APPARATUS AND METHOD FOR DETECTING PATHOGENIC

BACTERIA

CLAIM OF PRIORITY

[0001] This patent application claims the benefit of priority of U.S.

Provisional Patent Application Serial Number 62/621 ,272, filed January 24, 2018, which is hereby incorporated by reference herein in its entirety.

BACKGROUND

[0002] Monitoring water quality is an important health consideration. Many organizations, including municipal water suppliers and disaster-relief agencies need to ensure that the water supply is fit for human consumption. Monitoring water quality in medical and hospital settings can also be important. For example, measuring water quality of the rinsate from medical

instrumentation (e.g. endoscopes) provides an assessment of whether the device has been adequately disinfected. In each of these fields the primary customer needs the ability to detect small quantities of pathogens (as low as 1 CFU/100 inL of sample), in an amount of time that allows decisions to be made quickly concerning whether the water needs to be further treated or the medical device needs to be further cleaned before use in a patient. Moreover, minimizing the potential for false positive signals is important, so only measuring living bacteria (those capable of spreading infection) is desirable.

SUMMARY OF THE INVENTION

[0003] In various embodiments, a package is provided that includes an aqueous medium, a plurality of DNA primers including a first primer and a second primer, the first primer corresponding to a first nucleotide sequence from a target gene in a first species of human pathogenic bacteria, the second primer corresponding to a second nucleotide sequence from a target gene in a second species of human pathogenic bacteria, at least one DNA polymerase, wherein the primers are present in primer pairs (forward and reverse), and wherein the package includes at least one biochemically inert packaging material.

[0004] In various embodiments, a method of detecting bacteria includes separating bacteria from non-bacterial particles in a sample to provide a bacterial composition comprising viable bacteria and non-viable bacteria, treating the bacterial composition with a photoactive reagent that binds to dsDNA in nonviable bacteria to provide a treated composition, amplifying a target DNA sequence in viable bacteria in the treated composition using DNA polymerase and a plurality of DNA primers including a first primer and a second primer, the first primer corresponding to a first nucleotide sequence from a target gene in a first species of viable bacteria, the second primer rorresponding to a second nucleotide sequence from a target gene in a second species of viable bacteria; wherein the primers occur in primer pairs (forward and reverse) detecting bacteria based on the presence of amplified DNA and determining the number of bacteria based on the amount/quantity of amplified product.

[0005] In various embodiments, an apparatus for detecting pathogens includes: means for generating a non-uniform electric field, an inlet, a first fluid channel, at least two valves in fluid communication with the first fluid channel, at least two pumps in fluid communication with the first fluid channel and adapted to move a liquid, at least one analysis region in fluid communication with the first fluid channel, an outlet; and wherein the apparatus is adapted to detect an activity of DNA polymerase in the at least one analysis region.

[0006] In one embodiment, steps of separating bacteria from non- bacterial components in a sample, treating the bacteria with a reagent to inactive the non- viable bacteria DNA, amplifying the target DNA, and

detecting/quantitating the DNA/number of bacteria all in occur in one device (or they can occur in separate devices).

BRIEF DESCRIPTION OF THE FIGURES

[0007] The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments of the present invention.

[0008] FIG. 1 is a flowchart for cleaning an endoscope.

[0009] FIG. 2 is a schematic overview of a method, according to various embodiments.

[0010] FIG. 3 is a diagram of a method, according to various embodiments.

[0011] FIG. 4 is a schematic diagram showing amplification of a specific

DNA fragment using PCR, according to various embodiments. [0012] FIG. 5 includes a representation of a laboratory on card implantation, according to various embodiments.

[0013] FIG. 6 includes a flow chart of a method, according to various embodiments.

DETAILED DESCRIPTION OF THE INVENTION

[0014] Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0015] In this document, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B." In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section.

[0016] In the methods described herein, the acts can be carried out in any order without departing from the principles of the invention, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0017] The term "about" as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1 % of a stated value or of a stated limit of a range, and includes the exact stated value or range. [0018] The term "substantially" as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The term "substantially free of as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that the composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less.

[0019] As used herein, the term "pathogenic bacteria" means bacteria that can cause or lead to a disease state in mammals, such as humans, cats, dogs, pigs, cows, horses, and the like.

[0020] As used herein, the term "viable bacteria" refers to bacteria that are living.

[0021] As used herein, the term "non- viable bacteria" refers to dead bacteria, or portions of dead bacteria.

[0022] As used herein, the term "sterile" or "aseptic" refers to conditions that are free or substantially free (e.g., less than 1 CFU/ml) of bacteria, viruses, fragments of bacteria and/or viruses, and substances that can cause an immune response.

Consumable Package/Kit

[0023] In various embodiments, a package is provided that includes an aqueous medium, a plurality of DNA primers including a first primer and a second primer, the first primer corresponding to a first nucleotide sequence from a first target gene in a first species of human pathogenic bacteria, the second primer corresponding to a second nucleotide sequence from a second target gene in a second species of human pathogenic bacteria (primer are generally present in primer pairs (forward and reverse primers)), at least one DNA polymerase, and wherein the package is sealed from an atmosphere and includes at least one biochemically inert packaging material. Any number of primers can be included in the package, such that there can be third primer, a fourth primer, a fifth primer, a sixth primer, a seventh primer, an eighth primer, a ninth primer, and so on (primer pairs; such at that multiple genes and/or bacteria are tested for in the same reaction; multiplex). Thus, the total number of primers in the package is not particularly limited. The aqueous medium can be a sterile aqueous medium.

[0024] The package can also include probes, such as DNA probes, which can be from about 8 to about 30 nucleotide base pairs in length. The probes can be linked to a fluorophore moiety and a fluorescence quenching moiety. The fluorophore can be any suitable fluorophore, such as 5/6-carboxyfluorescein succinimidyl ester, and 5(6)-fluorescein isothiocyanate mixed isomer,

LightCycler ^® Red 610-N-hydroxysuccininnde ester, and Quasar® 570

(indocarbocyanines). The fluorescence quenching moiety can be any suitable fluorescence quencher, such as DDQ available from Eurogentec, Eclipse quenchers available from Epoch Biosciences, Iowa quenchers available from Integrated DNA Technologies, BHQ quenchers available from Biosearch Technologies, QSY quenchers are available from Molecular Probes. In various embodiments, the probe does not emit any light due to the presence of the fluorophore and quencher on the same probe. The probes, in various embodiments, attach to a target DNA sequence and are degraded by DNA polymerase as it synthesizes new DNA fragments. Upon degradation of the probe, the fluorophore can begin to emit light that can be detected. The package can also include DNA intercalating dyes, such as S YBR Green and EvaGreen, which can bind to dsDNA.

[0025] In various embodiments, the first primer and the second primer

(generally present in primer pairs; forward and revise primers) are each independently 15 to 70 base pairs long. Additional primers are also

independently 15 to 70 base pairs long. In various embodiments, the target gene is a pathogen-specific gene such as a virulence factor gene. The pathogen- specific gene can be a gene that uniquely identifies a particular bacterial species, subspecies, or strain. The target gene can also be any gene that can be targeted to specifically identify the species of bacteria of interest. In various embodiments, the target gene can correspond to a gene in Gram-positive and/or Gram-negative bacteria (e.g., Gram negative and Staphyllococcus aureus). In various embodiments, the first primer and the second primer each independently correspond to a nucleotide sequence from a first and second target gene, respectively, in E. coli, Staphyllcoccus aureus, Pseudomonas aeruginosa, or a first and second target gene, respectively in at least one human pathogenic bacterial species from a genus chosen from Enterococcus, Streptococcus, Klebsiella, Mycobacterium, Salmonella, or Shigella.

[0026] In various embodiments, the aqueous medium is a saline aqueous medium The water in the aqueous medium can be deionized water. The aqueous medium can include a concentration of one or more salts that are hypotonic, isotonic, or hypertonic relative to human blood. The aqueous medium can further include any suitable excipients, including buffers, stabilizing agents, surfactants, and the like, that do not damage or otherwise adversely affect the primers in the package.

[0027] In various embodiments, the biochemically inert material includes at least one of plastic, glass, metallic foil, and combinations thereof. The biochemically inert material can include the interior surface of the package in contact with the aqueous medium, or the exterior surface of the package in contact with atmosphere, and combinations thereof. The package can be configured to interface with a reaction chamber or other device or apparatus such that the contents of the package can be transferred without being exposed to atmosphere or light.

Method of Detecting Bacteria

[0028] In various embodiments, a method of detecting bacteria includes separating bacteria from non-bacterial particles in a sample to provide a bacterial composition comprising viable bacteria and non-viable bacteria, treating the bacterial composition with a photoactive reagent that binds to dsDNA in non- viable bacteria to provide a treated composition, amplifying a target DNA sequence in viable bacteria in the treated composition using DNA polymerase and a plurality of DNA primers including a first primer and a second primer (e.g., first and second primer pairs; forward and reverse primers comprising a pair), the first primer pair corresponding to a first nucleotide sequence from a first target gene in a first species of viable bacteria, the second primer corresponding to a second nucleotide sequence from a second target gene in a second species of viable bacteria; and detecting/determining the number of bacteria. The bacteria can be pathogenic, non-pathogenic, or a combination thereof.

[0029] Any number of primers (or primer pairs; forward and reverse) can be used in the method, such that there can be a third primer, a fourth primer, a fifth primer, a sixth primer, a seventh primer, an eighth primer, a ninth primer, and so on (so as to detect multiple genes in multiple species). Thus, the total number of primers that can be used is not particularly limited. The sample can include pathogenic, non-pathogenic, or a combination of pathogenic and nonpathogenic bacteria with a concentration of about 1 to about 30 cfu/mL, about 1 to about 25 cfu/mL, about 1 to about 20 cfu/mL, about 1 to about 15 cfu/mL, about 1 to about 10 cfu/mL, or about 1 to about 5 cfu/mL. The sample can include pathogenic bacteria with a concentration of about 1 to about 30 cfu/mL, about 1 to about 25 cfu/mL, about 1 to about 20 cfu/mL, about 1 to about 15 cfu/mL, about 1 to about 10 cfu/mL, or about 1 to about 5 cfu/mL. The sample can also include non-bacterial components, including fine particles, mammalian cells, blood, fecal matter, and the like. In various embodiments, the viable bacteria are viable pathogenic bacteria.

[0030] In various embodiments, the separating includes moving the sample through a non-uniform electric field. In various embodiments, the bacterial composition includes at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the viable and non-viable bacteria in the sample. The separating step can include dielectrophoresis (DEP) cell capture techniques to concentrate bacterial cells from any suitable sample, including endoscope rinsate. Under a non-uniform electric field, dielectric particles such as bacterial cells undergo a DEP force. The strength of the DEP force depends on the size and shape of the dielectric particles, frequency of the electric field, and electric conductivity of the solution. By controlling the frequency of the electric field, the movement of target dielectric particles (i.e. bacterial cells) can be manipulated. In flow through microfluidic channel under this electric field, bacterial cells experience both hydrodynamic (F _flow ) and DEP forces (F _DEP ).

When these two forces are at equilibrium, cells stop in the microfluidic channel (FIG. 2). By using this phenomenon, the field can be tuned to capture only bacterial cells in the DEP microfluidic channel, while other non-bacterial particles are not captured and can move via fluidic channels to other another region, such as a waste collection region.

[0031] In various embodiments, the separating includes a uniform or non-uniform magnetic field. Separating with a magnetic field can include labeling bacteria with magnetic materials, such as magnetic beads, and using the magnetic field to capture labelled bacteria. The separating can also include the use of gel electrophoresis techniques. Other techniques include paper filter, porous plastics, cross-linked hydrogels, hollow fiber, fine nonwoven and/or streptavidin bound to biotinylated antibodies.

[0032] In various embodiments, the sample can move with a flow rate from about 1 μL/min to about 2000 μL/min, about 5 μL/min to about 1800 μL/min, 10 μL/min to about 1600 μL/min, 20 μL/min to about 1400 μL/min, 30 μL/min to about 1200 μL/min, 40 μL/min to about 1100 μL/min, 50 μL/min to about 1000 μL/min, 60 μL/min to about 900 μL/min, 70 μL/min to about 800 μL/min, or 80 μL/min to about 700 μL/min. The flow can be less than about, greater than about, or equal to about 1 μL/min, 3 μL/min, 5 μL/min, 10 μL/min, 20 μL/min, 50 μL/min, 100 μL/min, 200 μL/min, 300 μL/min, 400 μL/min, 500 μL/min, 600 μL/min, 700 μL/min, 800 μL/min, 900 μL/min, 1000 μL/min, 1250 μL/min, 1500 μL/min, 1750 μL/min, 2000 μL/min, 2250 μL/min, or 2500 μL/min.

[0033] In various embodiments, the non-uniform field is an alternating field. The alternating field can have a frequency of about 0.01 MHz to about 10 MHz, about 0.1 MHz to about 10 MHz, about 0.5 MHz to about 8 MHz, about 1.0 MHz to about 6 MHz, or about 1.5 MHz to about 4 MHz. The frequency can be can be less than about, greater than about, or equal to about 0.01 MHz, 0.1 MHz, 0.5 MHz, 1.0 MHz, 2.0 MHz, 3.0 MHz, 4.0 MHz, 5.0 MHz, 6.0 MHz, 7.0 MHz, 8.0 MHz, 9.0 MHz, or 10.0 MHz. The non-uniform field can have a signal power of about 1 V to about 1000 V.

[0034] In various embodiments, the photoactive agent includes propidium monoazide (PMA). Other suitable photoactive agents that specifically bind to dsDNA can also be used (and/or other viability sorting agents/methods, such as propidium iodide, esterases). Upon photolysis, PMA dye becomes permanently fixed to the free DNA, which inhibits PCR. However, PMA cannot penetrate through bacterial cell membranes; therefore, PMA modification does not occur to the DNA in viable cells. Dead cells have disintegrated cell membranes, and therefore, PMA can access to and react with the DNA inside the dead cells. Treating the sample with PMA can include centrifuging the sample and exposing the sample and PMA to light having a wavelength of about 470 ran, although any suitable wavelength of light can be used that photo activates the PMA. In various embodiments, amplification occurs without purifying the treated composition. A treated composition can be amplified in the presence of the reaction products of propidium monoazide with DNA from non- viable bacterial cells. In various embodiments, the amplifying occurs in the absence of light. Amplification can take place in the absence of visible light, such as light having wavelengths of about 390 ran to about 700 ran.

[0035] In various embodiments, the first primer and the second primer (e.g., first and second primer pairs) each independently correspond to a nucleotide sequence from a target gene in Gram-positive or Gram-negative bacteria. In various embodiments, the first primer and the second primer pairs each independently correspond to a nucleotide sequence from a target gene in E. coli, Staphyllococcus aureus, Pseudomonas aeruginosa, or a target gene in at least one human pathogenic bacterial species from a genus chosen from

Enterococcus, Streptococcus, Klebsiella, Mycobacterium, Salmonella, or Shigella.

[0036] Suitable Enterococcus species include E. alcedinis, E.

aquimarinus, E. asini, E. avium, E. bulliens, E. caccae, E. camelliae, E.

canintestini, E. canis, E. casseliflavus, E. cecorum, E. columbae, E. devriesei, E. diestrammenae, E. dispar, E. durans, E. eurekensis, E. faecalis, E. faecium, E. gallinarum, E. gilvus, E. haemoperoxidus, E. hermanniensis, E. hirae, E.

italicus, E. lactis, E. lemanii, E. malodoratus, E. moraviensis, E. mundtii, E. olivae, E. pallens, E. phoeniculicola, E. plantarum, E. pseudoavium, E.

quebecensis, E. rqffinosus, E. ratti, E. rivorum, E. rotai, E. saccharolyticus, E. saigonensis, E. silesiacus, E. sulfitreus, E. solitarius, E. tenniHs, E. thailandicus,

E. ureasiticus, E. ureilyticus, E. viikkiensis, E. villorum, and E. xiangfangensis. [0037] Suitable Streptococcus species include 5. acidominimus, S.

agalactiae, S. alactolyticus, S. anginosus, S. australis, S. bovis, S. caballi, S. cameli, S. canis, S. caprae, S. castoreus, S. criceti, S. constellatus, S. cuniculi, S. danieliae, S. dentasini, S. dentiloxodontae, S. dentirousetti, S. devriesei, S.

didelphis, S. downei, S. dysgalactiae, S. entericus, S. equi, S. equinus, S. ferus, S. gallinaceus, S. gallolyticus, S. gordonii, S. halichoeri, S. halotolerans, S. henryi,

S. himalayensis, S. hongkongensis, S. hyointestinalis, S. hyovaginalis, S. ictaluri,

S. infantarius, S. infantis, S. lactarius, S. iniae, S. intermedins, S. lactarius, S. loxodontisalivarius, S. lutetiensis, S. macacae, S. marimammalium, S. mannotae, S. massiliensis, S. merionis, S. minor, S. milleri, S. mitis, S. moroccensis, S. mutans, S. oligofermentans, S. oralis, S. oricebi, S. oriloxodontae, S. orisasini, S. orisratti, S. orisuis, S. ovis, S. panodentis, S. pantholopis, S. parasanguinis, S. parasuis, S. parauberis, S. peroris, S. pharyngis, S. phocae, S. pneumoniae, S. pseudopneumoniae, S. pluranimalium, S. plurextorum, S. pneumoniae, S. porci, S. porcinus, S. porcorum, S. pseudopneumoniae, S. pseudoporcinus, S. pyogenes,

S. ratti, S. rifensis, S. rubneri, S. rupicaprae, S. salivarius, S. salivihxodontae, S. sanguinis, S. sinensis, S. sobrinus, S. tangierensis, S. thoraltensis, S. troglodytae,

S. troglodytidis, S. tigurinus, S. thermophilus, S. sanguinis, S. sobrinus, S. suis,

S. uberis, S. urinalis, S. ursoris, S. vestibuhris, S. viridans, and S.

zooepidemicus.

[0038] Suitable Klebsiella species include K. pneumoniae, K. terrigena,

K. oxytoca, K. variicola, and K. granulomatis.

[0039] Suitable Mycobacterium species include M.

tuberculosis complex, M. avium complex, M. gordonae clade, M. kansasii clade, M. nonchromogenicum/terrae clade, Mycolactone-producing mycobacteria, M. simiae clade, M. chelonae clade, M. fortuitum clade, M. mucogenicum clade, M. parafortuitum clade, and M. vaccae clade.

[0040] Suitable Salmonella species can include 5. bongori and 5.

enterica. Suitable 5. enterica subspecies include S. enterica subsp. enterica, S. enterica subsp. salamae, S. enterica subsp. arizonae, S. enterica subsp.

diarizonae, S. enterica subsp. houtenae, and 5. enterica subsp. indica. Suitable 5. enterica subsp. enterica serovars include Salmonella Choleraesuis, Salmonella Dublin, SalmoneUa Enteritidis, Salmonella Gallinarum, Salmonella Hadar, Salmonella Heidelberg, Salmonella Infantis, Salmonella Paratyphi, Salmonella Typhi, and Salmonella Typhiniurium,

[0041] Suitable Shigella species can include 5. boydii, S. dysenteriae, S. flexneri, and S. soniiei.

[0042] In various embodiments, the amplifying occurs in a microdroplet.

The microdroplet can include an oil, or a mixture of oils, for example, a fluorinated carrier oil or a mineral oil. The microdroplets can also include water mixed with an oil or mixture of oils. When microdroplets contain both water and oil, they can form a microdoplet that is a water-in-oil emulsion. In some embodiments, the DNA amplification occurs substantially in the water phase in the water-in-oil microdroplet emulsion. When the amplifying occurs in a microdroplet, the plurality of primers and the bacterial DNA are mixed together. The microdroplets can optionally include surfactants to decrease adsoiption at the droplet interface. The droplets including bacterial DNA can include other reagents, including DNA polymerase, deoxyribonucleotide triphosphate (dNTP), and a plurality of primers. In various embodiments, the microdroplet includes solvated pathogenic bacteria.

[0043] In various embodiments, the determining includes counting a number of microdroplets having DNA fragments amplified by the DNA polymerase. The counting can be conducted with a charge coupled device (CCD) or other devices that can detect light. To amplify/detect specific pathogens via presence of bacterial DNA and/or RNA, the polymerase chain reaction (PCR; including, qPCR or rt-PCR) or loop-mediated isothermal amplification (LAMP) can be used. In PCR and LAMP, a target DN A fragment is amplified by a DNA-replicating enzyme such as Taq DNA polymerase and Bst DNA polymerase, providing millions of copies from a single DNA fragment. This reaction does not need cultivation of bacteria, thereby providing the results quickly (less than 2 hours). The specificity of the PCR and LAMP reaction largely depends on the primer sequence (for example, primers to specifi gene can be designed based on the gene sequence; as a specific example,

Enterobacteriacea primers can include Forward primer rpOP If; 5'- ATGTTACAACCAAAGCGTACA-3' (SEQ ID NO: 1) and Reverse primer rpIP 185R; 5 ' -TTACCYTGACGCTT AACTGC-3" (SEQ ID NO: 2) (Takahashi et al. (2017)). Taq DNA polymerase initiates the PCR reaction by recognizing the binding of the plurality of primers to the template DNA from bacteria (FIG. 4). The method can also include using any of the DNA probes containing fluorescent moieties and fluorescence-quenching moieties described herein.

[0044] In various embodiments, the separating, treating, and amplifying occur in a device including: an inlet, a first fluid channel, at least two valves in fluid communication with the first fluid channel, at least two pumps in fluid communication with the first fluid channel and adapted to move a liquid, at least one reaction region in fluid communication with the first fluid channel, at least one analysis region in fluid communication with the first fluid channel, and an outlet. The valves can be any type of valve that can control fluid flow, such as electrically actuated valves, mechanically actuated valves, or combinations thereof. The control of fluid by a valve or valves can include stopping fluid flow or decreasing the amount of fluid flow. The analysis region can include a region where PCR amplification of bacterial DNA occurs, and where a fluorescent signal indicating that a PCR reaction is occurring can be detected by a charge coupled device or other light-detecting devices. The analysis region can also include at least one reaction region include a portion that is optically transparent to electromagnetic radiation. An optically transparent region can allow for electromagnetic radiation, such as light, to enter or leave the analysis region.

The portion that is optically transparent to electromagnetic radiation can include the walls or portions of the surface of the analysis region and can include materials such as glass or plastic.

[0045] In various embodiments, the at least one reaction region include a portion that is optically transparent to electromagnetic radiation. An optically transparent region can allow for electromagnetic radiation, such as light, to enter or leave the reaction region and photoactivate a photoactive agent, such as PMA, in the reaction region. The portion that is optically transparent to

electromagnetic radiation can include the walls or portions of the surface of the reactive region and can include materials such as glass or plastic. In various embodiments, treating occurs in the at least one reaction region. In various embodiments, the analysis region is in fluid communication with a second fluid channel, and wherein the second fluid channel is in fluid communication with at least one reagent region. In various, the reagent region includes DNA polymerase.

[0046] In various embodiments, the determining includes detecting visible electromagnetic radiation. In various embodiments, the detecting includes detecting fluorescence with a charge coupled device or other devices that can detect fluorescence intensity. In various embodiments, the device includes a microfluidic device.

Apparatus for Detecting Bacteria

[0047] In various embodiments, an apparatus for detecting pathogens includes: means for generating a non-uniform electric field, an inlet, first fluid channel, at least two valves in fluid communication with the first fluid channel, at least two pumps in fluid communication with the first fluid channel and adapted to move a liquid, at least one analysis region in fluid communication with the first fluid channel, an outlet; and wherein the device is adapted to detect an activity of DNA polymerase in the at least one analysis region. The means for generating a non-uniform electric field can include a function generator operably connected to an input of an AC power amplifier, and the output of the AC power amplifier is electrically connected to a, for example, printed circuit board (PCB) in which the non-uniform electric field is generated. The function generator can be configured to produce a sine wave output at any of the frequencies described herein. An exemplary commercial apparatus capable of generating non-uniform electric fields for separating bacteria through dielectrophoresis is the ELESTA device from AFI Corporation, Kyoto, Japan.

[0048] In various embodiments, an apparatus includes a first opposing channel and a second opposing channel, wherein the first and second opposing channels are in fluid communication with and perpendicular to the first fluid channel, and wherein the first and second opposing channels include an immiscible fluid that is immiscible with the fluid in the first fluid channel. In various embodiments, the apparatus include a microfluidic device.

Examples

[0049] Various embodiments of the present invention can be better understood by reference to the following Examples which are offered by way of illustration. The present invention is not limited to the Examples given herein. Materials and Methcxls

[0050] Bacterial strains. The bacterial strains listed in Table 1 were obtained from the Japan Collection of Microorganisms (JCM; Tsukuba, Japan), Research Institute for Microbial Diseases (RIMD) at Osaka University (Osaka, Japan), and BCCM/LMG (Ghent, Belgium). The bacteria were cultured in specific media recommended by the culture collection centers. The genomic DNA from Clostridium perfringens strains F4649 and NTCT 8239 were provided by Miyamoto (Tokushima Bunri University, Tokushima, Japan). Pseudogulbenkiania sp. strain NH8B was grown in R2A broth (W ako, Osaka, Japan). Pseudogulbenkiania sp. NH8B is a betaproteobacterium and is not considered to be a human pathogen. This strain was used as a candidate for process control during DNA extraction and qPCR.

[0051] Table 1 : Example bacterial strains and target genes for primer development.

[0052] Environmental samples. Human fecal samples were obtained from six healthy adults. Small aliquots (ca. 200 mg) were collected in 2-ml tubes and stored at -20°C until use. Pathogenic bacteria EHEC 0157:H7 strain Sakai, Salmonella enterica subsp. enterica serovar Typhiniurium JCM 1652,

Campylobacter jejuni JCM 2013, Clostridium perfringens JCM 1290, Legionella pneumophila JCM 7571, Listeria monocytogenes JCM 7671, Shigella flexneri RIMD 0509763, Vibrio cholerae RIMD 2203246, Vibrio parahaemolyticus JCM 2210633, and Pseudogulbenkiania sp. strain NH8B were cultured in liquid media until the optical density at 600 ran (OD600) was 0.2. Approximately 10 ⁷ cells from each bacterial culture were inoculated individually into the tubes including fecal samples (n = 6). The actual cell concentrations of the inocula were measured directly under fluorescence microscopy after 4' ,6-diamidino-2- phenylindole (DAPI) staining. The fecal samples inoculated with the serially diluted cell mixtures from 10 bacterial species were prepared at cell densities of approximately 10 ⁶ , 10 ⁵ , 104, 10 ³ , and 10 ² cells per tube (n = 6). The physicochemical characteristics of the water samples were as follows:

temperature, 10°C; pH 5.7; electrical conductivity, 11.5 S/m; suspended solids, 9.0 mg/liter; total nitrogen, 0.273 mg/liter; and total phosphorus, 0.364 mg/liter. EHEC strain Sakai and Pseudogulbenkiania sp. NH8B were grown as described above and inoculated into the water at different cell concentrations (10 ¹ to 10 ⁷ cells/liter). Pathogen-spiked or nonspiked water samples (5 liters) were filtered through 0.20-μm-ροre polyethersulfone membrane filters (Millipore), and bacterial cells were detached from the membrane by vigorously shaking in phosphate-buffered saline (PBS; pH 7.2) with 0.1% gelatin. The cells were centrifuged at 10,000 x g for 15 min and resuspended in 1 ml of PBS. The cells were transferred to 2-ml screw-cap tubes and centrifuged at 10,000 x g for 5 min. The cell pellets were stored at -20°C until use.

[0053] DNA extraction. DNA was extracted from pure bacterial cultures and fecal samples using a DNeasy blood and tissue kit (Qiagen) and a QIAamp DNAstool minikit (Qiagen), respectively.DNAwas also isolated from the cell pellets obtained from the water samples by using a PowerSoil DNA isolation kit (MoBio Laboratories, Carlsbad, CA), with a final elution volume of 50 μl. DNA samples were stored at -20°C until use.

[0054] Primers, probes, and plasmids for qPCR. Virulence factor genes or specific genes present in the target pathogens were used to design specific primers for qPCR. For quantification of the process control, the nosZ- like pseudogene from Pseudogulbenkiania sp. strainNH8B (GenBank accession no. NC_016002; locus tag, NH8B_3641) can be chosen, which was identified previously during its genome analysis. This gene encodes 61 amino acids, and it is too short to be a functional nitrous oxide reductase (NosZ). No nucleotide and amino acid sequences other than those of NH8B_3641 had an E value of <le-10 in the GenBank nt and nr databases, respectively.

[0055] The primers and probes were designed using the Universal Probe-

Library (UPL) Assay Design Center (Roche). The UPL probes were prevalidated short TaqMan probes that included locked nucleic acids, which were labeled with 6-carboxyfluorescein (6-FAM) at the 5' end and dark quencher near the 3' end. The nucleotide sequences of the target genes were obtained from GenBank and aligned using CLUSTALW. Primers and probes that perfectly matched the target sequences and mismatches in nontarget sequences were used for qPCR analysis. Existing TaqMan qPCR assays can be used for the detection and quantification of V. cholerae and V. parahaemolyticus. These TaqMan probes were labeled with 6-FAM and quencher with the minor grove binder (MGB; Applied Biosystems).

[0056] To generate the standard curves, PCR products from the target pathogens were cloned into pCR 2.1 -TOPO vector (Invitrogen) and transformed into E. coli DH5oc competent cells by electroporation (Bio-Rad) according to the manufacturer's instructions. Plasmids were isolated from the cloned E. coli cells using a GeneJET plasmid minikit (Fermentas), linearized by enzymatic digestion (BamH I; TaKaRa Bio), and purified using a FastGene gel/PCR extraction kit. DNA concentrations were fluorometrically measured using PicoGreen double- stranded DNA (dsDNA) quantification reagent (Molecular Probes, Eugene, OR). Serial dilutions (10 ⁰ to 10 ⁶ copies/μl) of the plasmid DNA were used to construct the standard curve for qPCR.

[0057] Conventional qPCR. Conventional qPCR was performed using an ABI Prism 7000 sequence detection system (Applied Biosystems). The reaction mixture (20 μl) included 2% FastStart universal probe master mix with

ROX (Roche), 900nM each forward and reverse primer, 400nM UPL probe, and

2 μl of the DNA template. PCR was performed in duplicate under the following conditions: initial annealing at 95°C for 10 min, followed by 40 cycles of 95 °C for 10 s, and 60°C for 1 min. ROX was used as a passive reference dye.

[0058] Microfluidic qPCR. To increase the template DNA yield, a preamplification (specific target amplification; STA) reaction was performed prior to the microfluidic qPCR. The STA reaction is a multiplex PCR, which employs the primers used for the microfluidic qPCR with a small number of PCR cycles (e.g., 10 to 14 cycles). In addition to the sample DNA, serial dilutions of the standard plasmid mixture (2 x 10 ⁰ to 2 x 10 ⁵ copies/μl) were amplified using the STA reaction to produce standard curves for the microfluidic qPCR.

[0059] The STA reaction mixture (10 μl) included 2% TaqMan Pre Amp master mix (Applied Biosystems), 0.2 μΜ each primer, and 3.2 μl of the DNA template. The reaction was performed using an ABI Prism 7000 sequence detection system under the following conditions: initial annealing at 95 °C for 10 min, followed by 14 cycles of 95°C for 10 s and 60°C for 4 min. After the STA reaction, the products (10 μl) were diluted 6-fold with 50 μl of TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8) and used in the microfluidic qPCR.

[0060] Unbiased amplification by the STA reaction was confirmed by conventional qPCR with a known amount of the template DNA. In this case the STA products were further diluted 10-fold with TE buffer (i.e., a 60- fold dilution of the original STA product). Microfluidic qPCR was performed in quadruplicate using a BioMark HD reader with a Dynamic Array 48.48 chip or 96.96 chip (Fluidigm) under the detection conditions for TaqMan/MGB chemistry. Aliquots (5 μl) of the sample premix (1% TaqMan universal PCR master mix [Applied Biosystems], 1 x GE sample loading reagent (Fluidigm), and 2.25 μl 6-fold diluted STA product) and 5 μl of the assay premix (1 x assay loading reagent (Fluidigm), 8 μΜ each primer, and 1 μΜ probe) were loaded into the 48.48 chip or the 96.96 chip (Fluidigm) and mixed using an IFC controller MX or HX (Fluidigm) for the 48.48 chip or the 96.96 chip, respectively, according to the manufacturer's instructions. After mixing, the microfluidic qPCR mixture included 800 nM each primer and 100 nM probe. The PCR was performed under the following conditions: 50°C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 70°C for 5 s, and 60°C for 1 min. ROX was used as a passive reference dye.

[0061] An example of the present subject matter can be in the form of a consumable "laboratory on a card" system. The system volume can be commensurate with that associated with the various reactions to take place.

[0062] Beginning with approximately 50 ml of water, assume that it includes at least 1 bacteria/ml contamination. Assume further that any larger particles (such as blood and other debris) are at a very low concentration, if present at all, since this sampling occurs after the device (such as an endoscope or other medical device) has been nominally cleaned and disinfected. It is reasonable to expect that some bacteria will be present at low concentrations.

[0063] An example can take the form of a lab on a card. One example of system 12 is shown in FIG. 5. The figure illustrates a plan or top view of portions of a card.

[0064] Arrow 10 denotes inflow of 50ml of rinsate of potentially contaminated water at port 14. This inflow is split evenly (through a fluid manifold) into 100 collection chambers, some of which are denoted here as chamber 15, in parallel arrangement. Each collection chamber 15 is 4mm long by 4mm wide and 32 microns deep, making a volume of 0.5 microliters (ul). A dielectrophoresis RF field is applied across each collection chamber 15. The dielectrophoresis RF field is at a strength of approximately 30V. Having split the 50 ml flow into one hundred chambers, the flowrate through each channel will be 800ul/hr, taking approximately 35-40 minutes.

[0065] The bacteria found in the rinsate will be attracted to one side of each collection chamber 15, while the depleted water will flow out of the channel leading out of the chamber to join with the outflow from the other collection chambers 15 in a common channel leading to valve 18. Valve 18 can be controlled to allow the depleted water to exit the card at a port, as shown at arrow 19, and disposed.

[0066] After removing the depleted water from the card, the

dielectrophoresis RF field can be powered off. Valve 18 can be controlled to direct the fluid toward propidium monoazide (PMA) mixing section 20 and flush through 50 microliters of water to be split among the collection chambers 15 to rinse the bacteria that will now be resuspended in the water rinsed through. The bacteria suspension then flows into PMA mixing section 20.

[0067] A needle pump, such as pump 22, and a suitable T-junction, can inject 25ul of PMA into the bacterial suspension as signified by arrow 24. The suspension then is mixed with the PMA through a microfluidic mixer section 20. Section 20 includes a plurality of vanes which induces a tortious fluid pathway.

[0068] Mixing section 20 can be a straight section (or with a turn to fit on the card) and is approximately 10 cm long. Protrusions or vanes in the channel induce turbulent mixing flow to allow the PMA to diffuse or penetrate the leaky membranes of the nonviable cells. Section 20 can have channel dimensions of 2 mm wide and depth of 0.5 mm The flowrate through the channel of section 20 will be 0.2 ul/s, which will give a facial velocity of about 0.21 mrn/s. Section 20 has walls 26 of light-opaque material.

[0069] Upon exiting PMA mixing section 20, the suspension flows into PMA light cure section. Light cure section includes light transparent wall segment 31 and emitter 30. Emitter 30 can include an LED lamp that emits light with 470-nm wavelength to cause the PMA to bind to the DNA of the nonviable cells. The light cure section can be 2.5 cm long to enable a two-minute light exposure. The width and depth dimensions of the light cure section can match those of mixing section 20 and is devoid of the mixing geometry.

[0070] Upon exit from the light cure section, the discharge is directed to

PCR section 40.

[0071] Immediately prior to the PCR section 40 is a T-junction and microinjection pump, such as pump 32, to introduce PCR reagent and oil, here denoted by arrow 34, to make microdroplets of the aqueous phase in the oil phase at region 36. A backflow preventer valve, denoted by arrow 38, prevents backflow of the aqueous/oil emulsion into the PMA light cure section.

[0072] The PCR cycling section 40 provides thermal cycling of the stream 50 times. The channel PCR cycling section 40 includes a serpentine path that engages and disengages with heater block 42.

[0073] The PCR cycling section 40 channel is 2mm wide and 0.5 mm deep. The flow rate will remain 0.2 ul/s, again yielding a facial velocity of 0.21 mm/s. The serpentine path of section 40 has an oscillatory path of period length 6.3 mm. There will be 50 repeats of the cycle in section 40 for a total channel length of 31 cm. Heater block 42 is configured to hold temperature at 95C, to heat the suspension and induce the polymerase activity. Downstream half of the PCR cycling section 40 is optical detector 50. Detector 50 can include a CCD camera or digital camera sensitive to fluorescence, to detect the signal generated by the PCR reaction and counted. The suspension will leave the channel after fluorescence detection and be disposed at discharge 52.

[0074] All or portions of system 12 can be fabricated on a card. The card can be fabricated using photolithography or other techniques used for microfluidic fabrication.

[0075] FIG. 6 illustrates method 600 according to one example.

[0076] At 610, rinsate from a device cleaning procedure is provided.

The device can include an endoscope.

[0077] At 620, method 600 includes separating bacteria. In one example, this includes performing dielectrophoresis. Dielectrophoresis can be performed by application of a modulated RF field. In one example, bacteria separation occurs in a plurality of collection chambers.

[0078] At 630, method 600 includes preparing a suspension of bacteria.

The suspension can be formed by rinsing from the collection chambers.

[0079] At 640, method 600 includes mixing the suspension with PMA.

Mixing can be performed in a microfluidic channel and can include passing the suspension through a tortious path having vanes or protrusions.

[0080] At 650, the mixed suspension is treated with light energy. The light energy can have a wavelength and amplitude to promote binding of DN A and nonviable cells.

[0081] At 660, method 600 includes performing PCR. This can entail introduction of a PCR reagent and oil using a microinjection pump.

[0082] At 670, this can include thermal treatment of the aqueous/oil emulsion.

[0083] At 680, method 600 includes counting based on the detected signal generated by the PCR reaction.

[0084] The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments of the present invention. Thus, it should be understood that although the present invention has been specifically disclosed by specific embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those of ordinary skill in the art, and that such modifications and variations are considered to be within the scope of embodiments of the present invention.

[0085] All referenced publications, patents and patent documents are intended to be incorporated by reference, as though individually incorporated by reference.