COMPOSITIONS AND METHODS FOR PROCESSING A BIOLOGICAL SAMPLE

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims the benefit of U. S. Provisional Patent Application Serial No. 62/184,514, filed on June 25, 2015 and titled "Compositions and Methods for Processing a Biological Sample," which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Traditionally, preparation and purification of biological samples has been done in the lab. However, epidemics such as the recent Ebola outbreak increase the demand for fast access to safe diagnostic testing and biological sample preparation. In some cases, this may involve untrained users that assist or replace scientifically trained professionals in the field. This poses challenges not only in minimizing contamination and ensuring safety of users, sterile treatment of samples, and accuracy of test results, but also in providing low-cost and portable and devices that preferably are not powered by electricity or are electricity free. Accordingly, safe, cost-efficient and reliable products and methods for biological sample preparation and diagnostic testing that can be used in the field by untrained or minimally trained users are urgently required.

SUMMARY OF THE INVENTION

As described below, aspects of the present invention feature systems, compositions, and methods for processing biological samples (e.g., purifying human blood derived leukocytes) using electricity-free tools without cross-contamination to obtain analytes (e.g., RNA) for downstream diagnostic applications. In particular, the systems, compositions, and methods are useful for handling biological samples containing or suspected of containing biohazardous, infectious, and/or pathogenic agents.

In one aspect, the invention provides a method for preparing a sample involving transferring a sample containing a cell or cell membrane into a syringe loaded with a buffer containing polyethylene oxide (PEO); and passing the sample through a filter.

In another aspect, the invention provides a method for isolating a cell from a sample, the method involving transferring a sample containing a cell into a syringe loaded with a buffer containing polyethylene oxide (PEO); and passing the sample through a filter having a pore size that captures the cell, thereby isolating the cell. In another aspect, the invention provides a method for isolating a leukocyte from a sample involving transferring a sample containing a leukocyte into a syringe loaded with a lysis buffer containing polyethylene oxide (PEO); and passing the lysed sample through a filter having a pore size that captures a leukocyte, thereby isolating a leukocyte.

In another aspect, the invention provides a method for obtaining leukocyte RNA from a sample involving transferring a sample containing a leukocyte into a syringe loaded with a lysis buffer containing polyethylene oxide (PEO); passing the lysed sample through a filter having a pore size that captures a leukocyte; passing a leukocyte lysis buffer through the filter; and collecting the flowthrough containing leukocyte RNA.

In one aspect, the invention provides a syringe holder having a groove for holding a syringe, a groove for holding a flange of the syringe, and one or more grooves for locking a plunger of the syringe at various positions.

In a related aspect, the invention provides a sample preparation kit containing a syringe holder according to any aspect of the invention; a detachable transfer device including a sleeve surrounding a needle and having an opening distal to the needle, where the needle is attached to the syringe or is fixed to the sleeve; and a sample preparation device including a filter attached to a sleeve having an opening distal to the filter, and a coupling attaching the filter to the tip of the syringe inside the sleeve.

In another aspect, the invention provides a method for transferring a sample into a syringe using a syringe holder involving providing a syringe fitted into a syringe holder having a groove for holding a flange of the syringe, and one or more grooves for locking a plunger at various positions; attaching to the syringe a detachable transfer device including a sleeve surrounding a needle, where the needle is attached to the syringe or is fixed to the sleeve; inserting into the sleeve a container containing a sample; and transferring the sample from the container into the syringe by puncturing the container.

In another aspect, the invention provides a method for sample purification involving providing a syringe fitted into a syringe holder having a groove for holding a flange of the syringe, and one or more grooves for locking the plunger at various positions; attaching to the syringe a detachable transfer device including a sleeve surrounding a needle, where the needle is attached to the syringe or is fixed to the sleeve; inserting into the sleeve a container containing a sample; transferring the sample from the container into the syringe by puncturing the container; affixing a sample preparation device onto the syringe, where the sample preparation device has a filter attached to a sleeve having an opening distal to the filter; and expelling the sample from the syringe through the filter, thereby purifying the sample.

In various embodiments of any aspect delineated herein, the sample is a blood sample. In certain embodiments, the lysis buffer is an erythrocyte lysis buffer. In particular embodiments, the flowthrough is collected in an RNase-free container.

In various embodiments of any aspect delineated herein, the sample, lysate, or buffer contains PEO. In various embodiments of any aspect delineated herein, the PEO has a molecular mass of at least about 100,000 g/mol (e.g., about 100,000 to about 8,000,000 g/mol). In various embodiments of any aspect delineated herein, one or more buffers (e.g., lysis buffer, wash buffer, etc.) has a PEO concentration between about 0.0001% and about 1%. In certain embodiments, one or more buffers (e.g., lysis buffer, wash buffer, etc.) has a PEO concentration between about 0.001% to about 1.0%. In particular embodiments, one or more buffers (e.g., lysis buffer, wash buffer, etc.) has a PEO concentration between about 0.01% and about 0.5%.

In various embodiments of any aspect delineated herein, the filter is washed by passing a wash buffer through the filter with a syringe after passing the sample through the filter. In various embodiments of any aspect delineated herein, the wash buffer contains polyethylene oxide (PEO). In various embodiments of any aspect delineated herein, the filter is dried by pushing air through the filter with a syringe after passing the wash buffer through the filter. In various embodiments of any aspect delineated herein, the filter has a pore size between about Ο. ΐμιτι and about 150 μιη. In various embodiments of any aspect delineated herein, the filter is made of glass fiber, nylon, polyvinylidene fluoride (PVDF), and/or cellulose acetate.

In various embodiments of any aspect delineated herein, a detachable transfer device is used. In certain embodiments, the detachable transfer device includes a sleeve surrounding a needle and having an opening distal to the needle, where the needle is attached to the syringe or is fixed to the sleeve. In various embodiments of any aspect delineated herein, the detachable transfer device includes an absorbent material positioned inside the sleeve to minimize exposure during transfer of the sample.

In various embodiments of any aspect delineated herein, a sample preparation device is used. In certain embodiments, the sample preparation device includes a filter attached to a sleeve having an opening distal to the filter, and a coupling attaching the filter to the tip of the syringe inside the sleeve. In various embodiments of any aspect delineated herein, the sample preparation device includes an absorbent material positioned inside the sleeve to minimize exposure during transfer of the contents of the syringe.

Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al, Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

The term "analyte" is is meant any compound under investigation using an analytical method. In particular embodiments, analytes include any nucleic acid molecule, polypeptide, carbohydrate, lipid, small molecule, marker, or fragments thereof. In certain embodiments, the analyte is associated with or indicates the presence of an infectious agent.

By "ameliorate" is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

By "decrease" is meant a negative alteration of at least about 5%, 10%, 25%, 50%, 75%, 85%, 90% or even by 100% of a reference value. "Detect" refers to identifying the presence, absence or amount of the analyte to be detected.

By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. In one embodiment, the disease is an infectious disease. Examples of diseases include e.g., Ebola, HIV/ AIDS, Influenza, Chagas Disease, Cholera, Tuberculosis, Malaria, Trypanosomiasis, Hepatitis C, Measles, SARS, yellow fever, Hepatitis C, Lassa virus, Dengue fever, West Nile, or bovine viral diarrhea.

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

By "healthcare professional" is meant any person providing medical care to a patient. Such persons include, but are not limited to, medical doctors, physician's assistants, nurse practitioners (e.g., an Advanced Registered Nurse Practitioner (ARNP)), nurses, residents, interns, medical students, and the like. Although various licensure requirements may apply to one or more of the occupations listed above in various jurisdictions, the term health care provider is unencumbered for the purposes of this patent application.

By "increase" is meant a positive alteration of at least about 5%, 10%, 25%, 50%, 75%, 85%, 90% or even by 100% of a reference value.

The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

By "isolated polynucleotide" is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least

75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

By "marker" is meant any protein or polynucleotide having an alteration (increase or decrease) in expression level or activity that is associated with a disease or disorder.

As used herein, "obtaining" as in "obtaining an agent" includes synthesizing, purchasing, or otherwise acquiring the agent.

By "polyethylene oxide (PEO)" is meant a polymer of ethylene oxide. In certain embodiments, the polyethylene oxide has a molecular mass at least about 100,000 g/mol

(e.g., about 100,000 to about 8,000,000 g/mol).

By "reference" is meant a standard or control condition.

A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.

By "specifically binds" is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.

Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.

Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

By "subject" is meant a mammal, including, but not limited to, a human or non- human mammal, such as a bovine, equine, canine, ovine, or feline. Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 provides a top view of a syringe holder relative in size to a metric ruler with a longitudinal indentation adapted and configured to hold a syringe, including a groove adapted and configured to hold the flange of the syringe, and a series of grooves, each of which is adapted and configured to lock the plunger of the syringe in position according to an embodiment of the invention.

Figure 2A shows a top view of a syringe containing sample preparation reagent A buffer attached to a transfer device according to an embodiment of this disclosure. Figure 2B shows a top view of a syringe containing sample preparation reagent A buffer placed into a syringe holder and attached to a transfer device before blood transfer according to an embodiment of this disclosure. A container containing blood is shown inserted into the sleeve of the transfer device.

Figure 2C shows a top view of a syringe containing sample preparation reagent A buffer mixed with blood placed into a syringe holder and attached to a transfer device after blood transfer according to an embodiment of this disclosure. A container containing blood is shown punctured with a needle of the transfer device. The ratio (v/v) of sample preparation reagent A buffer to blood in the syringe is set according to the position of the plunger locked in the syringe holder.

Figure 3A depicts a sample preparation device including a sleeve with an opening to insert a syringe at one end and adapted and configured at the other end with a twist lock fitting to connect the syringe to a filter according to an embodiment of this disclosure. In one embodiment, the separate components involved in making such a sample preparation device include: a syringe filter, a razor blade, a sleeve with a twist lock fitting cut off, and a cut-off twist lock fitting.

Figure 3B provides a cross sectional view of a sample preparation device including a sleeve with an opening to insert a syringe at one end and adapted and configured at the other end with a twist lock fitting to connect the syringe to a syringe filter according to an embodiment of this disclosure.

Figure 3C depicts a top view of a loaded syringe attached to a sample preparation device including a sleeve with an opening to insert the syringe at one end and adapted and configured at the other end with a twist lock fitting to connect the syringe to a syringe filter, and a liquid waste disposal container positioned to receive the contents of the syringe that are expelled through the filter when the plunger is depressed according to an embodiment of this disclosure.

DETAILED DESCRIPTION OF THE INVENTION

Aspects of the invention provide systems, compositions, and methods for processing biological samples (e.g., purifying human blood derived leukocytes) using electricity-free tools without cross-contamination to obtain an analyte (e.g., RNA) for downstream diagnostic applications. For example, the compositions of the invention can be stocked in mobile clinics, vehicles, schools or any place facilitating easy access for healthcare professionals to collect biological samples from patients. In particular, embodiments of this disclosure can be used by untrained or minimally trained users that assist or replace healthcare professionals in the field.

In addition, the systems, compositions, and methods are useful for handling biological samples containing or suspected of containing biohazardous, infectious, and/or pathogenic agents. Thus, in various aspects, the invention provides compositions and methods to safely and accurately detect and diagnose infectious diseases (e.g., Ebola, HIV/ AIDS, Influenza, Chagas Disease, Cholera, Tuberculosis, Malaria, Trypanosomiasis, Hepatitis C, Measles, SARS, yellow fever, Hepatitis C, Lassa virus, Dengue fever, West Nile, or bovine viral diarrhea) to inform treatment of subjects and assist in the prevention of epidemic outbreaks. During an epidemic outbreak, the compositions of the invention can be used by healthcare professionals to quickly process biological samples and to assess whether a subject has contracted an infectious disease. Devices for Biological Sample Processing

In one aspect, the invention provides a syringe holder to facilitate accurate volumetric measurement and to minimize the potential for contamination or exposure during sample transfer. Referring to Figure 1, the invention provides a syringe holder 100 with two ends and a longitudinal indentation 102 to fit the syringe, a half-rounded open frame 104 extending from the indentation 102 at one end of the syringe holder to hold a syringe tip, one groove 106 around the middle of the syringe holder to lock a syringe flange into place, and one or more grooves 108 to lock a plunger of a syringe in position at the other end of the syringe holder. The grooves 108 can be labeled according to an absolute volumetric scale or by relative numbers. The syringe holder 100 can be used with or adapted to syringes of varying sizes. In one embodiment, the syringe holder 100 is made or designed based on one or more syringe specifications or dimensions (e.g., syringe diameter, plunger length). In one embodiment the syringe holder is made out of plastic. This aspect of this disclosure can have a variety of embodiments. The syringe holder can be made of a polymer, glass, metal, ceramic, wood and/or any other material that is strong enough to hold a syringe and a syringe plunger in place.

In another aspect, the invention provides a detachable transfer device to facilitate biological sample transfer and to reduce or prevent contamination and/or exposure during sample transfer. Referring now to Figure 2A, the invention provides a detachable sample transfer device 200 (e.g., BD VACUTAINER® blood transfer device; Becton Dickinson, Franklin Lakes, NJ). A top view of a detachable transfer device 200 connected to a syringe 202 that can be pre-loaded with liquid content (e.g., a buffer, solution, or a biological liquid sample) is provided to show the different elements of the transfer device 200. The transfer device 200 includes a plastic sleeve 204, and an injection needle 208 at the bottom attached to the syringe 202 by a threaded coupling 210 (e.g., a Luer lock) on the syringe tip. In one embodiment, the plastic sleeve 204 is coated inside at the bottom 206 with absorbent material to avoid spills. The absorbent material or liner inside the sleeve can comprise any absorbent material (e.g., an absorbent polymer, synthetic fibers, natural fibers, paper, woven fabric, non- woven fabric, silicon, micronized silica particles, or any absorbent material). The absorbent material or liner can be impregnated with compounds to disinfectant or neutralize any unintentional sample release during sample transfer. The plastic sleeve 204 has an opening that allows insertion of collection containers (e.g., BD VACUTAINER® blood collection tube; Becton Dickinson, Franklin Lakes, NJ).

Referring now to Figure 2B, a top view of a detachable transfer device 200 (e.g., BD

VACUTAINER® blood transfer device; Becton Dickinson, Franklin Lakes, NJ) connected to a syringe 202 fitted into a syringe holder 100 prior to sample (e.g., blood) transfer is provided. The syringe 202 described herein can be pre-loaded with liquid content (e.g., a buffer, solution, or biological liquid sample). The syringe 202 is fitted into the syringe holder 100 such that the syringe barrel 212 is placed along the longitudinal indentation 102 of the syringe holder 100. A syringe barrel flange 214 is locked into the flange groove 106 and the syringe plunger 216 in turn is locked into one of the plunger grooves 108 to keep its liquid content from being accidentally released and to prevent the operator from over or under filling the syringe 202 for example, with blood, thereby altering the proper ratio of blood to lysis buffer. A collection container 218 (e.g., BD VACUTAINER® blood collection tube; Becton Dickinson, Franklin Lakes, NJ) is inserted into the sample transfer device 200 at a distance from the bottom of the plastic sleeve 204 such that the collection container 218 is not punctured by the injection needle 208.

Referring now to Figure 2C, the collection container 218 (e.g., BD VACUTAINER® blood collection tube; Becton Dickinson, Franklin Lakes, NJ) is shown fully inserted into the plastic sleeve 204 (e.g., BD VACUTAINER® blood transfer device; Becton Dickinson, Franklin Lakes, NJ, Figure 2A), of the detachable sample transfer device 200, so that the injection needle 208 pierces the septa of the collection tube 218 to allow a sample (e.g., blood) to flow into the syringe 202.

In yet another aspect, the invention provides a sample preparation device to separate a particle from a fluid in a biological sample (e.g., to purify leukocytes) and to reduce or prevent contamination and/or exposure during sample separation. Referring now to Figure 3A, the invention provides a sample preparation device 300. A top view of sample preparation device 300 and the individual elements involved in making the sample preparation device 300 are provided. The elements of the sample preparation device 300 include a plastic splash guard 302 (e.g., BD VACUTAINER®-based operator splash guard; Becton Dickinson, Franklin Lakes, NJ) with an opening at one side to insert a syringe. At the other end, the splash guard 302 is adapted and configured to connect to a filter 304 (e.g., WHATMAN® 5.0 μΜ nylon with glass microfiber filter (GMF); Cole-Parmer, Vernon Hills, IL; Sigma Aldrich, St. Louis, MO). The filter 304 attaches to the tip of the syringe, e.g., by a threaded coupling such as a Luer lock. In various embodiments of the invention, the filter 304 has a pore size of about 0.1 , 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 μιη. Filter 304 pore size can be selected based on use (e.g., large pore sizes can act as a pre-filter and small pore sizes can act to retain the targeted analyte). The filter 304 pore size can vary according to the properties and structure of the desired analyte to be retained, undesired debris and cell types, the sample matrix, and any interfering substances which may be present. In yet another embodiment of the invention, one or more filter 304 compositions can be employed in the same device. In one embodiment of the invention, the filter 304 composition is glass fiber, nylon, polyvinylidene fluoride (PVDF), or cellulose acetate. The filter 304 composition can be selected based on one or more of the chemical composition of the sample or buffer (e.g., a lysis buffer), a property of the target analyte (e.g., electrical charge of an analyte), separation of undesired debris, and/or operational pressure requirements. In still another embodiment of the invention, the filter 304 is round, square, oval, or any shape that supports the design and engineering requirements of the system. In various embodiments of the invention, the filter 304 has a diameter of about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, or 250 mm. In certain embodiments, the filter 304 has a diameter of about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mm. The filter 304 diameter can be selected based on one or more of the sample composition, initial sample volume, desired concentration or dilution effects, and/or desired final sample volume. In still another embodiment of the invention, the device may employ one or more filters in series. In another embodiment of the invention, filter 304 orientation may vary depending on the device design and the desired mode of operation (e.g., direct or tangential filtration with a recirculating or non-recirculating filtrate). In yet another embodiment of the invention, the filter 304 housing may be composed of a variety of thermoplastics, metals, and composites. The filter 304 housing can be selected based on one or more of the chemical composition of the sample or buffer (e.g., a lysis buffer), a property of the target analyte (e.g., electrical charge of an analyte), separation of undesired debris, and/or operational pressure requirements. In one embodiment of the invention, a twist lock fitting 306 at the bottom of the splash guard 308 has been cut off with a razor blade 310 and a filter 304 has been affixed to the open splash guard 308 with an appropriate glue or adhesive. In another embodiment of the invention, the filter 304 is connected to the bottom of the splash guard 308 by a threaded coupling (e.g., Luer lock) or an o-ring interference fitting. In yet another embodiment of the invention, the splash guard 302 is coated at the bottom 312 of the inside with an absorbent liner (i.e., an absorbent polymer, synthetic fibers, natural fibers, paper, woven fabric, non-woven fabric, silicon, micronized silica particles, or any absorbent material). In still another embodiment of the invention, the absorbent liner can be impregnated with compounds to disinfect or neutralize any unintentional sample release (e.g., from the inlet port). In still another embodiment of the invention, the absorbent liner can accelerate blood clotting.

Referring now to Figure 3B, a side view is provided showing the sample preparation device 300 fully assembled, including a plastic splash guard 308 (e.g., BD VACUTAINER®- based operator splash guard with twist lock fitting removed in Figure 3A), and a filter 304 (e.g., a WHATMAN® 5.0 M nylon with glass microfiber filter (GMF); Cole-Parmer, Vernon Hills, IL Sigma Aldrich, St. Louis, MO) mounted to the outside of the bottom of the splash guard 308 and extending into the splash guard 308 using an appropriate glue or adhesive. In one embodiment a twist lock fitting, a threaded coupling (e.g., Luer lock) or an o-ring interference fitting can be mounted to the inside of the bottom of the splash guard 308 around the extension of the filter 304 providing a connection for a syringe.

Referring now to Figure 3C, a side view is provided showing the sample preparation device 300 fully assembled with a syringe 314 containing a preparation sample inserted into a plastic splash guard 308 with the twist lock fitting removed as depicted in Figures 3A and 3B. The fully assembled sample preparation device 300 is facing down and the filter 304 (e.g., a WHATMAN ^® 5.0 M nylon with glass microfiber filter (GMF); Cole-Parmer of Vernon Hills, IL; Sigma Aldrich, St. Louis, MO, Figure 3A) is inserted into a waste disposal container 316 to allow for the syringe 314 content to be released into the liquid waste disposal container 316 without spilling.

Sample Preparation Methods

In various aspects, the invention provides sample preparation methods (e.g., including sample processing, sample transfer, and sample purification) employing the use of syringes and one or more devices of the invention. In one aspect, the invention provides a method involving transferring a sample comprising a cell or cell membrane into a syringe containing a buffer comprising polyethylene oxide (PEO), and passing the sample through a filter (e.g., syringe filter). Without being bound by theory, it has been found that polyethylene oxide (PEO) aids in the passage of crude lysate through a filter (e.g., a syringe filter). The invention is based at least in part on this discovery. Optionally, one or more wash steps may be used after passing the sample through the filter (e.g., to ensure passage of the entire sample through the filter, to remove residual agents or impurities, and the like). Optionally, one or more drying steps may be used after passing the sample through the filter or a wash buffer through the filter (e.g., to remove excess liquid and/or residual agents or impurities). In various embodiments, PEOs with a MW of about 100,000 to about 8,000,000 g/mol may be used, depending on the application and desired function. In various embodiments, one or more buffers (e.g., lysis buffer, wash buffer, etc.) comprise about 0.0001% to about 1% PEO. In certain embodiments, one or more buffers (e.g., lysis buffer, wash buffer, etc.) comprise about 0.001% to about 1.0% PEO. In particular embodiments, one or more buffers (e.g., lysis buffer, wash buffer, etc.) comprise about 0.01% to about 0.5% PEO.

In a further aspect, the method can be used for processing and/or obtaining an analyte from a sample containing lysed cells or that is a cell lysate. In the methods of the invention a cell lysate can be obtained by mixing the sample with a lysis buffer comprising polyethylene oxide (PEO) prior to passage through the filter. In some embodiments, the lysis buffer selectively lyses one cell type and not another. Depending on the sample type, origin, age, purity, etc., the initial selective lysis buffer may vary in composition to lyse a wide or narrow variety of undesired cell types and to deal with any interfering substances which may be present. After passing a sample through the filter, a wash buffer may be passed through the filter. The selection of suitable or appropriate wash buffers is within the knowledge of the skilled person. One or more subsequent lysis buffers may be used to lyse one or more cell types not previously lysed by previous contact with a lysis buffer.

The lysis and wash buffers of the invention can be varied in composition to lyse a wide or narrow variety of undesired cell types and to deal with any interfering substances that may be present. The composition of these buffers may include, for example and without limitation, polyethylene oxide (PEO), cationic, anionic, or non-ionic detergents, chaotropes, kosmotropes, salts, polymers, flow enhancers, anti-foaming agents, biologically relevant buffers, acids, bases, or a combination therein to selectively lyse some cell types (e.g., erythrocytes) while leaving others intact (e.g., leukocytes). In yet another embodiment of the invention, the final lysis buffer may vary in composition to produce a sample that is compatible with a variety of downstream analysis technologies (e.g., DNAble®, RNAble®, PCR, qPCR, RT-PCR, RT-qPCR, and immunoassay).

Buffer volumes at each step (e.g., initial lysis, wash, and final lysis) may depend upon the initial volume of the sample, the diameter and volume of the filter housing, requirements for the removal of interfering substances, or the desired final sample volume. In various embodiments of the invention, the buffer volumes for the initial lysis, wash, and final lysis are about 1, 2 ,3 , 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, or 10000 μΐ.

In another aspect, the invention provides a method for isolating a cell from a sample (e.g., a biological or environmental sample), involving transferring a sample comprising a cell into a syringe containing a buffer comprising polyethylene oxide (PEO); and passing the sample through a filter having a pore size that captures the cell, thereby isolating the cell. In a particular embodiment, the method involves selectively lysing at least one cell type, while another cell type remains unlysed or intact. The unlysed or intact cells are captured by the filter when the lysed sample is passed through the filter. The selection of a suitable or appropriate pore size is within the knowledge of the skilled person.

In a particular aspect, the method can be used to isolate a leukocyte from a biological sample (e.g., whole blood). In one embodiment, anti-coagulated whole blood is mixed with an erythrocyte lysis buffer (e.g., an isotonic buffer comprising a detergent, a hypotonic buffer) and the lysed sample is passed through a filter having a pore size that captures the leukocyte to isolate a leukocyte. Optionally, the filter having a captured leukocyte is washed and/or dried. In a further aspect, RNA may be obtained from the isolated leukocytes. A leukocyte lysis buffer is passed through the filter containing a captured leukocyte. RNA released by the lysis of the leukocyte is collected in an RNase-free container (e.g., a test tube). Optionally, the filter is washed and/or dried after passing the leukocyte lysis buffer through the filter.

In another aspect, the invention provides a method for transferring a sample into a syringe comprising the use of a syringe holder and a detachable transfer device of the invention. The syringe may be fitted into the syringe holder and/or attached to the detachable transfer device immediately prior to transfer of the sample. A container containing a sample is inserted into the sleeve of the detachable transfer device. The needle of the transfer device punctures the container to transfer the sample. In certain embodiments, the sample in the container is under vacuum. In particular embodiments of the invention, a syringe holder 100 is used. In particular embodiments of the invention, a detachable transfer device 200 is used.

In another aspect, the invention provides a method for sample purification comprising the use of a syringe holder, a detachable transfer device, and sample preparation device of the invention. The syringe may be fitted into the syringe holder and/or attached to the detachable transfer device immediately prior to transfer of the sample. A container containing a sample is inserted into the sleeve of the detachable transfer device. The needle of the transfer device punctures the container to transfer the sample. In certain embodiments, the sample in the container is under vacuum. After transfer of the sample into the syringe, a sample preparation device of the invention is affixed to the syringe (e.g., the tip). The sample is expelled from the syringe through the filter, thereby purifying the sample. In particular embodiments of the invention, a syringe holder 100 is used. In particular embodiments of the invention, a sample preparation device 200 is used. In particular embodiments of the invention, a sample preparation device 300 is used.

One or more methods of the invention may be combined or repeated to effect sample preparation (e.g., purification of a cell or analyte from a sample). In one example, instructions for a method to selectively purifying leukocytes from human blood incorporating one or more of the aforementioned methods is set forth at Table 1 .

Table 1. Instructions for syringe based selective purification of human blood derived leukocytes.

1. Obtain whole blood via venipuncture in an EDTA collection container (e.g., a

VACUTAINER®; Becton Dickinson, Franklin Lakes, NJ). Place syringe A pre-filled with 2.5 mL Buffer A (erythrocyte lysis buffer: sucrose, EGTA, Polyethylene Oxide MW 8xl0 ⁶ , and Igepal CA-630 in RNase-Free Water) attached to a blood transfer device (e.g., a BD VACUTAINER® blood transfer device; Becton Dickinson, Franklin Lakes, NJ) (see Figure 2A) into a syringe holder so that that plunger is locked in place (see Figure 2B). A 0.5 mL void is pulled in the syringe chamber (e.g., by vacuum).

Orient the syringe/holder assembly from step 2 so that the blood transfer device is facing upward. Insert the EDTA collection container containing patient specimen into the blood transfer device. Press the container into the blood transfer device so that the septa is pierced and blood is drawn downward (e.g., by vacuum) into the syringe containing Buffer A (see Figure 2C).

Remove the collection container containing the patient sample and set aside.

Remove the blood transfer device from the syringe and discard.

Attach the sample preparation device (see Figures 3A and 3B) to syringe A.

Incubate at room temperature for 3 minutes.

Slowly depress the plunger of syringe A to force the lysate through the filter (see Figure 3C).

Remove and discard syringe A.

Attach syringe B pre-filled with 5 mL Buffer B (wash buffer: sucrose, EGTA, Polyethylene Oxide MW 8x10 ⁶ , and Igepal CA-630 in RNase-Free Water).

Slowly depress the plunger of syringe B to wash the filter. Force the entire syringe contents though the filter, including the air.

Detach syringe B, aspirate 10 mL air, and reattach the syringe. Dry the membrane by forcing the air though the sample preparation device.

Detach and discard syringe B.

Attach syringe C pre-filled with 250 Buffer C (leukocyte lysis buffer: 1% Igepal CA-630 in RNase-Free Water). Depress the plunger, loading the filter housing with Buffer C.

Incubate for 2 minutes at room temperature.

Detach syringe C, aspirate 1 mL air. Reattach the syringe and depress the plunger, collecting the flowthrough in an RNase free 1.5 mL tube.

The sample is now ready for analysis (e.g., nucleic acid amplification and detection). Nucleic Acid Amplification Methods

Nucleic acid amplification technologies have provided a means of understanding complex biological processes, detection, identification, and quantification of biological organisms. Methods and compositions of the invention are useful for the identification of a target nucleic acid molecule in a test sample processed using the compositions and methods of the invention. The target sequence is amplified from virtually any sample that comprises a target nucleic acid molecule, including but not limited to samples comprising fungi, spores, viruses, or cells (e.g., prokaryotes, eukaryotes). Exemplary test samples include

environmental samples (e.g., water, extracts, suspensions), agricultural products (e.g., seeds) or other foodstuffs and their extracts. Exemplary test samples include biological samples, body fluids (e.g. blood, serum, plasma, amniotic fluid, sputum, urine, cerebrospinal fluid, lymph, tear fluid, feces, or gastric fluid), tissue extracts, organs, culture media (e.g., a liquid in which a cell, such as a pathogen cell, has been grown).

The polymerase chain reaction (PCR) is a common thermal cycling dependent nucleic acid amplification technology used to amplify DNA consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA using a DNA polymerase. Real-Time quantitative PCR (qPCR) is a technique used to quantify the number of copies of a given nucleic acid sequence in a biological sample. Currently, qPCR utilizes the detection of reaction products in real-time throughout the reaction and compares the amplification profile to the amplification of controls which contain a known quantity of nucleic acids at the beginning of each reaction (or a known relative ratio of nucleic acids to the unknown tested nucleic acid). The results of the controls are used to construct standard curves, typically based on the logarithmic portion of the standard reaction amplification curves. These values are used to interpolate the quantity of the unknowns based on where their amplification curves compared to the standard control quantities.

In addition to PCR, non-thermal cycling dependent amplification systems or isothermal nucleic acid amplification technologies exist including, without limitation:

Nicking Amplification Reaction, Rolling Circle Amplification (RCA), Helicase-Dependent Amplification (HDA), Loop-Mediated Amplification (LAMP), Strand Displacement Amplification (SDA), Transcription-Mediated Amplification (TMA), Self-Sustained

Sequence Replication (3SR), Nucleic Acid Sequence Based Amplification (NASBA), Single Primer Isothermal Amplification (SPIA), Q-β Replicase System, and Recombinase

Polymerase Amplification (RPA). Isothermal nicking amplification reactions have similarities to PCR thermocy cling. Like PCR, nicking amplification reactions employ oligonucleotide sequences which are complementary to a target sequences referred to as primers. In addition, nicking

amplification reactions of target sequences results in a logarithmic increase in the target sequence, just as it does in standard PCR. Unlike standard PCR, the nicking amplification reactions progress isothermally. In standard PCR, the temperature is increased to allow the two strands of DNA to separate. In nicking amplification reactions, the target nucleic acid sequence is nicked at specific nicking sites present in a test sample. The polymerase infiltrates the nick site and begins complementary strand synthesis of the nicked target nucleotide sequence (the added exogenous DNA) along with displacement of the existing complimentary DNA strand. The strand displacement replication process obviates the need for increased temperature. At this point, primer molecules anneal to the displaced complementary sequence from the added exogenous DNA. The polymerase now extends from the 3' end of the template, creating a complementary strand to the previously displaced strand. The second oligonucleotide primer then anneals to the newly synthesized complementary strand and extends making a duplex of DNA which includes the nicking enzyme recognition sequence. This strand is then liable to be nicked with subsequent strand displacement extension by the polymerase, which leads to the production of a duplex of DNA which has nick sites on either side of the original target DNA. Once this is synthesized, the molecule continues to be amplified exponentially through replication of the displaced strands with new template molecules. In addition, amplification also proceeds linearly from each product molecule through the repeated action of the nick translation synthesis at the template introduced nick sites. The result is a very rapid increase in target signal amplification; much more rapid than PCR thermocy cling, with amplification results in less than ten minutes.

Nicking Amplification Assays

The invention provides for the detection of target nucleic acid molecules amplified in an isothermal nicking amplification assay, as are known in the art (e.g., DNAble®,

RNAble®) See, for example, U.S. Patent Application Publication Nos. 20150104788 and 20130280706, each of which is incorporated herein in its entirety. Polymerases useful in the methods described herein are capable of catalyzing the incorporation of nucleotides to extend a 3' hydroxyl terminus of an oligonucleotide (e.g., a primer) bound to a target nucleic acid molecule and/or a 3' hydroxyl terminus at a nick site in a double-stranded DNA molecule in conjunction with strand displacement activity. Such polymerases also lack or have substantially reduced 5'-3' exonuclease activity and may include those that are thermophilic. DNA polymerases useful in methods involving primers having 2' -modified nucleotides in the primer region comprising the six 3 '-terminal nucleotides include derivatives and variants of the DNA polymerase I isolated from Bacillus stearothermophilus, also classified as

Geobacillus stearothermophilus, and from closely related bacterial strains, isolates and species comprising the genus Geobacillus, which lack or have substantially reduced 5'-3' exonuclease activity and have strand-displacement activity. Exemplary polymerases include, but are not limited to the large fragments of Bst DNA polymerase I, Gst DNA polymerase I, Gka DNA polymerase I. To amplify or detect an RNA target, a reverse transcriptase is further added to the reaction.

A nicking enzyme or agent binds double-stranded DNA and cleaves one strand of a double-stranded duplex. A nicking enzyme that cleaves the top stand (the strand comprising the 5 '-3 ' sequence of the nicking agent recognition site) may be used in a nicking amplification reaction. In a particular embodiment, the nicking enzyme cleaves the top strand only and 3 ' downstream of the recognition site. In exemplary embodiments, the reaction comprises the use of a nicking enzyme that cleaves or nicks downstream of the binding site such that the product sequence does not contain the nicking site. Using an enzyme that cleaves downstream of the binding site allows the polymerase to more easily extend without having to displace the nicking enzyme. Ideally, the nicking enzyme is functional under the same reaction conditions as the polymerase. Exemplary nicking enzymes include, but are not limited to, N.Bst9I, N.BstSEI, Nb.BbvCI(NEB),

Nb.Bpul OI(Fermantas), Nb.BsmI(NEB), Nb.BsrDI(NEB), Nb.BtsI(NEB), Nt.AlwI(NEB), Nt.BbvCI(NEB), Nt.Bpul OI(Fermentas), Nt.BsmAI, Nt.BspD6I, Nt.BspQI(NEB),

Nt.BstNBI(NEB), and Nt.CviPII(NEB). Sequences of nicking enzyme recognition sites are provided at Table 2.

Table 2. Nicking enzyme recognition sequences

-GAGTCNNNNNiNN-

N.Bst9I

-CTCAGNNNNN · NN-

-GAGTCNNNNNiNN-

N.BstSEI

-CTCAGNNNNN · NN-

-CCTCA ^« GC

Nb.BbvCI(NEB)

-GGAGT† CG

-CCTNA ^« GC

Nb.Bpul OI(Fermantas)

-GGANT† CG

-GAATG · CN

Nb.BsmI(NEB)

-CTTAC†GN

-GCAATG ·ΝΝ

Nb.BsrDI(NEB)

-CGTTAC†NN

-GCAGTG ·ΝΝ

Nb.BtsI(NEB)

-CGTCAC†NN

-GGATCNNNNiN

Nt.AlwI(NEB)

-CCTAGNNNN'N

-CCiTCAGC

Nt.BbvCI(NEB)

-GG · AGTCG

-CCiTNAGC

Nt.BpulOI(Fermentas)

-GG · ANTCG

-GTCTCNiN

Nt.BsmAI

-CAGAGN ·Ν

-GAGTCNNNNiN

Nt.BspD6I

-C CAGNNNN'N

-GCTCTTCNi

Nt.BspQI(NEB)

-CGAGAAGN

-GAGTCNNNNiN

Nt.BstNBI(NEB)

-C CAGNNNN'N

-lCCD

Nt.CviPII(NEB)

- GGH Nicking enzymes also include engineered nicking enzymes created by modifying the cleavage activity of restrictuion endonucleases (NEB Expressions July 2006, vol 1.2). When restriction endonucleases bind to their recognition sequences in DNA, two catalytic sites within each enzyme for hydrolyzing each strand drive two independent hydrolytic reactions which proceed in parallel. Altered restriction enzymes can be engineered that hydrolyze only one strand of the duplex, to produce DNA molecules that are "nicked" (3 ^' -hydroxyl, 5 ^' - phosphate), rather than cleaved. Nicking enzymes may also include modified CRISPR/Cas proteins, Transcription activator-like effector nucleases (TALENs), and Zinc-finger nucleases having nickase activity.

A nicking amplification reaction typically comprises nucleotides, such as, for example, dideoxyribonucleoside triphosphates (dNTPs). The reaction may also be carried out in the presence of dNTPs that comprise a detectable moiety including but not limited to a

32 33 125 35

radiolabel (e.g., P, P, I, S) an enzyme (e.g., alkaline phosphatase), a fluorescent label (e.g., fluorescein isothiocyanate (FITC)), biotin, avidin, digoxigenin, antigens, haptens, or fluorochromes. The reaction further comprises certain salts and buffers that provide for the activity of the nicking enzyme and polymerase.

Advantageously, the nicking amplification reaction is carried out under substantially isothermal conditions where the temperature of the reaction is more or less constant during the course of the amplification reaction. Because the temperature does not need to be cycled between an upper temperature and a lower temperature, the nicking amplification reaction can be carried out under conditions where it would be difficult to carry out conventional PCR. Typically, the reaction is carried out at about between 35 °C and 90 °C (e.g., about 35, 37, 42, 55, 60, 65, 70, 75, 80, or 85 °C). Advantageously, it is not essential that the temperature be maintained with a great degree of precision. Some variability in temperature is acceptable.

Sets of primers for amplification reactions are selected having AAG's < -15 - -30 kcal/mole or more. The performance characteristics of amplification reactions may be altered by increasing the concentration of one or more oligonucleotides (e.g., one or more primers and/or probes) and/or their ratios. In various embodiments, concentration of a primers is 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 nM or more. Melt temperature (Tm) and reaction rate modifiers may also be used to lower the melting temperature of the

oligonucleotides, such as (but not limited to) ethylene glycol and glycerol. In addition, DNA polymerase reaction rate modifiers (such as dNTP and magnesium concentration) may be used to alter the reaction rate to lead to a greater quantification precision.

This invention also provides methods of monitoring a nicking amplification reaction in real time. Detectable polynucleotide probes are used for the detection of target nucleic acid molecules or amplicons thereof in a nicking amplification reaction. In various embodiments, the oligonucleotide probe comprises an oligonucleotide having a nucleic acid sequence that is substantially complementary to a target nucleic acid sequence and having fluorescent reporter and quenching molecule covalently attached to opposite 5' and 3' ends of the oligonucleotide, respectively (e.g., separated by at least about 10-25 nucleotides). The fluorescent reporter and quencher molecules are selected to be any interacting fluorophore and quencher pair or FRET donor-acceptor pair known in the art. The oligonucleotide probe is capable of forming a stem-loop hairpin structure by hybridization when the oligonucleotide is not bound to the target nucleic acid molecule. In this conformation, the quenching molecule absorbs the excitation energy from the fluorescent reporter. When the

oligonucleotide probe binds the target nucleic molecule, the fluorescent reporter and quenching molecule are separated so that the excitation energy from the fluorescent reporter is detectable. Detectable oligonucleotide probes of the invention can be synthesized with a variety of fluorophores (e.g., FAM, HEX) and quenching molecules (e.g., 5IabRQ, BHQ-1).

In certain embodiments, the detectable polynucleotide probe is non-amplifiable, comprising at least one polymerase-arresting molecule (e.g., incapable of supporting polymerase extension utilizing the detectable oligonucleotide probe as a target). Such polymerase-arresting molecules that prevent or reduce the illegitimate amplification of a detectable polynucleotide probe include a C3-spacer, damaged DNA bases, or other spacer moiety. The detectable oligonucleotide probe may comprise one or more modified nucleotide bases (e.g., 2' Fluoro and 2'OMe nucleotides) having enhanced binding affinity to a complementary nucleotide. Detectable oligonucleotide probes of the invention can be synthesized with a variety of modifications and may be designed to hybridize with virtually any target sequence.

Kit

One aspect of this disclosure provides a sample purification kit including syringe holder 100, a sample transfer device 200, and a sample preparation device 300 as disclosed herein and instructions for use. The syringe holder 100 and the transfer device 200 and sample preparation device 300 can be combined in a sample purification kit as described herein.

The instructions for use (see e.g., Table 1) can be in written or electronic form and can include, for example, instructions on how to transfer the liquid sample (e.g., blood) using the syringe holder 100 and the transfer device 200, how to purify the sample using the sample preparation device 300, and general or specific instructions (e.g., word descriptions, illustrations, symbols) regarding methods of using the kit.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as,

"Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989);

"Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987);

"Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

Example 1. A method for selective purification of human blood derived leukocytes.

Epidemics such as the recent Ebola outbreak demand fast access to safe and reliable diagnostic tests and methods for biological sample preparation that can be used in the field. The present invention provides a method to purify human blood derived leukocytes to obtain RNA for downstream diagnostic applications in six steps without cross-contamination using electricity-free tools. A blood sample obtained from a patient was transferred into a syringe filled with reagent buffer A (erythrocyte lysis buffer: sucrose, EGTA, Polyethylene Oxide MW 8xl0 ⁶ , and Igepal CA-630 in RNase-Free Water) to break down cell membranes in the sample. Secondly, the sample was incubated for 3 minutes at room temperature and passed through the filter. Without being bound to a particular theory, leukocytes were captured by the filter, while erythrocytes and non-leukocyte cells in the sample were lysed and their membranes solubilized. Next, the filter was washed by attaching a syringe containing reagent buffer B (wash buffer: sucrose, EGTA, Polyethylene Oxide MW 8xl0 ⁶ , and Igepal CA-630 in RNase-Free Water) and passing reagent B through the filter. To dry the filter, air was pushed through the filter. Following this step another syringe filled with reagent buffer C (leukocyte lysis buffer: 1% Igepal CA-630 in RNase-Free Water) was attached and the filter was loaded with reagent buffer C by depressing the plunger of the syringe. Finally, after 2 minutes of incubation at room temperature, air was pushed through the filter to collect the flowthrough in an RNase free tube. Without being bound to a particular theory, the leukocytes were lysed, thereby releasing RNA. The RNA or any other analyte in the flowthrough can be used for downstream diagnostic applications. In various embodiments, the biological sample to be prepared can be one or more of blood, serum, breast milk, amniotic fluid, saliva, mucus, lymph, cerebrospinal fluid, pericardial fluid, peritoneal fluid, semen, gastric acid, feces, bile and/or urine. Example 2. A kit for preparation of biological samples (e.g. human blood derived leukocytes)

Epidemics in remote areas often require cost-efficient, electricity-free and reliable products that can be safely used in the field by untrained or minimally trained users. Rapid, point of need detection of epidemics, such as Ebola is required to effect interventions to prevent its spread. A kit was generated for safe, user-friendly and contamination-free preparation of biological samples to obtain RNA for downstream diagnostic applications and testing. The kit contained instructions for use (see Table 1), a syringe holder 100 (Figures 1, 2B and 2C), a 5 mL syringe, a 10 mL syringe with Luer lock closure, a 1 mL syringe with Luer lock closure, 2.5 mL erythrocyte lysis buffer A (sucrose, EGTA, Polyethylene Oxide MW 8x106, and Igepal CA-630 in RNase-Free Water), 5 mL wash buffer B (sucrose, EGTA, Polyethylene Oxide MW 8x10 ⁶ , and Igepal CA-630 in RNase-Free Water), 0.25 mL leukocyte lysis buffer C (1% Igepal CA-630 in RNase-Free Water), a detachable transfer device 200 (e.g., BD VACUTAINER ^® blood transfer device; Becton Dickinson, Franklin Lakes, NJ, Figure 2A), a sample preparation 300 device containing a VACUTAINER ^® -based operator splash guard 308 (e.g., BD VACUTAINER ^® ; Becton Dickinson, Franklin Lakes, NJ, Figure 3A), modified by attaching it to a filter 304 (e.g., WHATMAN ^® 5.0 M nylon with glass microfiber filter (GMF); Cole-Parmer, Vernon Hills, IL; Sigma Aldrich, St. Louis, MO), a container for liquid waste disposal 316 (Figure 3B), and an RNase free 1.5 mL tube. Using the kit according to the instructions provided, RNA or any other analyte can be purified for downstream diagnostic applications. In various embodiments, the biological sample prepared using the kit can be one or more of blood, serum, breast milk, amniotic fluid, saliva, mucus, lymph, cerebrospinal fluid, pericardial fluid, peritoneal fluid, semen, gastric acid, feces, bile and/or urine.

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.