytic device is configured to accept a mobile electronic device. In some embodiments, the analytic device comprises one or more excitation sources. In some embodiments, the one or more excitation sources are disposed onto a carriage. In some embodiments, the analytic device is configured to heat or cool the milk sample. In some embodiments, the analytic device comprises a heating block to heat the milk sample. In some embodiments, the analytic device comprises a fan to cool the milk sample. In some

embodiments, the mobile electronic device comprises a user display. In some embodiments, the analytic device comprises a processor configured to control one or more processes relating to analysis of the milk sample. In some embodiments, the time period is less than 3 hours. In some embodiments, the time period is less than 2 hours. In some embodiments, the time period is less than 1 hour.

[0025] Another aspect of the present disclosure provides a system for assaying for a presence of one or more microorganisms in a milk sample from a cow or a collection of cows, comprising: a sample preparation device configured to process the milk sample under conditions sufficient to extract one or more nucleic acids from the one or more microorganisms, thereby yielding a processed sample; and a mobile diagnostic device configured to subject the processed sample to conditions sufficient to (i) assay for a presence of the one or more nucleic acids, which presence of the one or more nucleic acids is indicative of the presence of the one or more microorganisms in the milk sample, and (ii) output a report that identifies the one or more microorganisms in the milk sample. In some embodiments, the system further comprises a sample extraction device, wherein the sample extraction device is configured to transfer the milk sample between two different containers. In some embodiments, the sample extraction device comprises a housing defining an internal fluid passage having a distal open end and a proximal open end. In some embodiments, the sample extraction device further comprises a porous medium within the fluid passage between the distal open end and the proximal open end. In some embodiments, the sample preparation device comprises at least one container to store or receive a reagent. In some embodiments, the at least one container comprises or is configured to accept one or more conduits. In some embodiments, the sample preparation device comprises one or more electrical or mechanical components to perform one or more sample processing operations. In some embodiments, the one or more electrical or mechanical components comprise computers, circuits, motors, actuators, or any combination thereof. In some embodiments, the mobile diagnostic device comprises one or more excitation sources. In some embodiments, the one or more excitation sources are disposed onto a carriage. In some embodiments, the mobile diagnostic device is configured to heat or cool the milk sample. In some embodiments, the mobile diagnostic device comprises a heating block to heat the milk sample. In some embodiments, the mobile diagnostic device comprises a fan to cool the milk sample. In some embodiments, the mobile diagnostic device comprises a processor configured to control one or more processes relating to analysis of the milk sample. In some embodiments, the sample preparation device is configured to process the milk sample and (ii) the mobile diagnostic device is configured to assay for the presence of the one or more nucleic acids and output the report, within 5 miles of a location at which the milk sample is collected from the cow or the collection of cows.

[0026] Another aspect of the present disclosure provides a kit for assaying for a presence or amount of one or more microorganisms in a milk sample from a cow or a collection of cows, comprising: (a) a cartridge; and (b) at least one nucleic acid molecule having a sequence selected from Tables 1-4. In some embodiments, the at least one nucleic acid molecule is a primer or a probe. In some embodiments, the system further comprises one or more reagents. In some embodiments, the one or more reagents comprise a reagent necessary for sample processing. In some embodiments, the one or more reagents comprise a reagent necessary for performing nucleic acid amplification. In some embodiments, the one or more reagents comprise a lysis buffer, an enzyme, dNTPs, or any combination thereof. In some embodiments, the kit further comprises a detectable label. In some embodiments, the detectable label is a fluorophore.

[0027] Another aspect of the present disclosure provides a kit for assaying for a presence of one or more microorganisms in a milk sample from a cow or a collection of cows, comprising: (a) reagents comprising at least one nucleic acid molecule having a sequence selected from Tables 1- 4, and (b) instructions that direct a user to use the reagents to subject the processed sample to conditions sufficient to (i) process the milk sample under conditions sufficient to extract one or more nucleic acids from the one or more microorganisms, thereby yielding a processed sample, and (ii) use the reagents to assay for the presence of the one or more nucleic acids to yield a report indicative of the presence of the one or more microorganisms, in a time period of less than 4 hours. In some embodiments, the at least one nucleic acid molecule is a primer. In some embodiments, the reagents comprise a plurality of nucleic acid molecules having sequences selected from Tables 1-4. In some embodiments, the reagents comprise a plurality of nucleic acid molecules having sequences of Tables 1-4. In some embodiments, the reagents further comprise polymerizing enzymes. In some embodiments, the reagents further comprise a lysis buffer, a polymerizing enzyme, dNTPs, or any combination thereof. In some embodiments, the reagents further comprise detectable labels. In some embodiments, the detectable labels are fluorophores.

[0028] Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.

[0029] Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

[0030] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure.

Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

[0031] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also“figure” and“FIG.” herein), of which: [0033] FIGs. 1A-1B show amplification (FIG. 1A) and standard (FIG. IB) curves for gram positive bacteria primers and probes using tenfold serial dilutions from 50,000 to 50 genome equivalents;

[0034] FIGs. 2A-2B show amplification (FIG. 2A) and standard (FIG. 2B) curves for Mycoplasma primers and probes using tenfold serial dilutions from 50,000 to 50 genome equivalents;

[0035] FIGs. 3A-3B show amplification (FIG. 3A) and standard (FIG. 3B) curves for Prototheca primers and probes using tenfold serial dilutions from 50,000 to 50 genome equivalents;

[0036] FIGs. 4A-4B show amplification (FIG. 4A) and standard (FIG. 4B) curves for Staphylococcus aureus primers and probes using tenfold serial dilutions from 50,000 to 50 genome equivalents;

[0037] FIGs. 5A-5B show amplification (FIG. 5A) and standard (FIG. 5B) curves for Streptococcus agalactiae primers and probes using tenfold serial dilutions from 50,000 to 50 genome equivalents;

[0038] FIG. 6A shows deoxyribonucleic acid (DNA) amplification curves for 10,000 and 10 colony forming units (CFU) of Mycoplasma spiked into a milk sample; FIG. 6B shows ribonucleic acid (RNA) amplification curves for 10,000 and 10 colony forming units (CFU) of Mycoplasma spiked into a milk sample;

[0039] FIG. 7 schematically illustrates a sample extraction device;

[0040] FIG. 8 shows a sample preparation device;

[0041] FIG. 9 shows a portable analytic device including a carriage for analyzing a sample;

[0042] FIG. 10 schematically illustrates portable analytic device incorporating a mobile electronic device; [0043] FIG. 11 schematically illustrates a sample preparation process;

[0044] FIG. 12 shows a computer system that is programmed or otherwise configured to implement methods of the present disclosure herein; and

[0045] FIGs. 13A-13D show amplification curves for gram positive bacteria (FIG. 13A), Staphylococcus aureus (FIG. 13B), Prototheca (FIG. 13C), and Mycoplasma (FIG. 13D).

DETAILED DESCRIPTION

[0046] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0047] Whenever the term“at least,”“greater than,” or“greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term“at least,”“greater than” or“greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0048] Whenever the term“no more than,”“less than,” or“less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term“no more than,”“less than,” or“less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0049] The present disclosure provides methods, systems, and kits for sample processing and/or analysis. A method for analyzing a sample for the presence or absence of a microorganism or pathogen and/or determining an amount of a microorganism or pathogen in a sample may comprise obtaining the sample from an animal or environment, processing the sample under conditions to separate or extract a nucleic acid from the microorganism or pathogen, and assaying for the presence of the nucleic acid. A portable and/or mobile device may be used to perform the assaying operation. A report identifying the microorganism or pathogen and/or an amount of microorganism or pathogen in the sample may also be provided (e.g., on a display of an analytic and/or mobile device). A sample may derive from a biological or an environmental source such as milk or a surface associated with milk collection. In some examples, the method may be carried out in less than four hours and/or within five miles of the site from which the sample was collected.

Samples

[0050] A sample for analysis by the methods described herein may derive from a biological or an environmental source. The biological source may be, for example, an animal such as a cow, sheep, or goat. The biological source may be a human, such as a human working with or around one or more animals.

[0051] The sample may be obtained invasively (e.g., tissue biopsy) or non-invasively (e.g., venipuncture, swab, or milk collection). For example, the sample may be collected by milking a bovine, ovine, or caprine animal or by collecting a material from a teat or udder of the animal. A milk sample may be collected, for example, on a dairy farm, such as in a milking or housing facility, a bam, or outside. The milk sample may be collected manually or using a mechanical apparatus.

[0052] A sample may comprise a tissue or fluid sample from a subject, such as saliva, semen, blood (e.g., whole blood), serum, synovial fluid, tear, urine, milk (e.g., breast milk), colostrum, amniotic fluid, bile, or plasma. A tissue sample may comprise, for example, a skin sample or tumor sample. A sample may be obtained from a portion of an organ of a subject. In some cases, a milk sample for analysis or processing may comprise processed milk (e.g., milk from which solids have already been removed, and/or on which other processing such as pasteurization has been performed). A milk sample may derive from a single animal or a collection of animals (e.g., batch milk, such as milk prepared for processing or distribution). A milk sample may derive from an animal or collection of animals that is healthy or appears to be healthy (e.g., potentially affected by subclinical mastitis) or from an animal or collection of animals that appear to be affected by clinical mastitis.

[0053] A sample may be an environmental sample. For example, the sample may include water, soil, hay, feed, fecal maher, begging, manure, or other materials. A sample may be collected from a surface, such as a surface of a tool, machine, vessel, glove, or structure. An

environmental sample may be collected in or from a location at which an animal may be housed, sheltered, milked, processed, fed, or cleaned. For example, a sample may be collected in or from a bam, farm, factory, milking facility, or storage facility. A sample may also be collected in or from a location in which milk is stored or processed, such as a milk silo or refrigeration facility.

A sample may comprise water from, for example, a lake, stream, river, estuary, bay, ocean, trough, well, silo, or hose. An environmental sample may be associated with a particular animal, collection of animals, or location. For example, a sample collected from a surface of machinery used for milking may, upon processing and analysis, provide a source of information for a collection of animals milked using that machinery. An environmental sample may be associated with an animal or a collection of animals that is healthy or appears to be healthy (e.g., potentially affected by subclinical mastitis) or from an animal or collection of animals that appear to be affected by clinical mastitis. [0054] A sample may have any suitable volume or quantity. For example, a sample may comprise at least about 1 nanobter (nl), 2 nl, 5 nl, 10 nl, 20 nl, 50 nl, 100 nl, 200 nl, 500 nl, 1 microbter (pl), 2 mΐ, 5 mΐ, 10 mΐ, 20 mΐ, 25 mΐ, 50 mΐ, 100 mΐ, 200 mΐ, 300 mΐ, 400 mΐ, 500 mΐ, 600 mΐ, 700 mΐ, 800 mΐ, 900 mΐ, 1 milliliter (ml), 2 ml, 5 ml, 10 ml, 20 ml, 50 ml, 100 ml, or more than about 100 ml of a material. In some cases, a sample may comprise from about 1 mΐ to about 50 mΐ, such as from about 1 mΐ to about 25 mΐ, or from about 10 mΐ to about 25 mΐ (e.g., about 20 mΐ). In some cases, a sample may comprise from about 100 mΐ to about 1 ml, such as from about 200 mΐ to about 700 mΐ (e.g., about 500 mΐ).

[0055] A sample may include one or more microorganisms, such as one or more pathogens.

Such pathogens may include one or more members selected from the group consisting of bacteria, yeast and viruses. For example, a sample may include one or more microorganisms selected from the group consisting of gram negative bacteria (e.g., Escherichia cob [E. cob], Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas aeruginosa, Citrobacter, Enterobacter, Serratia marcescens, and Pasteurella), gram positive bacteria (e.g., Staphylococcus spp., Staphylococcus aureus [S. aureus], Staphylococcus chromogenes [S. chromogenes],

Staphylococcus epidermidis [S. epidermidis], Staphylococcus hyicus [S. hyicus], Staphylococcus simulans [S. simulans], Streptococcous uberis [S. uberis], Streptococcus agalactiae [S.

agalactiae], Streptococcous dysgalactiae [S. dysgalactiae], Streptococcous faecalis [S. faecabs], Streptococcous faecium [S. faecium], Corynebacterium bovis, Bacillus bcheniformis, Bacillus cereus, Trueperella pyogenes, Enterococcus spp., Enterococcus faecabs, Enterococcus faecium, Peptoniphilus indobcus, Arcanobacterium pyogenes, Lactococcus lactis, and Nocardia), Mycoplasma (e.g., Mycoplasma spp., Mycoplasma alkalescens, Mycoplasma bovis, Mycoplasma bovigenitalium, Mycoplasma califomicum, Mycoplasma canadense, Mycoplasma arginine, Mycoplasma dispar, and Mycoplasma bovihimis), Prototheca (e.g., Prototheca spp., Prototheca zopfii, Prototheca wickerhamii, and Prototheca blaschkeae), and yeast (e.g., Candida albicans and Cyrptococcus neoformans). For example, a sample may include one or more

microorganisms selected from the group consisting of Mycoplasma, Prototheca, Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, and gram positive bacteria.

[0056] A sample may include one or more cells and/or nucleic acids. In some cases, a sample may include one or more cells and/or nucleic acids deriving from one or more different sources. For example, a sample may include cells from an animal from which it was collected and from one or more microorganisms or pathogens. Alternatively, a sample may be a cell-free sample (e.g., a plasma sample comprising cell-free analytes or nucleic acids). A sample may comprise an in-vitro sample or an ex-vivo sample.

[0057] Nucleic acids may be circulating and/or cell-free nucleic acid fragments or may derive from cells. For example, a nucleic acid may be derived from a eukaryotic cell, a prokaryotic cell, or a non-cellular source (e.g., viral particles). A nucleic acid may refer to a substance whose molecules consist of many nucleotides linked in a chain. Non-limiting examples of a nucleic acid include deoxyribonucleic acid (DNA), genomic DNA, plasmid DNA, cDNA, ribonucleic acid (RNA), micro RNA (miRNA), an artificial nucleic acid analog (e.g., a peptide nucleic acid, a morpholino oligomer, a locked nucleic acid, a glycol nucleic acid, or a threose nucleic acid), and chromatin. A nucleic acid may be double stranded or single stranded. A sample may comprise a nucleic acid that may be intracellular. Alternatively, a sample may comprise a nucleic acid that may be extracellular (e.g., cell-free). Combining a sample with a lysing agent (e.g., an enzyme such as a protease or lysozyme) or subjecting a sample to a mechanical process such as mixing, bead beating, or vortexing may cause a cell in the sample to release one or more samples including one or more nucleic acids. A sample may comprise a nucleic acid (e.g., chromatin) that may be fragmented. Sample preparation

Container

[0058] A sample may undergo processing in preparation for assaying or analysis (e.g., as described herein). A sample may be provided or obtained in a container (e.g., a vessel, well, column, vial, or tube). For example, a sample may be collected directly from an animal (e.g., by milking the animal) and into a container. The sample may be collected directly from the animal and provided in the container without any processing (e.g., purification, extraction or filtration). As an alternative, the sample may be collected directly form the animal and processed prior to performing an assay disclosed herein. The sample may be processed using an extraction device that separates nucleic acid molecules from other material in the sample, such as cellular material.

[0059] A container may have any useful size and capacity. For example, a container may hold at least about 1 nl, 2 nl, 5 nl, 10 nl, 20 nl, 50 nl, 100 nl, 200 nl, 500 nl, 1 microliter (pl), 2 mΐ, 5 mΐ, 10 mΐ, 20 mΐ, 25 mΐ, 50 mΐ, 100 mΐ, 200 mΐ, 300 mΐ, 400 mΐ, 500 mΐ, 600 mΐ, 700 mΐ, 800 mΐ, 900 mΐ,

0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, or more of a fluid, such as a liquid. Such fluid may include a sample from a subject from which the fluid is collected. The sample may included or be suspected of including a pathogen.

[0060] A container may also have any useful shape. For example, a container may be substantially cylindrical or substantially rectangular and comprise an opening at one end and/or in a side of the container. For example, a container may be a cylindrical tube with an opening at its top, or a cylindrical tube with an opening along the length of the cylinder (e.g., in an upper or bottom portion of the tube). One end of a container may be flat, rounded, or tapered. For example, a container with a substantially cylindrical geometry may have a conical bottom rather than a flat bottom. [0061] A container may comprise any useful material including a plastic, a polymer, or a glass.

A material may be selected based upon its compatibility with various fabrication techniques, e.g., photolithography, wet chemical etching, laser ablation, air abrasion techniques, LIGA, reactive ion etching (RIE), injection molding, embossing, and other techniques. A material may also be selected for its compatibility with the full range of conditions to which a container and/or sample preparation device may be exposed, including extremes of pH, temperature, salt concentration, and application of electric fields. Accordingly, a container may comprise, e.g., silica based substrates, such as glass, quartz, silicon or poly silicon. In the case of a semi-conductive material, it may be useful to provide an insulating coating or layer, e.g., silicon oxide, over the material. A container and/or sample preparation device may comprise a polymeric material such as polymethylmethacrylate (PMMA), polycarbonate, polytetrafluoroethylene (TEFLON™), polyvinylchloride (PVC), polydimethylsiloxane (PDMS), polysulfone, and the like. Such polymeric substrates may be readily manufactured using fabrication techniques; using molding techniques, such as injection molding, embossing or stamping; or by polymerizing the polymeric precursor material within the mold. Such polymeric materials may be useful due to, for example, ease of manufacture, low cost and disposability, as well as general inertness to most extreme reaction conditions. A polymeric material may include a treated surface, e.g., a derivatized or coated surface, to enhance its utility in a container and/or sample preparation device, e.g., provide enhanced fluid direction.

[0062] A container may comprise a transparent portion (e.g., a window) and/or an opaque portion. A container may be reusable or consumable (e.g., designed for a single use or a small number of uses).

[0063] A container may include an opening through which sample is provided to the container. The opening may be disposed in the top or upper portion of the container. The opening may be closable and may include or be configured to accept a cap, cover, seal, or lid. A seal may be formed of a single material (e.g., aluminum) or a composition of two or more materials. For example, a seal may be formed of a material that comprises a tri-layer of an aluminum, adhesive layer and polypropylene layer. In some cases, the seal material may allow a syringe (e.g., a plastic syringe) to penetrate the seal. A seal may adhere to a container at temperatures of a minimum of lO°C up to and including 54°C, and maintain a seal for at least about 1 month, 6 months, 12 months, 24 months, 36 months, 48 months, 60 months, or more months.

[0064] A container may also include structural features useful in the processing of a sample. Structural features useful in the processing of a sample may include one or more openings, ports, protrusions, or intrusions. For example, a container may include a second opening disposed in the bottom or lower portion of the container. Such an opening may be useful for removing solid material from the sample after phase separation (e.g., filtration, sedimentation, and/or another process, as described herein). The opening may be operatively couplable to a pressure or vacuum source via, for example, a port configured to accept a conduit to or from the pressure or vacuum source to facilitate the transfer of a fluid or solid material from one position to another.

A pressure or vacuum source may comprise a pump, a house vacuum system, a compressor, or a compressed gas source. For example, a vacuum may be used to remove solid material disposed in the bottom portion of the container from the container (e.g., to move the solid material to a waste chamber), and/or a fluid material may be transferred from the container to, for example, another container. An opening may also be used to add a material to the container including a sample. For example, pressure may be used to effect fluid flow from another container including a reagent (e.g., as described herein) into a container including a sample. An opening of the container may be closable, e.g., using a manually and/or automatically actuatable valve. [0065] A container may include multiple ports or channels for addition or removal of a sample or other materials. For example, a container may comprise one or more sample introduction ports.

A sample may also be introduced to a container with a pipette or syringe.

[0066] A container may also include a filter and/or a filter support configured to support a filter that may be used to separate solid material from a sample. A filter may comprise any material capable of capturing a target (e.g., a nucleic acid) from a sample. A filter may be organic or inorganic; may be metal (e.g., copper or silver) or non-metal; may be a polymer or may not be a polymer; may be conducting, semiconducting or nonconducting (insulating); may be reflecting or nonreflecting; may be porous or nonporous; etc. A filter as described above may be formed of any suitable material, including metals, metal oxides, semiconductors, polymers (particularly organic polymers in any suitable form including woven, nonwoven, molded, extruded, cast, etc.), silicon, silicon oxide, and composites thereof. A number of materials (e.g., polymers) suitable for use as filters in the present disclosure may be used. Suitable materials for use as filters include, but are not limited to, polycarbonate, gold, silicon, silicon oxide, silicon oxynitride, indium, tantalum oxide, niobium oxide, titanium, titanium oxide, platinum, iridium, indium tin oxide, diamond or diamond-like film, acrylic, styrene-methyl methacrylate copolymers, ethylene/ aery lie acid, acrylonitrile-butadiene-styrene (ABS), ABS/poly carbonate,

ABS/polysulfone, ABS/polyvinyl chloride, ethylene propylene, ethylene vinyl acetate (EVA), nitrocellulose, nylons (including nylon 6, nylon 6/6, nylon 6/6-6, nylon 6/9, nylon 6/10, nylon 6/12, nylon 11 and nylon 12), polyacrylonitrile (PAN), polyacrylate, polycarbonate, polybutylene terephthalate (PBT), poly(ethylene) (PE) (including low density, linear low density, high density, cross-linked and ultra-high molecular weight grades), poly(propylene) (PP), cis and trans isomers of poly(butadiene) (PB), cis and trans isomers of poly(isoprene), polyethylene terephthalate) (PET), polypropylene homopolymer, polypropylene copolymers, polystyrene (PS) (including general purpose and high impact grades), polycarbonate (PC), poly(epsilon-caprolactone) (PECL or PCL), poly(methyl methacrylate) (PMMA) and its homologs, poly(methyl acrylate) and its homologs, poly(lactic acid) (PLA), poly(gly colic acid), polyorthoesters, poly(anhydrides), nylon, polyimides, polydimethylsiloxane (PDMS), polybutadiene (PB), polyvinylalcohol (PVA), polyacrylamide and its homologs such as poly(N-isopropyl acrylamide), fluorinated polyacrylate (PFOA), poly(ethylene-butylene) (PEB), poly(styrene-acrylonitrile) (SAN),

polytetrafluoroethylene (PTFE) and its derivatives, polyolefin plastomers, fluorinated ethylene- propylene (FEP), ethylene-tetrafluoroethylene (ETFE), perfluoroalkoxy ethylene (PFA), polyvinyl fluoride (PVF), polyvinylidene fluoride (PVDF), polychlorotrifluoroethylene (PCTFE), polyethylene-chlorotrifluoroethylene (ECTFE), styrene maleic anhydride (SMA), metal oxides, glass, glass wool, silicon oxide or other inorganic or semiconductor material (e.g., silicon nitride), compound semiconductors (e.g., gallium arsenide, and indium gallium arsenide), and combinations thereof.

[0067] Examples of filters include polypropylene, polystyrene, polyethylene, dextran, nylon, amylases, glass, natural and modified celluloses (e.g., nitrocellulose), polyacrylamides, agaroses and magnetite. A filter may comprise silica or glass because of its great chemical resistance against solvents, its mechanical stability, its low intrinsic fluorescence properties, and its flexibility of being readily functionalized. In an example, a filter is formed of silicon oxide (e.g., glass).

[0068] A filter material may be modified with one or more different layers of compounds or coatings that serve to modify the properties of the surface. For example, a filter may further comprise a coating material on the whole or a portion of the surface of the filter. For example, the coating material may be nitrocellulose, silane, thiol, disulfide, or a polymer. When the material is a thiol, the filter may comprise a gold-coated surface and/or the thiol comprises hydrophobic and hydrophilic moieties. When the coating material is a silane, the filter comprises glass and the silane may present terminal moieties including, for example, hydroxyl, carboxyl, phosphate, glycidoxy, sulfonate, isocyanato, thiol, or amino groups. Alternatively, the coating material may be a derivatized monolayer or multilayer having covalently bonded linker moieties. For example, the monolayer coating may have thiol (e.g., a thioalkyl selected from the group consisting of a thioalkyl acid (e.g., l6-mercaptohexadecanoic acid), thioalkyl alcohol, thioalkyl amine, and halogen containing thioalkyl compound), disulfide or silane groups that produce a chemical or physicochemical bonding to the filter. The attachment of a monolayer to a filter may also be achieved by non-covalent interactions or by covalent reactions.

[0069] A filter coating may comprise at least one functional group (e.g., after attachment to the filter). Examples of functional groups on a monolayer coating include, but are not limited to, carboxyl, isocyanate, halogen, amine or hydroxyl groups. These reactive functional groups on a coating may be activated by standard chemical techniques to corresponding activated functional groups on the monolayer coating (e.g., conversion of carboxyl groups to anhydrides or acid halides, etc.). Examples of activated functional groups of a filter coating for covalent coupling to terminal amino groups include anhydrides, N-hydroxysuccinimide esters or other common activated esters or acid halides; anhydride derivatives for coupling with a terminal hydroxyl group; hydrazine derivatives for coupling onto oxidized sugar residues of a linker compound; or maleimide derivatives for covalent attachment to thiol groups of a linker compound. To produce a derivatized coating, at least one terminal carboxyl group on the coating may be activated to an anhydride group and then reacted, for example, with a linker compound. Alternatively, the functional groups on a coating may be reacted with a linker having activated functional groups (e.g., N-hydroxysuccinimide esters, acid halides, anhydrides, and isocyanates) for covalent coupling to reactive amino groups on the coating. [0070] A container may include a paddle, propeller, stirring rod, a collection of beads, or other mechanical element. For example, a container may include one or more protrusions into the body of the container. Such elements may be useful, for example, for promoting lysis or phase separation by a sample upon agitation or mixing of the container.

[0071] A container may also include a mechanical key element such as a groove or protrusion to facilitate placement or securing of the container within a support, housing, and/or a device for sample preparation or analysis. For example, a substantially cylindrical container may comprise a protrusion disposed at one end along the length of the container and the support, housing, and/or device for sample preparation or analysis may comprise a corresponding indentation configured to accept the container in a particular orientation. In another example, a container may comprise one or more notches or grooves in its bottom portion that are configured to stabilize the container within a support, housing, or device. Stabilizing features may prevent release of the container or sample spillage during, for example, a phase separation process.

[0072] A container (e.g., a sample container) may include one or more reagents prior to the addition of a sample. For example, a sample container may comprise a powder, packet, liquid, coating, or crystal including one or more reagents. Alternatively or in addition, a reagent may be added to a sample container after a sample has been added. A reagent may be, for example, a lysis buffer, a wash buffer, a drying agent (e.g., a drying buffer), or an elution buffer. Non limiting examples of buffers include NP-40 lysis buffer, Radio Immunoprecipitation Assay (RIP A) lysis buffer, sodium dodecyl sulfate (SDS) lysis buffer, Ammonium-Chloride-Potassium (ACK) lysing buffer, volatile chemicals (e.g., acetone and ethanol), EDTA, Tris-HCl, and water.

[0073] A container may comprise one or more buffers useful for analyzing a sample according to the Boom Method. In accordance with the Boom method, a biological sample is lysed and/or homogenized by mixing the biological sample with detergent in the presence of protein degrading enzymes. The chaotropic agents and silica or silica coated beads are mixed with the lysed biological sample. The chaotropic agents disrupt and denature the structure of nucleic acids by interfering with the macromolecular interactions mediated by non-covalent forces, such as hydrogen bonding, van der Waals forces, and hydrophobic interactions, for example. In the presence of the chaotropic agents, water is removed from the phosphate groups of the nucleic acids, exposing them and allowing hydrophobic bonding to the silica, such as silica or silica coated beads. Protein, cellular debris, and other substances in the biological samples do not bond to the silica and are retained in the solution. The silica beads are washed several times to remove non-nucleic acid materials, such as proteins, lipids, cellular constituents, including cellular molecules, and other substances found in biological samples. Silica coated magnetic beads may be used to assist in the separation of the nucleic acids bound to the silica coating from the solution, via a magnetic field or magnet. The nucleic acids are then eluted from the silica or silica coated beads into a buffer by decreasing the concentration of the chaotropic agents. The elution buffer may be pure water or Tris EDTA ("TE") buffer, for example.

Sample extraction device

[0074] A sample extraction device may be used to transfer a fluid sample, or a portion thereof, between containers. A sample extraction device may comprise a housing defining an internal fluid passage having a distal open end and a proximal open end. The housing may comprise a plastic tubing having a proximal end and a distal end, a proximal fitting inserted into the proximal end of the plastic tubing, and a distal fitting inserted into the distal end of the plastic tubing, where the proximal fitting has a first end surface within the plastic tubing, and the distal fitting has a second end surface within the plastic tubing, and where the proximal fitting defines a proximal fluid passage through the proximal fitting and the distal fitting defines a distal fluid passage through the distal fitting. Facing end surfaces of the proximal distal portion and the distal end portion may be spaced to define a chamber in fluid communication with the proximal fluid passage and the distal fluid passage. A porous medium may be disposed within the chamber.

[0075] The sample extraction device may comprise a porous medium within the fluid passage between the distal open end and the proximal open end. The porous medium may be configured to allow fluid flow in the fluid passage to flow around the porous medium, toward the proximal open end of the housing, when fluid is drawn from the distal open end toward the proximal open end; allow fluid in the fluid passage to flow toward the distal open end of the housing, through the porous medium when the fluid is forced toward the distal open end; and capture a target (e.g., a nucleic acid) in the porous medium from the fluid when fluid is forced through the porous medium. The fluid passage may be configured to allow the porous medium to move in a first direction from a first position to a second position, toward the proximal end, when fluid is drawn into the device, allowing fluid flow around the porous medium, and to allow the porous medium to move from the second position to the first position to cause fluid to flow toward the distal open end. The porous medium may be supported by an internal surface of the housing. The internal surface may also support a porous support that is configured to allow passage of a fluid through the support without bending. The porous medium may comprise a compliant material (e.g., glass fibers) configured to bend, at least in part, to allow the fluid to flow around the porous medium (e.g., a peripheral bendable region or a central bendable region). The housing of the sample extraction device may facilitate the bending of a region of a porous medium. The sample extraction device may further comprise a member between a protruding portion of a porous medium that is free to move toward the protruding portion when fluid is drawn into the device, and move away from the protruding portion when fluid is forced toward the distal open end. The proximal open end of a sample extraction device may be couplable to a pressure source (e.g., as described herein).

[0076] FIG. 7 shows a side view of an example of a sample extraction device comprising an extraction unit 101 coupled to a syringe 102 via a female port 104 that receives a tip of syringe 102. Female port 104 may be part of a Luer fitting. Extraction unit 101 and syringe 102, which may both comprise clear tubing, are transparent. The internal configuration of extraction unit 101 and syringe 102 are therefore shown. Syringe 102 comprises a tubular body 106 defining a passage 107 and a plunger 108 having a first proximal end 110 extending outside the tubular body and second distal end 112 movable within the passage. The first end 110 of

plunger 108 may have a flat, wide surface 114 and the second end 112 of plunger 108 may include a seal.

[0077] The sample extraction device may be used in combination with a syringe. The proximal open end of a sample extraction device may be configured to receive a tip of a syringe. The distal end of a sample extraction device may comprise a tube that extends out of the distal portion that is configured for insertion into a fluid sample to be drawn into the housing.

[0078] Additional details of sample extraction devices and uses thereof may be found, for example, in U.S. Patent Applications Numbers 14/530,449 and 15/682,675, each of which is entirely incorporated herein by reference.

Sample preparation devices

[0079] A container may be a component of a sample preparation device. A sample preparation device may include one or more containers or chambers. For example, a sample preparation device may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, or more than 100 chambers. In one example, a sample preparation device includes 5 chambers. Containers or chambers of a sample preparation device may be integrated into the structure of the sample preparation device and/or be removable from the sample preparation device. For example, a sample preparation device may be configured to accept one or more assay tubes.

[0080] Containers or chambers of a sample preparation device may be useful for different purposes. For example, a container may be used to store or process a sample, to store a buffer or reagent for sample processing, to serially mix a sample with a buffer or reagent to process a sample, to store waste, or to analyze a sample. One or more containers of a sample preparative device may be fluidly connected to one another, e.g., as described herein. A sample preparation device may include more than one container into which more than one sample may be added. In another example, a sample preparation device may include a first container into which a sample may be added and one or more additional containers or chambers where the sample, or a portion or component thereof, may be transferred. Alternatively, or in addition, a sample preparation device may include a first container into which a sample may be added and one or more additional containers or chambers where one or more reagents may be stored. For example, a reagent from a second container may be added to a first container including the sample as a component of a sample preparation process. Containers of a sample preparation device may or may not have the same size, shape, capacity, and features. For example, a container into which a sample is added (e.g., a sample chamber) may have a greater volume than a container for storing a reagent or a container for containing a portion of the sample. Similarly, a container into which a sample is added may have additional features (e.g., as described herein) that may not be useful in another container of a sample preparation device.

[0081] Containers of a sample preparation device may have any useful size, capacity, and shape, e.g., as described herein. Containers of a sample preparation device may have different dimensions, capacity, geometry, or configuration based on their designated purpose. For example, a container for processing a sample may be larger than a container for storing a reagent. Similarly, a sample preparation device comprising a container may comprise any useful material, e.g., as described herein.

[0082] One or more containers of a sample preparation device may comprise a seal. In some cases, a chamber of a sample preparation device may be permanently sealed. For example, a sample preparation device may comprise a waste chamber, and the waste chamber may be permanently sealed. A sample preparation device may comprise a lid capable of covering or sealing one or more containers of the sample preparation device.

[0083] One or more containers of a sample preparation device may include one or more openings, ports, protrusions, or intrusions, e.g., as described herein. An opening or port may be closable, e.g., using a manually and/or automatically actuatable valve. A valve may be actuated by various approaches. Such approaches include pneumatic actuation, such as with the aid of positive pressure or negative pressure from a source of positive pressure or negative pressure, respectively. Positive pressure may be provided using one or more compressors. Negative pressure may be provided using one or more pumps. In another approach, a valve may be actuated using electrothermal heating. For example, a valve may be a shape memory valve. A shape memory valve may refer to any type of valve that comprises a material that“remembers” its original shape and is capable of returning to its pre-deformed shape when heated. In some cases, the shape memory valve may comprise a nitinol or Nickel Titanium wire that actuates a seal during contraction upon electrothermal heating. In other cases, the shape memory valve may comprise a copper-aluminum-nickel wire that actuates a seal during contraction upon electrothermal heating. In yet another approach, valves may be actuated using electromechanical approaches. For example, the valve may be a solenoid valve. An electromechanical valve may refer to any type of valve that is controlled by an electric current (e.g., through a solenoid). In the case of a two-port valve, flow may be switched on or off. In the case of a three-port valve, outflow may be switched between any or both of the one or more outlet ports.

[0084] A sample preparation device including a sample container may include or be configured to accept one or more conduits. A conduit may comprise a fluid flow path that is configured to direct or transfer a fluid from one point to another. A conduit may be a channel or a plurality of channels. For example, a conduit may be a microfluidic channel or a plurality of microfluidic channels. A conduit may comprise an opening for connecting with a pump or valve to regulate the flow of a liquid (e.g., a buffer or a sample) along the conduit. A conduit may fluidly connect one or more containers of a sample preparation device, e.g., to allow a sample, reagent, or waste material to pass between the containers. Positive or negative pressure may be used to effect fluid transfer between containers, e.g., as described herein.

[0085] A conduit of a sample preparation device may have various dimensions. A cross- sectional area of a conduit may be at least about 0.01 mm 2 , 0.05 mm 2 , 0.1 mm 2 , 0.2 mm 2 , 0.3 mm 2 , 0.4 mm 2 , 0.5 mm 2 , 0.6 mm 2 , 0.7 mm 2 , 0.8 mm 2 , 0.9 mm 2 , 1.0 mm 2 , 1.1 mm 2 , 1.2 mm 2 , 1.3 mm 2 , 1.4 mm 2 , 1.5 mm 2 , 1.6 mm 2 , 1.7 mm 2 , 1.8 mm 2 , 1.9 mm 2 , 2.0 mm 2 , 3.0 mm 2 , 4.0 mm 2 , 5.0 mm , 10 mm , 15 mm , 20 mm , 25mm , or greater than 25 mm . A shape of a cross section of a conduit may be a triangle (e.g., an equilateral triangle, an isosceles triangle, a scalene triangle, a right triangle, an acute triangle, or an obtuse triangle), a square, a rectangle, a diamond, a rhombus, a parallelogram, a kite, a trapezoid, a pentagon, a hexagon, a heptagon, an octagon, a nonagon, a decagon, a shape having greater than 10 sides, a circle, an oval, an egg shape, or a droplet shape.

[0086] A conduit may be defined by the structure of a sample preparation device. In one example, a sample preparation device includes a top portion, a bottom portion, and an interior portion, where the interior portion substantially defines the conduits of the sample preparation device. The top portion may include one or more containers (e.g., a sample chamber, a reagent storage chamber, and a waste chamber), the interior portion may comprise a conduit or network of conduits, and the bottom portion may comprise one or more adapters, openings, or caps to couple the sample preparation device to, e.g., an analytical device, a pump, or a valve. A conduit opening, e.g., in a bottom portion of a sample preparation device, may allow the sample preparation device to dock in a device or system comprising pumps, valves, and/or electronic components to perform processing and/or analysis on a sample contained therein. A conduit opening may also allow for different containers of a sample preparation device to be fluidly connected, via their respective conduits, depending on the process being performed. For example, a sample preparation device may comprise one or more containers including reagents useful for different processes of components thereof. A sample preparation device may be configured such that reagents are transferrable between their one or more containers and a container including a sample. For example, a reagent from a first reagent chamber may be transferred to a container including a sample, followed by a reagent from a second reagent chamber. Alternatively, a reagent from a first reagent chamber may be transferred to a second container and mixed with a second reagent from a second reagent chamber. The mixture of the first and second reagents may then be transferred to a container including a sample. A sample preparation device may also comprise a conduit that is fluidly connected to each container of the sample preparation device. For example, a sample preparation device may comprise two or more chambers that are connected to a common or primary conduit via separate secondary conduits. For example, a first conduit fluidly connected to a first chamber and a second conduit fluidly connected to a second chamber may both fluidly connect to a primary conduit. Any number of secondary conduits, each of which may be fluidly connected to a container, may be fluidly connected to a primary conduit. A valve may be used to restrict flow to or from one or more specific secondary conduits.

[0087] A container of a sample preparation device may include multiple ports or channels for addition or removal of a sample or other materials. For example, a sample preparation device including a sample container may comprise one or more sample introduction ports. A sample may also be introduced to a container of a sample preparation device with a pipette or syringe.

[0088] A sample preparation device (e.g., comprising one or more containers) may comprise a housing or system including a button or housing display. Alternatively, a sample preparation device may be configured for placement in a housing or system including a button or housing display. A housing or system associated with a sample preparation device may also include one or more processors, pumps, valves, displays, actuators, mechanical elements, circuitry, or another other useful component.

[0089] One or more containers (e.g., chambers) of a sample preparation device may include a reagent for analyzing or processing a sample, e.g., as described herein.

[0090] A sample preparation device may be capable of processing a sample. For example, a sample preparation device may comprise one or more electrical and/or mechanical components (e.g., computers, circuits, motors, actuators, robotic components, or other components) to perform one or more sample processing operations such as adding a sample to a sample container, transferring a sample or a portion thereof to another container or device, transferring a reagent from a reagent chamber to a sample container, or agitating a sample or reagent within a container. For example, a sample preparation device may be capable of actuating a valve and/or applying a positive or negative pressure to cause a fluid to be transferred from one container to another. A sample preparation device may be capable of performing one or more sample processing operations automatically, e.g., upon initiation of a process or program by a user via a button or user display.

[0091] FIG. 8 shows a top view of an example sample preparation device. The sample preparation device includes a sample container 804, reagent containers 802, conduits 801 connecting various components of the device, openings 803 for connecting with a pump or valve to regulate flow of a liquid (e.g., a buffer or sample) along a conduit, a waste chamber 806, and additional containers (e.g., assay tubes) 807 to, for example, accept aliquots of a sample.

Sample processing

[0092] The present disclosure provides methods for processing a sample, e.g., before analysis of the sample. Sample processing may comprise preparing a sample for an amplification reaction. For example, a sample may be processed to lyse cells, remove solid or other materials, denature proteins and/or nucleic acids, dilute a sample, buffer the sample to a particular pH, or any combination thereof.

[0093] Some or all processing of a sample may take place in the container or device in which it is obtained or provided. For example, a sample may be collected directly from an animal (e.g., by milking the animal) into a container, and the sample may be processed in that container.

Alternatively, a sample may be obtained or provided in a first container and subsequently transferred to a second container or a set of containers for processing. Some or no processing may be performed in the first container. For a sample preparation device including multiple sample chambers, multiple samples may be processed simultaneously or in succession. Multiple samples may be processed independently from one another (e.g., under different processing conditions). Alternatively, multiple samples may be processed using the same or substantially the same processing conditions. [0094] A sample may be processed (e.g., partially or completely) and subsequently divided (e.g., aliquoted) for subsequent processing, analysis, or assaying. A sample may be divided into 2, 3,

4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 384, 500, 1000, 1064, or any other useful number of aliquots. For example, a sample comprising about 500 pl of milk (e.g., freshly collected or previously collected, such as thawed, previously frozen milk) may be processed and then divided into smaller quantities (e.g., about 20 mΐ quantities) for assaying and analysis. Aliquots of a sample may be transferred to one or more containers of a sample preparation device, e.g., as described herein. For example, a sample may be provided in a first container (e.g., a sample container) of a sample preparation device for processing and subsequently divided into one or more containers (e.g., wells, plates, vials, tubes, or other containers or devices) for subsequent processing and analysis. The one or more containers for subsequent processing or analysis may be integrated into a sample preparation device or may be separate from a sample preparation device. For example, aliquots of a processed sample may be transferred to one or more assay tubes that are removable from a sample preparation device. These assay tubes may be used in, e.g., an analytical device for analyzing a sample.

[0095] Sample processing may comprise phase separation. A sample may comprise solid and/or liquid material. A sample may also comprise an emulsion or dispersion. For example, a sample may comprise solids (e.g., solids suspended in a solution) that may be separated out from a liquid portion of the sample via one or more of a precipitation process, an extraction process, a clarification process, a crystallization process, a sedimentation process, a centrifugation process, a fluid flow process (e.g., a continuous separation process), a mechanical agitation process (e.g., bead beating), and a filtration process (e.g., filtration through a fine mesh or sieve). Solid material in a sample may be separated from liquid material by, for example, shaking or vortexing the sample, allowing solid materials to settle out of the solution, and filtering the sample. For example, a bead beating device may be used to bead beat a sample for several minutes (e.g., five minutes). A lysis buffer (e.g., as described herein) may be added to the sample during or prior to a mechanical agitation process). Separation of solid and liquid materials (e.g., phase separation) may involve subjecting a sample to one or more chemical or temperature changes instead of, or in addition to, mechanical processing (e.g., vortexing, bead beating, centrifugation, filtration, or shaking). For example, an acid or rennet may be added to a sample to precipitate solids (e.g., milk solids). Alternatively, or in addition, heating or cooling a sample may facilitate separation of solid material from the sample (e.g., precipitation). A separation process may be performed manually, in a semi-automated fashion, or automatically. In one example, a sample is added to a sample preparation device (e.g., as described herein) and solids are automatically separated from the sample. The automatic phase separation process may involve one or more temperature changes and/or mechanical processes. Solid sample material may be retained for processing and analysis. In some cases, solid sample material may be discarded and only liquid sample material may be retained for processing and analysis. For example, a milk sample obtained from a cow (e.g., by milking or by obtaining a previously frozen milk sample) may include solid material such as fat, cells, hair, dust, and other materials. One or more of these materials may be separated from the fluid portion of the milk sample by one or more processes described above.

[0096] Sample processing may comprise lysing one or more cells to release a component of the cells, such as a nucleic acid. Lysing may be achieved by combining the sample with one or more lysing agents or buffers. A lysis buffer may include, for example, one or more salts, solubilizing agents, buffers, reducing agents, or detergents. For example, a lysis buffer may include one or more components selected from the non-limiting group consisting of Tris-HCl, HEPES-NaOH, sodium dihydrogen phosphate, disodium hydrogen phosphate, NaCl, EDTA, Triton X-100, and sodium dodecyl sulfate (SDS). Alternatively or in addition, cell lysis may be achieved by a mechanical process such as mixing, vortexing, bead beating, or centrifugation. For example, a sample may be subjected to bead beating and combined with a lysis buffer serially or

simultaneously. In one example, a 500 pl sample is combined with 1 ml of a lysis buffer (e.g., a 3M or 6M lysis buffer). One or more additional reagents may be combined with the sample with a lysis buffer. For example, a carrier ribonucleic acid (RNA) and/or a solvent (e.g., ethanol solution) may be combined with a sample and a lysis buffer.

[0097] Sample processing may comprise heating a sample. A container including a sample may be positioned in proximity to a heater to provide heat to the sample and/or the container. A heater or heating block may be a component of a sample preparation device. For example, a sample preparation device may comprise a heating block (e.g., resistive heater) capable of transferring thermal energy to a sample. A thermal unit (e.g., heater) controlling the heat of the heating block may be a component of the sample preparation device or may be separate from the sample preparation device. A heating block may comprise any useful material. Non-limiting examples of materials that may be used to construct a heating block include aluminum, concrete, glass, quartz, steel, iron, nickel, zinc, copper, brass, silver, tin, gold, and any combination thereof. A heating block may be formed of an alloy. For example, a heating block may be constructed using steel. The higher the specific heat capacity of the material, the more energy may be required to raise the temperature of the material. Accordingly, a heating block may comprise a material with a specific heat capacity (at 25°C, as measured in Joules per gram per °C, e.g.,

J/g°C) of less than about 2 J/g°C, 1.5 J/g°C, 1 J/g°C, 0.9 J/g°C, 0.8 J/g°C, 0.7 J/g°C, 0.6 J/g°C, 0.5 J/g°C, 0.45 J/g°C, 0.4 J/g°C, 0.35 J/g°C, 0.3 J/g°C, 0.25 J/g°C, 0.2 J/g°C, 0.15 J/g°C, 0.1 J/g°C, 0.05 J/g°C, or 0.01 J/g°C. For example, a heating block may comprise a material having a specific heat capacity of less than about 1 J/g°C at 25°C. A heating block may also comprise one or more fins to increase a surface area of the heating block and provide better heat dissipation from the heating block. The greater the volume of the material used to construct the heating block, the more energy may be required to raise the temperature of the heating block.

Accordingly, a volume of a material used to construct a heating block may be less than about 20 cubic centimeters, 15 cubic centimeters, 10 cubic centimeters, 9 cubic centimeters, 8 cubic centimeters, 7 cubic centimeters, 6 cubic centimeters, 5 cubic centimeters, 4 cubic centimeters, 3 cubic centimeters, 2 cubic centimeters, 1 cubic centimeter, 0.9 cubic centimeters, 0.8 cubic centimeters, 0.7 cubic centimeters, 0.6 cubic centimeters, 0.5 cubic centimeters, 0.4 cubic centimeters, 0.3 cubic centimeters, 0.2 cubic centimeters, or 0.1 cubic centimeters. For example, a volume of a material used to construct a heating block may be less than about 0.5 cubic centimeters.

[0098] Sample processing may comprise combining a sample with one or more reagents. A reagent may be selected from the non-limiting group consisting of a wash buffer, detergents, a drying buffer, and an elution buffer. A sample may be washed with one or more wash buffers. For example, a sample may be washed with a protein wash, a wash buffer, and a drying wash. A buffer or wash may include one or more solvents, salts, and detergents. For example, a buffer or wash may include phosphate buffered saline (PBS), PBS-T, Tris-buffered saline (TBS), TBS-T, NaCl, Tween 20, Tris/HCl, or any other useful component. A sample may be dried (e.g., air dried) following washing. A drying process may comprise pumping air into a container including a sample using, e.g., a syringe.

[0099] After a sample has been substantially processed, it may be combined with an elution buffer. For example, a sample processed in a sample preparation column may be eluted from the column using an elution buffer. The elution buffer may be used to extract a target (e.g., a nucleic acid) from a filter into the elution buffer. For example, a nucleic acid trapped on a filter may be released from the filter by an elution buffer. Mechanical agitation or air bubbles generated by positive pressure in a sample chamber may be used to distribute the elution buffer in a sample chamber to enhance extraction of the target from a filter.

[00100] FIG. 11 shows an exemplary process flow for preparing a sample (e.g., a milk sample) for analysis. In operation 1101, solid material is separated from the sample, e.g., as described herein. Phase separation may comprise, for example, bead beating the sample (e.g., using a hand-held bead beating device) precipitating solids by adding an acid or rennet, and/or filtering out solids with a fine mesh. In operation 1102, the sample is combined with a lysis buffer. In some instances, a lysis buffer may be added to the sample during the phase separation process. For example, a lysis buffer may be added to the sample during a bead beating process to facilitate separation of solids from the sample. In one example, 1 ml of lysis buffer (e.g., a 6M or 3M lysis buffer combined with 50% EtOH and carrier RNA) is added to a 500 pl sample of milk.

Combination of the sample with a lysis buffer may be accompanied by or followed by a mixing or mechanical agitation operation. For example, positive pressure may be applied (e.g., using a pump and/or a syringe) to introduce air bubbles to a sample chamber. This may cause the lysis buffer to become more evenly distributed throughout the sample container to enhance lysis of cells including within the sample. As described herein, the sample container may include a filter capable of capturing a nucleic acid from the sample. Negative pressure may be applied (e.g., using a pump, as described herein) to draw the sample and lysis buffer through the filter, thereby capturing targets (e.g., nucleic acids) on the filter. Negative pressure may also be used to draw waste material (e.g., solids) into a waste container.

[00101] In some cases, lysis buffer may be added to a container prior to adding the sample. For example, a sample may undergo a phase separation process in a first container and then be transferred (e.g., manually or automatically) to a second container including a lysis buffer. This may prevent loss of target nucleic acids within the sample (e.g., due to adhesion along the wall of a sample container).

[00102] In operation 1103, the sample (e.g., including nucleic acids captured on a filter) is combined with one or more washes or buffers. For example, the sample may be washed with a protein wash, a washing buffer, and a drying wash. As in operation 1102, vacuum may be applied to draw materials through the sample container and, e.g., over the filter including nucleic acids. The washes may remove unwanted material from the sample and prepare the sample for further analysis. In some cases, heat may be applied (e.g., using a heating block or pad disposed along an outer surface of the sample container) to remove a residual wash or buffer such as a drying buffer (e.g., through vaporization). This may reduce contamination of a target by a drying agent.

[00103] In operation 1104, an elution buffer is pumped into the sample container, thereby extracting nucleic acids from the filter into the elution buffer. As in operation 1102, positive pressure or mechanical agitation may be used to enhance extraction of the target from the filter. The processed sample may subsequently be transferred from the sample container to, e.g., one or more other containers (e.g., assay tubes) for subsequent analysis. The transfer process may be automated or performed manually.

Sample Assaying

[00104] The present disclosure provides methods for assaying a sample (e.g., a milk sample) for a presence of a nucleic acid. The presence of a nucleic acid may be indicative of a presence of, e.g., a microorganism or pathogen in the sample. For example, the methods provided herein may be useful for assaying a milk sample (e.g., a milk sample from a cow) for the presence of one or more microorganisms associated with mastitis. The one or more microorganism or pathogen may include, for example, a microorganism selected from the group consisting of gram negative bacteria (e.g., Escherichia coli [E. coli], Klebsiella pneumoniae, Klebsiella oxytoca,

Pseudomonas aeruginosa, Citrobacter, Enterobacter, Serratia marcescens, and Pasteurella), gram positive bacteria (e.g., Staphylococcus spp., Staphylococcus aureus [S. aureus], Staphylococcus chromogenes [S. chromogenes], Staphylococcus epidermidis [S. epidermidis], Staphylococcus hyicus [S. hyicus], Staphylococcus simulans [S. simulans], Streptococcous uberis [S. uberis], Streptococcus agalactiae [S. agalactiae], Streptococcous dysgalactiae [S. dysgalactiae],

Streptococcous faecalis [S. faecalis], Streptococcous faecium [S. faecium], Corynebacterium bovis, Bacillus licheniformis, Bacillus cereus, Trueperella pyogenes, Enterococcus spp., Enterococcus faecalis, Enterococcus faecium, Peptoniphilus indolicus, Arcanobacterium pyogenes, Lactococcus lactis, and Nocardia), Mycoplasma (e.g., Mycoplasma spp., Mycoplasma alkalescens, Mycoplasma bovis, Mycoplasma bovigenitalium, Mycoplasma califomicum, Mycoplasma canadense, Mycoplasma arginine, Mycoplasma dispar, and Mycoplasma bovihimis), Prototheca (e.g., Prototheca spp., Prototheca zopfii, Prototheca wickerhamii, and Prototheca blaschkeae), and yeast (e.g., Candida albicans and Cyrptococcus neoformans). For example, the microorganism or pathogen may be, for example, a microorganism selected from the group consisting of Mycoplasma, Prototheca, Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, and gram positive bacteria.

[00105] The method may comprise obtaining the milk sample (e.g., from a cow); processing the milk sample under conditions sufficient to separate or extract one or more nucleic acids from one or more microorganisms or pathogens to yield a processed sample (e.g., as described herein); and subjecting the processed sample to conditions sufficient to assay for a presence of the one or more nucleic acids (e.g., as described herein). The method may also comprise outputting a report that identifies a microorganism or pathogen in the milk sample. [00106] The method may be performed over a short time period (e.g., less than about 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour). The processing and/or assaying operations may be performed at the same or substantially the same location where the milk sample is collected. For example, a sample may be collected in a milking facility, refrigeration facility, or storage facility (e.g., a tank or silo), and analysis may be performed within the milking facility, refrigeration facility, or storage facility. Alternatively, a sample may be collected in a milking facility, refrigeration facility, or storage facility, and analysis may be performed in another facility in proximity to the milking facility, refrigeration facility, or storage facility where the sample was collected such as in a bam, storage facility, milking facility, refrigeration facility, housing facility, house, or other location. All or some of the processing and assaying operations may be performed in the same or a different location. Processing and/or assaying may also be performed outside. Sample collection may be performed at a first location and sample processing, assaying, and reporting may be performed at a second location that is within about 10 miles of the first location. The second location may be within about 9 miles, 8 miles, 7 miles, 6 miles, 5 miles, 4 miles, 3 miles, 2 miles, or 1 mile of the first location.

Performance of the method in a short period of time and/or in proximity to the site of sample collection may allow practitioners to rapidly interpret the results of an assay and use these results to make decisions regarding, for example, livestock activity. For instance, a result indicating the presence of one or more microorganisms or pathogens associated with mastitis may be used to inform a decision regarding whether to treat an animal or separate an animal from a collection of animals.

[00107] A sample may be processed using various assays. One or more assays may be performed in the container in which a sample is collected or provided or any other container. In some cases, an amplification reaction may be performed on a sample included in a sample preparation device, e.g., as described herein. In other cases, aliquots of sample may be transferred from a sample container to one or more other containers. For example, aliquots of sample may be transferred to a set of assay tubes or wells. The assay tubes or wells may be compatible with, or a component of, an analytic device useful for analyzing a sample, e.g., as described herein.

Analytic Device

[00108] Assaying may be performed in an analytic device. Alternatively, an assayed sample may be provided in an analytic device for analysis. For example, a nucleic acid amplification reaction may be performed separate from an analytic device, and the assayed sample may then be interrogated with the analytic device to, e.g., measure an endpoint signal corresponding to an amplified product. In other examples, assaying may be monitored using the analytic device to provide substantially real time measurements. For instance, real-time PCR may be performed in an analytic device.

[00109] An analytic device may include or be configured to accept one or more containers (e.g., assay tubes or wells, as described herein). For example, an analytic device may be configured to accept 2, 3, 4, 5, 6, 7, 8, 9, 10, or more containers including a sample, or a portion thereof. In this manner, multiple samples, or aliquots thereof, may be analyzed serially or in parallel. Each container included in or used in conjunction with an analytic device may be used to perform a different assay. For example, one or more different reagents (e.g., as described herein) may be added to each container of an analytic device. A first container may be used to perform a first assay while a second container may be used to perform a second assay. The first container may include a first set of primers and probes specific to a first microorganism or pathogen, or collection thereof, while the second container may include a second set of primers and probes specific to a second microorganism or pathogen, or collection thereof. Similarly, a third container may include a third set of primers and probes specific to a third microorganism or pathogen, or collection thereof. In one example, an analytic device is configured to accept three containers, and each container may be used to perform a different assay.

[00110] An analytic device may comprise a housing. A housing may comprise at least one button capable of, upon actuation, affecting the operability of the analytic device (e.g., powering on/off the device or connecting the analytic device to other devices). An analytic device may comprise 1, 2, 3, 4, 5, or more buttons. For example, an analytic device may comprise 4 buttons. Each button may correspond to a different function or feature of the analytic device. In some cases, pairs of buttons may correspond to the same function or feature of the analytic device. For example, an analytic device may include a button to increase a value, zoom level, volume, or other characteristic as well as a button to decrease the same value, zoom level, volume, or other characteristic. A button may be disposed anywhere on the housing. A button may comprise a physical mechanism (e.g., a depressible, slidable, or rotatable mechanism) or a touch-sensitive feature or mechanism. A touch-sensitive mechanism may be a touch-sensitive virtual mechanism (e.g., a virtual button). A virtual mechanism may be virtually depressible, virtually slidable, or virtually rotatable, thereby giving the illusion of a physical button. An analytic device may comprise or be configured to accept a mobile electronic device communicatively coupled with a wireless connection to the analytic device, and the mobile electronic device may comprise one or more virtual buttons. Depression of a virtual button of the mobile electronic device may transmit a signal from the mobile electronic device to the analytic device, thereby affecting, e.g., a thermocy cling program or other process, as described herein. A connection between an analytic device and a mobile electronic device may comprise a one-way or two-way wired or wireless connection, such as a WiFi connection, a Bluetooth connection, a Bluetooth LE connection, an ANT+ connection, or a Gazell connection. [00111] An analytic device may comprise a user display (e.g., as a component of a mobile electronic device) capable of displaying a report that identifies a presence of a target in a sample. For example, a user display may display a report indicating the presence of a particular microorganism or pathogen (e.g., bacterium) in a sample. The user display may also display a report indicating an absolute or relative quantity of a particular microorganism or pathogen (e.g., bacterium) in the sample. The amount may be calculated, e.g., based on an amount of emission at a particular wavelength or over a particular range of wavelengths measured corresponding to a sample.

[00112] An analytic device may be portable. For example, an analytic device including a housing may be able to be easily carried or moved. A size, weight and/or shape of the housing and/or other components may affect the portability of the analytic device. An analytic device may have an ergonomically shaped housing of a size that enables a user to hold the analytic device with one or two hands. For example, the housing may comprise a gripping region, e.g., a portion of the housing that is gripped by the user when the user holds the analytic device.

[00113] An analytic device may include or be configured to accept a mobile electronic device.

For example, an analytic device may comprise a dock configured to support a camera-enabled mobile electronic device.

[00114] An analytic device may be configured to heat or cool a sample within a container (e.g., an assay tube or well). An analytic device may be configured to raise or lower the temperature of a container using a heating block controlled by a thermal unit, as described herein. An analytic device may also include one or more fans configured to move air to cool the surface of a heating block.

[00115] An analytic device may comprise one or more excitation sources. An excitation source may be disposed on a carriage (e.g., a moving carriage, as described herein) and may be configured to deliver excitation energy to a sample (e.g., a sample in an assay tube or well) through an excitation filter and a light path. For an analytic device comprising a moving carriage, a single excitation source disposed on the carriage may be configured to deliver excitation energy to two or more samples (e.g., two or more samples in two or more containers) through the same excitation filter and light path (e.g., as the moving carriage aligns the excitation source and light path with different assay tubes containing different samples). An excitation source may comprise a Light Emitting Diode (LED) or an organic LED (OLED) or an array of LEDs or OLEDs (e.g., a set of single-color LEDs or OLEDs). An excitation source may have any useful size, shape, wavelength, or other characteristic. An excitation source may be a high power LED that may emit greater than or equal to about 1 mW of excitation energy. An array of excitation sources may be constructed and arranged in any configuration. For example, the excitation sources in an array may be arranged linearly along the axis of movement of a moving carriage.

[00116] An analytic device may comprise one or more optical components. For example, an analytic device may include one or more optical filters (e.g., an emission filter or an excitation filter) and one or more lenses. Filters may be optical bandpass filters (e.g., optical interference films) having a bandpass at a frequency that may be optimal for one or more of (i) an excitation wavelength of a fluorophore or dye, and (ii) an emission wavelength of a fluorophore or dye. A filter may substantially attenuate non-bandpass frequencies to prevent transmission of undesirable light. One or more lenses may be used to direct, re-direct, focus, disperse, or collimate excitation or emission energy. For example, a lens may be used to focus excitation energy onto a sample (e.g., a sample in an assay tube or well). In another example, a lens may be used to collimate excitation energy from an excitation source. Non-limiting examples of lenses that may be used include a biconvex lens, a plano-convex lens, a positive meniscus lens, a negative meniscus lens, a plano-concave lens, a biconcave lens, a Fresnel lens, a cylindrical lens, a lenticular lens, and a gradient index lens. For example, a Fresnel lens may be used to collimate excitation energy from an excitation source and direct the excitation energy into a light path. A Fresnel lens may be made much thinner than a comparable plano-convex lens, in some cases taking the form of a flat sheet, which may be advantageous for producing a portable analytic device.

[00117] A light path of an analytic device may comprise an open space of a particular geometry and volume. The space may be defined by a container or guide such as a pipe. A light path (e.g., a light pipe) may be constructed using any useful material. Non-limiting examples of materials that may be used to construct a light path (e.g., a light pipe) include glass, silica, fluorozirconate, fluoroaluminate, chalcogenide, plastic, PMMA, polystyrene, silicone resin, and any combination thereof.

[00118] An analytic device may comprise a carriage (e.g., a static or moving carriage). A moving carriage may be configured to translate along a predetermined path (e.g., via a track or groove) to bring an excitation source and/or optical filter in alignment with a light path that provides excitation energy from an excitation source to a container. A moving carriage may be used to shift a light path aligning with a first light source and a sample container to a second light source and a second sample container. Similarly, a moving carriage may be used to shift a sample from aligning with a first light path to align with a second light path. The inclusion of a moving carriage may thus allow multiple assay tubes to share light paths and associated components such as optical filters (e.g., excitation and emission filters). For example, a first sample aliquot contained within a first assay tube or well may be interrogated using excitation energy provided along a first light path of the moving carriage. The moving carriage may subsequently translate along a predetermined path and be repositioned such that it is configured to provide excitation energy along a second light path to a second sample aliquot contained within a second assay tube or well. In some instances, the second light path may be the same as the first light path.

[00119] A moving carriage may be configured to move (e.g., using an electric motor, motor with a cam, magnetic levitation, spring, or other components) from a first or original position to a final position, making one or more stops at specified positions between the original and final positions. The path between the original and final positions may be a linear path and may comprise one or more grooves, tracks, or rails along which a moving carriage may travel. The path between the original and final positions may comprise one or more specified positions at which a moving carriage may stop (e.g., via a manual or automated control, as described herein). The one or more specified positions may correspond to the positions of one or more containers or seats or housings therefor in an analytic device. A specified position may comprise a mechanical component such as a key to facilitate positioning of a moving carriage in the specified position (e.g., beneath a sample container).

[00120] An analytic device may comprise a detector. A detector may be configured to receive emission energy from a sample (e.g., a sample or aliquot thereof in a container), and possibly through an emission filter. Accordingly, the detector may comprise any suitable photodetector, such as, for example, an optical detector, a photoresistor, a photovoltaic cell, a photo diode, a phototube, a photomultiplier tube, a charge coupled device (CCD) camera, a complementary metal oxide semiconductor (CMOS), or any combination thereof. Emission energy may be produced by any suitable source, such as, for example, by the excitation of a component of a sample in a container (e.g., an excitable fluorophore). A detector may be configured to selectively receive emission energy from a sample (e.g., energy of a particular wavelength or intensity). A detector may comprise a plurality of detectors (e.g., a series of photodetectors, each configured to receive a light beam having a different wavelength than the light beams received by the other photodetectors).

[00121] An analytic device may comprise one or more additional components such as circuitry or a power supply. Circuitry may comprise a circuit board, e.g., within a housing of an analytic device. A power supply may be a self-contained power supply such as a battery. A power supply may provide power to at least one component of an analytic device such as a thermal unit, a fan, a processor, a moving carriage, a mobile electronic device, or an excitation source.

[00122] An analytic device may also include a processor configured to control one or more processes relating to analysis of a sample. For example, a processor may control heating or cooling of a sample, or a portion thereof (e.g., by instructing a thermal unit to heat or cease heating, or a fan to cool or cease cooling, a heating block in contact with a container including the sample, or portion thereof); addition of reagents to a container; mechanical mixing of components inside a sample container; or any other process relating to the analysis of a sample, as described herein.

[00123] In one example, an analytic device may comprise, for example, a housing; a heating block; a heating unit in thermal communication with the heating block and configured to provide thermal energy to a container (e.g., a container including a sample or portion thereof); and a light path (e.g., a light pipe) comprising an optical filter (e.g., an excitation filter or an emission filter) or a carriage (e.g., a moving carriage) comprising one or more light paths, where each light path comprises an optical filter and is configured to provide an excitation energy from an excitation source to a container. An analytic device including a carriage may also include a power supply, an excitation source, a processing unit configured to communicate with one or more components of the analytic device, and/or a user device. The user device may comprise or be a component of a computer, as described herein. The analytic device may also include a detector capable of detecting emission from a sample. The emission detected may be indicative of the presence or absence of a target (e.g., a target nucleic acid) within a sample. FIG. 9 shows an analytic device 902 including seats for a plurality of containers, a moving carriage 903, and heating blocks 904. FIG. 9 also shows a sample preparation device 901 configured for use with the analytic device.

[00124] The present disclosure provides a method of analyzing a sample (e.g., a milk sample) containing or suspected of containing one or more microorganisms or pathogens comprising providing the sample containing or suspected of containing the one or more microorganisms or pathogens; processing the sample under conditions sufficient to separate or extract one or more nucleic acids from the one or more microorganisms or pathogens, thereby yielding a processed sample; providing an analytic device; placing the processed sample, or a portion thereof, into an assay tube in the analytic device; subjecting the processed sample, or portion thereof, to conditions sufficient to assay for a presence of the one or more nucleic acids (e.g., by providing thermal energy to the processed sample, or portion thereof), where a presence of the one or more nucleic acids is indicative of a presence of the one or more microorganisms or pathogens in the sample; directing an excitation source through a light path of the analytic device (e.g., a light path of a moving carriage of the analytic device) to expose the processed sample, or portion thereof, to excitation energy; and detecting emission from the processed sample, or portion thereof, wherein the emission is indicative of the presence or absence of microorganisms or pathogens in the sample.

[00125] In another example, an analytic device may comprise a housing; a dock configured to receive a camera-enabled, mobile electronic device; a controllable assay chamber within the housing that is configured to support at least one container (e.g., assay tube or well); an excitation source; and a processing device configured to communicate with the mobile electronic device and to control at least one condition of the container based, at least in part, on instructions received from the mobile electronic device. Such a device may also be referred to as a“mobile diagnostic device.” One or both of the housing and the dock may comprise a window positioned such that, when the mobile electronic device is received in the dock, the camera of the mobile electronic device is positioned to capture images through the window. In this manner, the mobile electronic device may be a detector capable of, e.g., monitoring an amplification reaction.

[00126] FIG. 10 shows a perspective, partial cross-sectional, partial breakaway view of an analytic device including a mobile electronic device 206 in a docking station 204, and a lid 228 in an open position. In an upper portion of the analytic device, a chamber 230 is provided between a front wall (not shown) and a plenum wall 233. A rear assay tube 234c is shown and a middle assay tube 234b is shown in cross-section. The top 254 of assay tube 234c is also shown. A blower or fan 236 is positioned below the assay tubes. A battery 238 is positioned below the chamber 230. A battery charging circuit 239 is below fan 236. Assay tubes 234c and 234b are in plenum behind a plenum wall 233. Lid 228 in this example is connected to a rear rectangular wall 222 by a hinge. A ledge 248 may be used to maintain lid 228 in a closed position and a magnet 246 is provided in a recess in ledge 248 to maintain lid 228 in a closed position. The analytic device includes air vents 262 through rear wall 222 and air inlet vents 263 in side walls. The bottom of docking station 204 includes extensions 218 to tilt the analytic device when the device rests on a flat surface.

[00127] The present disclosure provides a method of analyzing a sample (e.g., a milk sample) containing or suspected of containing one or more microorganisms or pathogens comprising providing the sample containing or suspected of containing the one or more microorganisms or pathogens; processing the sample under conditions sufficient to separate one or more nucleic acids from the one or more microorganisms or pathogens, thereby yielding a processed sample; providing an analytic device comprising a dock configured to receive a camera-enabled mobile electronic device; placing the processed sample, or a portion thereof, into a container in the analytic device; subjecting the processed sample, or portion thereof, to conditions sufficient to assay for a presence of the one or more nucleic acids (e.g., by providing thermal energy to the processed sample, or portion thereof), where a presence of the one or more nucleic acids is indicative of a presence of the one or more microorganisms or pathogens in the sample; directing an excitation source through a light path to expose the processed sample, or portion thereof, to excitation energy; and detecting emission from the processed sample, or portion thereof, wherein the emission is indicative of the presence or absence of microorganisms or pathogens in the sample.

[00128] Additional details of analytic devices may be found, for example, in U.S. Patent Number 9,579,655, and U.S. Application Number 15/436,080, each of which is herein incorporated by reference in its entirety.

Nucleic Acid Amplification

[00129] An assay for analyzing a sample may include nucleic acid amplification. For example, any type of nucleic acid amplification reaction may be used to amplify a target nucleic acid and generate an amplified product. Moreover, amplification of a nucleic acid may be linear, exponential, or a combination thereof. Amplification may be emulsion-based or may be non emulsion based. Non-limiting examples of nucleic acid amplification methods include reverse transcription, primer extension, polymerase chain reaction (PCR), ligase chain reaction, asymmetric amplification, rolling circle amplification, and multiple displacement amplification (MDA).

[00130] A target nucleic acid may be, for example, a DNA or RNA, or a fragment thereof, such as a DNA or RNA from a microorganism or pathogen (e.g., a bacterium). An amplified product may comprise DNA. In cases where a target RNA is amplified, DNA may be obtained by reverse transcription of the RNA and subsequent amplification of the DNA may be used to generate an amplified DNA product. The amplified DNA product may be indicative of the presence of the target RNA in the biological sample. In cases where a target DNA is amplified, various DNA amplification methods may be employed. Non-limiting examples of DNA amplification methods include PCR, variants of PCR (e.g., real-time PCR, allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR, emulsion PCR, dial-out PCR, helicase-dependent PCR, nested PCR, hot start PCR, inverse PCR, methylation-specific PCR, miniprimer PCR, multiplex PCR, nested PCR, overlap-extension PCR, thermal asymmetric interlaced PCR, touchdown PCR), and ligase chain reaction (LCR). DNA amplification may be linear or exponential. DNA amplification may be achieved with nested PCR, which may improve the sensitivity of detecting amplified DNA products. In some cases, nucleic acid amplification is isothermal. Non-limiting examples of isothermal nucleic acid amplification methods include helicase-dependent amplification, nicking enzyme amplification, recombinase polymerase amplification, loop-mediated isothermal amplification, and nucleic acid sequence based amplification.

[00131] Nucleic acid amplification reactions may be conducted, for example, by including reagents necessary for each nucleic acid amplification reaction in a reaction vessel to obtain a reaction mixture and subjecting the reaction mixture to conditions necessary for each nucleic amplification reaction. Reagents may include, for example, primers, probes, polymerases, enzymes, lyophilization reagents, deoxyribonucleotides, washes, and elution buffers. Reverse transcription amplification and DNA amplification may be performed sequentially, such as, for example, performing reverse transcription amplification on RNA to generate complementary DNA (cDNA), and subsequently subjecting the cDNA to DNA amplification (e.g., PCR) to amplify the cDNA. [00132] A sample, or a portion thereof, including or suspected of including a nucleic acid may be subjected to conditions sufficient to assay the sample for the presence of the nucleic acid.

Nucleic acid amplification reactions may be conducted on multiple samples or aliquots of a single sample serially or in parallel. For example, a processed sample may be divided into multiple aliquots for analysis. Each aliquot of the sample may undergo the same or a different analysis. For example, each aliquot of the sample may be combined with one or more different reagents directed to a given target, such as, for example, a primer having sequence

complementarity with a target sequence. Amplification products may be detected optically, such as using fluorophores. Fluorophore-labeled primers or hybridization probes and/or fluorescent dyes that bind to DNA may be excited, and an emitted fluorescence detected. Fluorescence emission from a dye may also be detected. In some instances, the ratio of a fluorophore emission to a dye emission may be calculated.

Primers and Probes

[00133] A primer or probe used in an amplification reaction may comprise a fluorophore and/or a quencher. For example, a primer or probe may be a dual hybridization primer or probe. A three- dimensional structure of an unbound primer may be such that a quencher is in close enough proximity to a fluorophore to prevent excitation of the fluorophore and/or the detection of an emission signal from the fluorophore. A primer may be a Scorpion primer.

[00134] A fluorescent DNA dye, such as SYBR Green I or BRYT Green, may be added to a mixture containing a target nucleic acid and at least one amplification primer. An amplification primer may be a linear single-stranded oligonucleotide that is extendable by a DNA polymerase. In some cases, an amplification primer or probe may be labeled with a fluorophore that is excitable. Upon performing an amplification reaction, such as, e.g., a polymerase chain reaction that includes annealing and extending the primer, the fluorophore (e.g., of the amplification primer or probe or a dye) may be excited and an emission detected either during the amplification (real-time detection) or following completion of amplification (either an end-point detection at the conclusion of the amplification reaction or during a subsequent thermal analysis (melting curve)). Unincorporated primers, probes, or dyes may not fluoresce.

[00135] A wide range of fluorophores and/or dyes may be used in primers according to the present disclosure. Available fluorophores include, but are not limited to, ATT0647N, coumarin, fluorescein, tetrachlorofluorescein, hexachlorofluorescein, Lucifer yellow, rhodamine, BODIPY, tetramethylrhodamine, Cy3, Cy5, Cy7, eosine, Texas red, SYBR Green I, SYBR Gold, 5-FAM (also called 5-carboxyfluorescein; also called Spiro(isobenzofuran-l(3H), 9'- (9H)xanthene)-5 -carboxylic acid, 3',6'-dihydroxy-3-oxo-6-carboxyfluorescein); 5-Hexachloro- Fluorescein ([4,7,2',4',5',7'-hexachloro-(3',6'-dipivaloyl-fluoresceinyl )-6-carboxylic acid]); 6- Hexachloro-Fluorescein ([4,7,2',4',5',7'-hexachloro-(3',6'-dipivaloylfluoresceinyl) -5-carboxylic acid]); 5-Tetrachloro-Fluorescein ([4,7,2',7'-tetra-chloro-(3',6'-dipivaloylfluoresceinyl)-5- carboxylic acid]); 6-Tetrachloro-Fluorescein ([4,7,2',7'-tetrachloro-(3',6'- dipivaloylfluoresceinyl)-6-carboxylic acid]); 5-TAMRA (5-carboxytetramethylrhodamine;

Xanthylium, 9-(2,4-dicarboxyphenyl)-3,6-bis(dimethyl-amino); 6-TAMRA (6- carboxytetramethylrhodamine; Xanthylium, 9-(2,5-dicarboxyphenyl)-3,6-bis(dimethylamino); EDANS (5-((2-aminoethyl)amino)naphthalene-l -sulfonic acid); l,5-IAEDANS (5-((((2- iodoacetyl)amino)ethyl)amino)naphthalene-l -sulfonic acid); DABCYL (4-((4- (dimethylamino)phenyl) azo)benzoic acid) Cy5 (Indodicarbocyanine-5) Cy3 (Indo- dicarbocyanine-3); and BODIPY FL (2,6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza- s-indacene-3-proprionic acid), Quasar-670 (Bioreseach Technologies), CalOrange (Bioresearch Technologies), Rox, as well as suitable derivatives thereof. For example, a fluorophore may be selected from the group consisting of Texas Red, Cy5, and FAM. Combination fluorophores such as fluorescein-rhodamine dimers, may also be suitable. Fluorophores may be chosen to absorb and emit in the visible spectrum or outside the visible spectrum, such as in the ultraviolet or infrared ranges. Suitable quenchers may also include DABCYL and variants thereof, such as DABSYL, DABMI and Methyl Red. Fluorophores may also be used as quenchers, because they tend to quench fluorescence when touching certain other fluorophores. Quenchers may be either chromophores such as DABCYL or malachite green, or fluorophores that may not fluoresce in the detection range when the probe is in the open conformation.

[00136] Allele-discriminating probes useful according to the present disclosure may also include probes that bind less effectively to a target-like sequence, as compared to a target sequence. The change in the level of fluorescence in the presence or absence of a target sequence compared to the change in the level of fluorescence in the presence or absence of a target-like sequence, may provide a measure of the effectiveness of binding of a probe to a target or target-like sequence.

[00137] Primer and/or probe sets directed to a target nucleic acid may be utilized to conduct a nucleic acid amplification reaction. Primer sets may comprise one or more primers. For example, a primer set may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more primers.

Similarly, probe sets may comprise one or more probes, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more probes. A primer or probe set may comprise primers or probes directed to different amplified products or different nucleic acid amplification reactions. For example, a primer or probe set may comprise a first primer or probe necessary to generate a first strand of nucleic acid product that is complementary to at least a portion of the target nucleic acid and a second primer or probe complementary to the nucleic acid strand product necessary to generate a second strand of nucleic acid product that is complementary to at least a portion of the first strand of nucleic acid product. For example, a primer or probe set may be directed to a target RNA. The primer or probe set may comprise a first primer or probe that may be used to generate a first strand of nucleic acid product that is complementary to at least a portion the target RNA. In the case of a reverse transcription reaction, the first strand of nucleic acid product may be DNA. The primer or probe set may also comprise a second primer or probe that may be used to generate a second strand of nucleic acid product that is complementary to at least a portion of the first strand of nucleic acid product. In the case of a reverse transcription reaction conducted with DNA amplification, the second strand of nucleic acid product may be a strand of nucleic acid (e.g., DNA) product that is complementary to a strand of DNA generated from an RNA template.

[00138] A plurality of containers including a plurality of samples or sample aliquots may include the same primers or primer sets, or different primers or primer sets. Similarly, a plurality of containers including a plurality of samples or sample aliquots may include the same probes or probe sets, or different probes or probe sets. Each container may be directed to a different target, or at least a subset of the containers may be directed to the same target. Any suitable number of primer or probe sets may be used. For example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more primer and/or probe sets may be used. Where multiple primer or probe sets are used, one or more primer or probe sets may each correspond to a particular nucleic acid amplification reaction or amplified product. For example, a sample may be divided into 2, 3, 4, 5, or more aliquots, and each aliquot may include a different primer or probe set. This may facilitate, for example, the detection of different target nucleic acids within a single sample.

[00139] Primers and probes may be specific to particular microorganisms or pathogens, such as a microorganism or pathogen associated with mastitis. For example, a primer or probe may be specific to a microorganism or pathogen selected from the group consisting of gram negative bacteria (e.g., Escherichia coli [E. coli], Klebsiella pneumoniae, Klebsiella oxytoca,

Pseudomonas aeruginosa, Citrobacter, Enterobacter, Serratia marcescens, and Pasteurella), gram positive bacteria (e.g., Staphylococcus spp., Staphylococcus aureus [S. aureus], Staphylococcus chromogenes [S. chromogenes], Staphylococcus epidermidis [S. epidermidis], Staphylococcus hyicus [S. hyicus], Staphylococcus simulans [S. simulans], Streptococcous uberis [S. uberis], Streptococcus agalactiae [S. agalactiae], Streptococcous dysgalactiae [S. dysgalactiae],

Streptococcous faecalis [S. faecalis], Streptococcous faecium [S. faecium], Corynebacterium bovis, Bacillus licheniformis, Bacillus cereus, Trueperella pyogenes, Enterococcus spp., Enterococcus faecalis, Enterococcus faecium, Peptoniphilus indolicus, Arcanobacterium pyogenes, Lactococcus lactis, and Nocardia), Mycoplasma (e.g., Mycoplasma spp., Mycoplasma alkalescens, Mycoplasma bovis, Mycoplasma bovigenitalium, Mycoplasma califomicum, Mycoplasma canadense, Mycoplasma arginine, Mycoplasma dispar, and Mycoplasma bovihimis), Prototheca (e.g., Prototheca spp., Prototheca zopfii, Prototheca wickerhamii, and Prototheca blaschkeae), and yeast (e.g., Candida albicans and Cyrptococcus neoformans). For example, a primer or probe may be specific to a microorganism or pathogen selected from the group consisting of Mycoplasma, Prototheca, Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, Escherichia coli, and gram positive bacteria. A probe may be specific to one or more microorganisms or pathogens and may comprise a fluorophore, as described herein. For example, a probe specific to gram positive bacteria may comprise a Texas Red fluorophore. A probe specific to Streptococcus agalactiae and Staphylococcus aureus may comprise a Cy5 fluorophore. A probe specific to Mycoplasma and Prototheca may comprise a FAM fluorophore.

Amplification reactions

[00140] Any suitable type and number of nucleic acid amplification reactions may be conducted. In some cases, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleic acid amplification reactions are conducted. [00141] A nucleic acid amplification reaction may be isothermal or may involve thermal cycling. For example, polymerase chain reaction (PCR) may comprise thermal cycling (e.g., cycling the temperature of a sample in a container). Performing PCR may involve making a series of repeated temperature changes (e.g., thermal cycles) with each series (e.g., cycle) including 1, 2,

3, 4, or more discrete temperature operations. Thermal cycling may be preceded by a single temperature operation at a higher temperature (e.g., >90°C). Temperatures used and the length of time they are applied in each cycle may vary based on, for example, the enzyme used for DNA synthesis, the concentration of bivalent ions and nucleotides (dNTPs) in the reaction, and the melting temperature (Tm) of one or more primers. The individual operations of an amplification reaction such as PCR may comprise initialization, denaturation, annealing, and/or

extension/elongation. An amplification reaction may also comprise or be preceded by a reverse transcription operation in which a complementary DNA (cDNA) is produced from an RNA. Initialization may be used for DNA polymerases that require heat activation (e.g.,“hot start” PCR). Initialization may comprise heating a sample (e.g., a sample in an assay tube) to a high temperature (e.g., 94-96°C [20l-205°F) or 98°C [208°F], if thermostable polymerases are used), which may be maintained for about 1-10 minutes (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes). Denaturation may comprise heating (e.g., to 94-98°C [20l-208°F]) a sample (e.g., a sample in an assay tube) for a given time, such as between about 1 second and 5 minutes, 5 seconds and 5 minutes, or 30 seconds and 5 minutes. This may result in DNA melting, or denaturation, of a double-stranded DNA template by breaking hydrogen bonds between complementary bases, yielding two single-stranded nucleic acids (e.g., templates). Annealing may comprise lowering the temperature of a sample (e.g., a sample in an assay tube) to, e.g., 50- 65°C (l22-l49°F) for a given time, such as between about 1 second and 5 minutes, 5 seconds and 5 minutes, or 30 seconds and 5 minutes, thereby allowing annealing of one or more primers to each of the single-stranded nucleic acid templates. At least two different primers may be included in the reaction mixture, including one for each of the two single-stranded nucleic acid templates containing a target region. The primers may be single-stranded nucleic acids themselves. Conditions suitable for effective extension/elongation may depend on the DNA polymerase used. Extension/elongation comprises synthesizing a new DNA strand

complementary to a single-stranded nucleic acid template by adding, in the presence of a DNA polymerase, free dNTPs from a reaction mixture that are complementary to the template in the 5'- to-3' direction and condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxy group at the end of the nascent (elongating) DNA strand. The time required for extension/elongation may depend on the DNA polymerase used and/or on the length of the DNA target region to amplify.

[00142] Denaturation, annealing, and extension/elongation may constitute a single thermal cycle. Multiple cycles may be required to amplify a nucleic acid target to a detectable level.

[00143] Any useful number of thermal cycles may be performed. For example, at least about 1 thermal cycle, 2 thermal cycles, 3 thermal cycles, 4 thermal cycles, 5 thermal cycles, 6 thermal cycles, 7 thermal cycles 8 thermal cycles, 9 thermal cycles, 10 thermal cycles, 11 thermal cycles, 12 thermal cycle, 13 thermal cycles, 14 thermal cycles, 15 thermal cycles, 16 thermal cycles, 17 thermal cycles, 18 thermal cycles 19 thermal cycles, 20 thermal cycles, 25 thermal cycles, 30 thermal cycles, 35 thermal cycles, 40 thermal cycles, 45 thermal cycles, 50 thermal cycles, 100 thermal cycles, or more thermal cycles may be performed. Thermal cycling may be carried out by regulating the heat of a heating block of an analytic device.

[00144] In one example, a target nucleic acid (e.g., a target RNA or DNA) may be extracted or released from a biological sample during heating phases of nucleic acid amplification. In the case of a target RNA, for example, the biological sample comprising the target RNA may be heated and the target RNA released from the biological sample. The released target RNA may begin reverse transcription (via reverse transcription amplification) to produce complementary DNA. The complementary DNA may then be amplified.

[00145] A DNA polymerase may be used in an amplification reaction. Any suitable DNA polymerase may be used, including commercially available DNA polymerases. A DNA polymerase may refer to an enzyme that is capable of incorporating nucleotides to a strand of DNA in a template bound fashion. Non-limiting examples of DNA polymerases include Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Expand polymerases, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, Pho polymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tih polymerase, Tfi polymerase, Platinum Taq polymerases, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragment, and variants, modified products, and derivatives thereof. A “hot start” polymerase may be used, e.g., in an amplification reaction. For certain“hot start” polymerases, a denaturation operation at about 94°C - 95°C for a time period from about 30 seconds to 10 minutes, 1 minute to 10 minutes, or 2 minutes to 10 minutes, may be required, which may change the thermal profile based on different polymerases.

[00146] A lysis agent may also be used during sample processing. The lysis agent may be used to release a nucleic acid, such as a DNA or an RNA, from a biological particle, such as, for example, a cell or viral particle. Any suitable lysis agent may be used, including commercially available lysis agents. Non-limiting examples of lysis agents include Tris-HCl, EDTA, detergents (e.g., Triton X-100, SDS), lysozyme, glucolase, proteinase E, viral endolysins, exolysins zymolose, Iyticase, proteinase K, endolysins and exolysins from bacteriophages, endolysins from bacteriophage PM2, endolysins from the B. subtilis bacteriophage PBSX, endolysins from Lactobacillus prophages Lj928, Lj965, bacteriophage 15 Phiadh, endolysin from the Streptococcus pneumoniae bacteriophage Cp-I, bifunctional peptidoglycan lysin of

Streptococcus agalactiae bacteriophage B30, endolysins and exolysins from prophage bacteria, endolysins from Listeria bacteriophages, holin-endolysin, cell 20 lysis genes, guanidine thiocyanate, guanidine hydrochloride, and combinations thereof. In some cases a buffer may comprise a lysis agent (e.g., a lysis buffer), as described herein. An example of a lysis buffer is sodium hydroxide (NaOH).

Other processes

[00147] Assays may also be performed to measure other characteristics of a sample. In some instances, an assay, measurement, or assessment may be performed prior to processing of the sample. For example, a visual assessment of a milk sample may be performed to assess the qualities of milk (e.g., wateriness, thickness, and ropiness). An assay may also be performed to measure a quantity of cells within a sample. For example, leukocytes within a sample may be quantified. Cell counting may be performing using a coarse technique, such as the California Mastititis Test, or using a more robust cell counting test. Obtaining a somatic cell count of a milk sample may involve combining a milk sample with an anionic surfactant, agitating the sample, and observing gel formation. Information (e.g., results) associated with these or other assays performed on a sample may be outputted in a report, e.g., along with information associated with an amplification assay, generated on a user display.

Kits

[00148] Provided herein are kits for processing and/or assaying samples. A kit may include one or more sample processing cartridges and/or one or more reagents. The kit may include instructions for processing a sample. The cartridge may be configured to interface with a system of the present disclosure. [00149] The one or more reagents may include lysis buffer, wash buffer, a drying agent, and an elution buffer. For example, the one or more reagents can comprise NP-40 lysis buffer, Radio Immunoprecipitation Assay (RIP A) lysis buffer, sodium dodecyl sulfate (SDS) lysis buffer, Ammonium-Chloride-Potassium (ACK) lysing buffer, volatile chemicals (e.g., acetone and ethanol), EDTA, Tris-HCl, phosphate-buffered saline (PBS), hexamethyldisilazane (HMDS), trichlorotrifluoroethane (Freon 113), tetramethylsilane (TMS), PELDRI II, and water. The one or more reagents may include chaotropic salts, such as guanidine thiocyanate and guanidine hydrochloride. In some cases, the one or more reagents comprise lithium chloride. The one or more reagents can be stable when stored at room temperature. The one or more reagents can be stable for at least one year, at least two years, at least three years, at least four years, at least five years, or more when stored at room temperature.

[00150] The one or more reagents may comprise dry agents. The dry agents can be stable when stored at room temperature. The dry agents can be stable for at least one year, at least two years, at least three years, at least four years, at least five years, or more when stored at room temperature.

[00151] The instructions may be in physical (e.g., printed) or electronic form. The instructions may be in print media. As an alternative, the instructions may be accessible by a user on the Internet, such as through a uniform resource locator.

[00152] A kit may comprise one or more reagents for assaying a sample. For example, a kit may comprise one or more reagents for nucleic acid amplification. The one or more reagents for nucleic acid amplification include, but are not limited to, primers, dNTPs, enzymes, and buffers. In some cases, the kit comprises reagents necessary for reverse transcription amplification and DNA amplification. [00153] A kit may comprise one or more primers. A kit may comprise one or more primer sets. For example, a primer set directed to a target RNA may be contained in the kit. The primer set may comprise a first primer that may be used to generate a first strand of nucleic acid product that is complementary to at least a portion the target RNA. In the case of a reverse transcription reaction, the first strand of nucleic acid product may be DNA. The primer set may also comprise a second primer that may be used to generate a second strand of nucleic acid product that is complementary to at least a portion of the first strand of nucleic acid product. In the case of a reverse transcription reaction conducted with DNA amplification, the second strand of nucleic acid product may be a strand of nucleic acid (e.g., DNA) product that is complementary to a strand of DNA generated from an RNA template. The kit can comprise any suitable number of primer sets. For example, the kit can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more primer sets. Where multiple primer sets are included in a kit, one or more primer sets may each correspond to a particular nucleic acid amplification reaction or amplified product.

[00154] The kit may comprise a DNA polymerase. Any suitable DNA polymerase may be included in the kit, including commercially available DNA polymerases. A DNA polymerase may refer to an enzyme that is capable of incorporating nucleotides to a strand of DNA in a template bound fashion. Non-limiting examples of DNA polymerases include Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Expand polymerases, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, Pho polymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tih polymerase, Tfi polymerase, Platinum Taq polymerases, Hi-Fi polymerase, Tbr polymerase, Tfi polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragment, and variants, modified products, and derivatives thereof. A “hot start” polymerase may be used, e.g., in an amplification reaction. For certain“hot start” polymerases, a denaturation step at about 94°C - 95°C for about 2 minutes to 10 minutes may be required, which may change the thermal profile based on different polymerases.

[00155] The kit can comprise a reagent cartridge. The reagents used for assays (e.g., thermocy cling reactions or nucleic acid amplifications) can be provided in the reagent cartridge. The reagent cartridge can be premixed or prepacked. The reagent cartridge can be prepacked and ready for use. The reagent cartridge can be designed for different targets, for example, by containing primers specific for a given target or given targets. For example, the reagent cartridge can be designed for targeting microorganisms that cause a disease. In some embodiments, the reagent cartridge is designed for targeting nucleic acids from one or more microorganisms that cause fever or flu. In some embodiments, the reagent cartridge is designed for targeting nucleic acids from one or more viruses that cause fever or flu. In some embodiments, the reagent cartridge is designed for targeting nucleic acids from one or more microorganisms that cause an infectious disease. In some embodiments, the reagent cartridge is designed for targeting one or more microorganisms present in a sample. In some embodiments, the reagent cartridge is designed for targeting one or more microorganisms present in an environmental sample. The reagent cartridge can comprise a chamber for sample loading.

[00156] The reagent cartridge can be stable and have a long shelf life. For example, the reagent cartridge can be stable at ambient condition or have a shelf life of at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months,

5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, or 30 months. For another example, the reagent cartridge can be stable at ambient condition or have a shelf life of at least 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 4 years, 5 years, or longer.

[00157] In some cases, the reagent used for assays can be divided into two parts, a dry part and a wet (e.g., liquid) part. The dry part can be provided in a reagent cartridge as described herein. The wet part can be provided in a separate container in the kit. The dry part and the wet part can be mixed in the analytic device when performing an assay.

[00158] In some embodiments, the wet part can be provided in a reagent cartridge as described herein. The dry part can be provided in a separate container. The dry part and the wet part can be mixed in the analytic device when performing an assay.

[00159] In some embodiments, both the dry part and the wet part can be provided in a reagent cartridge without contacting or mixing with each other. In some embodiments, both the dry part and the part can be provided in separate reagent cartridges.

[00160] A kit may comprise a detectable label, for example, a fluorescent dye or a fluorophore. In some cases, the kit comprises one or more primers labeled with a detectable label. In some cases, the kit comprises a fluorophore. In some cases, the kit further comprises a quencher. Available fluorophores include ATT0647N, coumarin; fluorescein; tetrachlorofluorescein; hexachlorofluorescein; Lucifer yellow; rhodamine; BODIPY; tetramethylrhodamine; Cy3; Cy5; Cy7; eosine; Texas red; SYBR Green I; SYBR Gold; 5-FAM (also called 5-carboxyfluorescein; also called Spiro(isobenzofuran-l(3H), 9'-(9H)xanthene)-5-carboxylic acid, 3',6'-dihydroxy-3- oxo-6-carboxyfluorescein); 5-Hexachloro-Fluorescein ([4,7,2',4',5',7'-hexachloro-(3',6'- dipivaloyl-fluoresceinyl)-6-carboxylic acid]); 6-Hexachloro-Fluorescein ([4, 7, 2', 4', 5', 7'- hexachloro-(3',6'-dipivaloylfluoresceinyl)-5-carboxylic acid]); 5-Tetrachloro-Fluorescein ([4,7,2',7'-tetra-chloro-(3',6'-dipivaloylfluoresceinyl)-5-c arboxylic acid]); 6-Tetrachloro- Fluorescein ([4,7,2',7'-tetrachloro-(3',6'-dipivaloylfluoresceinyl)-6-ca rboxylic acid]); 5-TAMRA (5-carboxytetramethylrhodamine; Xanthylium, 9-(2,4-dicarboxyphenyl)-3,6-bis(dimethyl- amino); 6-TAMRA (6-carboxytetramethylrhodamine; Xanthylium, 9-(2,5-dicarboxyphenyl)-3,6- bis(dimethylamino); EDANS (5-((2-aminoethyl)amino)naphthalene-l -sulfonic acid); 1,5- IAEDANS (5 -((((2-iodoacetyl)amino)ethyl)amino)naphthalene-l -sulfonic acid); DABCYL (4- ((4-(dimethylamino)phenyl) azo)benzoic acid) Cy5 (Indodicarbocyanine-5) Cy3 (Indo- dicarbocyanine-3); BODIPY FL (2,6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s- indacene-3-proprionic acid); Quasar-670 (Bioreseach Technologies); CalOrange (Bioresearch Technologies); and Rox as well as suitable derivatives thereof. Combination fluorophores such as fluorescein-rhodamine dimers may also be suitable. Fluorophores may be chosen to absorb and emit in the visible spectrum or outside the visible spectrum, such as in the ultraviolet or infrared ranges. Suitable quenchers may also include DABCYL and variants thereof, such as DABSYL, DABMI and Methyl Red. Fluorophores may also be used as quenchers, because they tend to quench fluorescence when touching certain other fluorophores. Quenchers may be chromophores such as DABCYL or malachite green, or fluorophores that may not fluoresce in the detection range.

[00161] A kit may comprise a probe. For example, the kit can comprise an allele-discriminating probe. Allele-discriminating probes include probes that bind less effectively to a target-like sequence, as compared to a target sequence. The change in the level of fluorescence in the presence or absence of a target sequence compared to the change in the level of fluorescence in the presence or absence of a target-like sequence may provide a measure of the effectiveness of binding of a probe to a target or target-like sequence.

[00162] A kit may comprise a filter. The filter may be a disposable filter. The filter may be configured to or may be used to remove particulates from a sample. For example, the filter can be configured to or can be used to remove large particulates from a milk sample. In some cases, the filter can be used to remove particulates from a sample prior to processing through the nucleic acid sample preparation device. The filter can include pore sizes of at least about 0.1 micrometers (pm), 0.2 pm, 0.3 pm, 0.4 pm, 0.5 pm, 0.6 pm, 0.7 pm, 0.8 pm, 0.9 pm, 1 pm, 10 pm, 20 pm, 30 pm, 40 pm, 50 pm, 100 pm, 200 pm, 300 pm, 400 pm, 500 pm, 1000 pm, 2000 pm, 3000 pm, 4000 pm, 5000 pm, or greater. The pore sizes may range from 0.2 pm to 0.8 pm, from 0.8 pm to 1 pm, from 1 pm to 2 pm, from 2 pm to 5 pm, from 5 pm to 10 pm, from 10 pm to 20 pm, from 20 pm to 40 pm, from 40 pm to 60 pm, from 60 pm to 80 pm, from 80 pm to 100 pm, from 100 pm to 200 pm, from 200 pm to 400 pm, from 400 pm to 600 pm, from 600 pm to 800 pm, from 800 pm to 1,000 pm, from 1,000 pm to 1,200 pm, or from 1,200 pm to 1,500 pm. The filter can be formed of various materials, such as a polymeric material, a metal- containing material, a semiconductor material, an insulating material, and/or a composite material. The filter can be formed of, without limitation, one or more materials selected from the group consisting of polypropylene, polyethylene, polycarbonate, gold, silicon, silicon oxide, silicon oxynitride, indium, tantalum oxide, niobium oxide, titanium, titanium oxide, platinum, iridium, indium tin oxide, diamond or diamond-like film, acrylic, styrene-methyl methacrylate copolymers, ethylene/acrylic acid, acrylonitrile-butadiene-styrene (ABS), ABS/poly carbonate, ABS/polysulfone, ABS/polyvinyl chloride, ethylene propylene, ethylene vinyl acetate (EVA), nitrocellulose, nylons (including nylon 6, nylon 6/6, nylon 6/6-6, nylon 6/9, nylon 6/10, nylon 6/12, nylon 11 and nylon 12), polyacrylonitrile (PAN), polyacrylate, polycarbonate, polybutylene terephthalate (PBT), poly(ethylene) (PE) (including low density, linear low density, high density, cross-linked and ultra-high molecular weight grades), poly(propylene) (PP), cis and trans isomers of poly(butadiene) (PB), cis and trans isomers of poly(isoprene), polyethylene terephthalate) (PET), polypropylene homopolymer, polypropylene copolymers, polystyrene (PS) (including general purpose and high impact grades), polycarbonate (PC), poly(epsilon-caprolactone) (PECL or PCL), poly(methyl methacrylate) (PMMA) and its homologs, poly(methyl acrylate) and its homologs, poly(lactic acid) (PLA), poly(gly colic acid), polyorthoesters, poly(anhydrides), nylon, polyimides, polydimethylsiloxane (PDMS), polybutadiene (PB), polyvinylalcohol (PVA), polyacrylamide and its homologs such as poly(N-isopropyl acrylamide), fluorinated polyacrylate (PFOA), poly(ethylene-butylene) (PEB), poly(styrene-acrylonitrile) (SAN),

polytetrafluoroethylene (PTFE) and its derivatives, polyolefin plastomers, fluorinated ethylene- propylene (FEP), ethylene-tetrafluoroethylene (ETFE), perfluoroalkoxy ethylene (PFA), polyvinyl fluoride (PVF), polyvinylidene fluoride (PVDF), polychlorotrifluoroethylene (PCTFE), polyethylene-chlorotrifluoroethylene (ECTFE), styrene maleic anhydride (SMA), metal oxides, glass, glass wool, silicon oxide or other inorganic or semiconductor material (e.g., silicon nitride), compound semiconductors (e.g., gallium arsenide, and indium gallium arsenide), and combinations thereof.

Computer systems

[00163] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 12 shows a computer system 1201 that is programmed or otherwise configured to process and/or assay a sample. The computer system 1201 may regulate various aspects of sample processing and assaying of the present disclosure, such as, for example, activation of a valve or pump to transfer a reagent or sample from one chamber to another or application of heat to a sample (e.g., during an amplification reaction). The computer system 1201 may be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device may be a mobile electronic device.

[00164] The computer system 1201 includes a central processing unit (CPU, also“processor” and“computer processor” herein) 1205, which may be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 1201 also includes memory or memory location 1210 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 1215 (e.g., hard disk), communication interface 1220 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1225, such as cache, other memory, data storage and/or electronic display adapters. The memory 1210, storage unit 1215, interface 1220 and peripheral devices 1225 are in communication with the CPU 1205 through a communication bus (solid lines), such as a motherboard. The storage unit 1215 may be a data storage unit (or data repository) for storing data. The computer system 1201 may be operatively coupled to a computer network (“network”) 1230 with the aid of the communication interface 1220. The network 1230 may be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 1230 in some cases is a telecommunication and/or data network. The network 1230 may include one or more computer servers, which may enable distributed computing, such as cloud computing. The network 1230, in some cases with the aid of the computer system 1201, may implement a peer-to-peer network, which may enable devices coupled to the computer system 1201 to behave as a client or a server.

[00165] The CPU 1205 may execute a sequence of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 1210. The instructions may be directed to the CPU 1205, which may

subsequently program or otherwise configure the CPU 1205 to implement methods of the present disclosure. Examples of operations performed by the CPU 1205 may include fetch, decode, execute, and writeback.

[00166] The CPU 1205 may be part of a circuit, such as an integrated circuit. One or more other components of the system 1201 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC). [00167] The storage unit 1215 may store files, such as drivers, libraries and saved programs. The storage unit 1215 may store user data, e.g., user preferences and user programs. The computer system 1201 in some cases may include one or more additional data storage units that are external to the computer system 1201, such as located on a remote server that is in

communication with the computer system 1201 through an intranet or the Internet.

[00168] The computer system 1201 may communicate with one or more remote computer systems through the network 1230. For instance, the computer system 1201 may communicate with a remote computer system of a user. Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung®

Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device,

Blackberry®), or personal digital assistants. The user may access the computer system 1201 via the network 1230.

[00169] Methods as described herein may be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 1201, such as, for example, on the memory 1210 or electronic storage unit 1215. The machine executable or machine readable code may be provided in the form of software. During use, the code may be executed by the processor 1205. In some cases, the code may be retrieved from the storage unit 1215 and stored on the memory 1210 for ready access by the processor 1205. In some situations, the electronic storage unit 1215 may be precluded, and machine-executable instructions are stored on memory 1210.

[00170] The code may be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or may be compiled during runtime. The code may be supplied in a programming language that may be selected to enable the code to execute in a pre compiled or as-compiled fashion. [00171] Aspects of the systems and methods provided herein, such as the computer system 1201, may be embodied in programming. Various aspects of the technology may be thought of as “products” or“articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code may be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk.

“Storage” type media may include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine“readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[00172] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[00173] The computer system 1201 may include or be in communication with an electronic display 1235 that comprises a user interface (UI) 1240 for providing, for example, a current stage of processing or assaying of a sample (e.g., a particular operation, such as a lysis operation, that is being performed). Examples of UTs include, without limitation, a graphical user interface (GUI) and web-based user interface.

[00174] Methods and systems of the present disclosure may be implemented by way of one or more algorithms. An algorithm may be implemented by way of software upon execution by the central processing unit 1205.

EXAMPLES

Example 1. Processing a milk sample. [00175] 500 mΐ of milk is provided in a sample container. The milk may be from a freshly milked cow or collection of cows or frozen stored milk. The milk may be provided in a sample preparation column comprising a filter or may be transferred to the sample preparation column during processing. The milk sample is processed to remove solids and other unwanted materials and to prepare the sample for subsequent analysis. Processing the milk sample may comprise phase separation via bead beading, precipitation, filtration, or a combination thereof, as described herein. The milk sample is then combined with 1 ml of a lysis buffer. The solution is then mixed and pumped through the sample preparation column using a 1 ml syringe. The column is then washed one or more times with a protein wash, a wash buffer, and a drying wash. The column is then air dried using a syringe. Nucleic acids trapped on the filter of the sample preparation column are then eluted using 125 mΐ to 1 ml of an elution buffer.

Example 2. Assaying a milk sample.

[00176] A processed milk sample (e.g., a milk sample processed according to Example 1) is provided. One or more portions of the processed sample are transferred to a well (e.g., a well of a three well strip) for analysis. Each portion of the processed sample may be transferred to a separate well, and each well may include necessary reagents to perform a real time polymerase chain reaction, including lyophilized master mix, primers, and probes. The master mix includes polymerase, nucleotides, and buffering salts. The master mix for RNA targets also includes a reverse transcriptase enzyme. The primer and probe sets of each well are specific to one or more particular microorganisms (e.g., pathogens such as bacteria). Tables 1-4 include details of primers and probes specific to, for example, gram positive bacteria, Staphylococcus aureus, Streptococcus agalactiae, Mycoplasma, and Prototheca. Table 1. Primers and probes for gram positive bacteria, Staphylococcus aureus, and

Streptococcus agalactiae.

Table 2. Primers and probes for Mycoplasma.

Table 3. Primers and probes for Prototheca.

Table 4. Additional primers and probes.

[00177] Amplification reactions are carried out in each well to provide amplified product corresponding to a nucleic acid of each microorganism (e.g, pathogen such as a bacterium). The presence of amplification signal for a given well is thus indicative of the presence or absence of a particular microorganism in the well and thus in the original sample. Information regarding the presence or absence of a particular microorganism may be used to, for example, evaluate a sample for mastitis.

[00178] FIGs. 1A-1B show amplification (FIG. 1A) and standard (FIG. IB) curves for gram positive bacteria primers and probes using tenfold serial dilutions from 50,000 to 50 genome equivalents. FIGs. 2A-2B show amplification (FIG. 2A) and standard (FIG. 2B) curves for Mycoplasma primers and probes using tenfold serial dilutions from 50,000 to 50 genome equivalents. FIGs. 3A-3B show amplification (FIG. 3A) and standard (FIG. 3B) curves for Prototheca primers and probes using tenfold serial dilutions from 50,000 to 50 genome equivalents. FIGs. 4A-4B show amplification (FIG. 4A) and standard (FIG. 4B) curves for Staphylococcus aureus primers and probes using tenfold serial dilutions from 50,000 to 50 genome equivalents. FIGs. 5A-5B show amplification (FIG. 5A) and standard (FIG. 5B) curves for Streptococcus agalactiae primers and probes using tenfold serial dilutions from 50,000 to 50 genome equivalents.

[00179] To test this method, 10,000 and 10 colony forming units (CFU) of Mycoplasma is spiked into a milk sample and amplification reactions performed as described herein. FIGs. 6A-6B show corresponding deoxyribonucleic acid (DNA; FIG. 6A) and ribonucleic acid (RNA; FIG. 6B) amplification curves. The 10 CFU sample in the DNA experiment did not amplify.

[00180] Similarly, FIGs. 13A-13D show amplification curves for gram positive bacteria (FIG. 13A), Staphylococcus aureus (FIG. 13B), Prototheca (FIG. 13C), and Mycoplasma (FIG. 13D). These curves were generated by extracting DNA from a sample (e.g., according to Example 1) of unpasteurized milk inoculated with 1,000 CFU/ml of the respective targets.

[00181] Several aspects are described with reference to example applications for illustration. Unless otherwise indicated, any embodiment may be combined with any other embodiment. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the features described herein. A skilled artisan, however, will readily recognize that the features described herein may be practiced without one or more of the specific details or with other methods. The features described herein are not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and/or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the features described herein.

[00182] Some inventive embodiments herein contemplate numerical ranges. When ranges are present, the ranges include the range endpoints. Additionally, every sub range and value within the range is present as if explicitly written out. The term“about” or“approximately” may mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example,“about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively,“about” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, within 5- fold, or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term“about” meaning within an acceptable error range for the particular value may be assumed.

[00183] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.