Quantitative pathogen detection method

Abstract

A method for quantifying the presence of a pathogen in a sample is described herein. The method may include steps of enrichment and concentrating of the pathogen and a statistical method for determining the initial quantity of pathogen in the sample based on the quantity measured after enrichment and concentrating.

Description

Attorney Docket No.: 234994-563211 QUANTITATIVE PATHOGEN DETECTION METHOD CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to and the benefit of U.S. ProvisionalApplication No.63 / 572,438, filed April 1, 2024, the contents of which are herein incorporated by reference in their entirety. TECHNICAL FIELD

[0002] The present invention relates to a method for quantifying the presence of a pathogenin a sample, such as a food sample or environmental sample. BACKGROUND

[0003] To ensure food safety, regulatory agencies such as the United States Department ofAgriculture’s Food Safety and Inspection Service promulgate pathogen-reduction performance standards for pathogens (e.g. Salmonella and Campylobacter) in meat, poultry, egg products, and corresponding processing environments. Some such pathogen-reduction standards apply presence / absence criteria, while others require quantitative information on the pathogen. Many food samples have low numbers of pathogens, which will not be detected by conventional agar plating method for quantitative detection. The most probable number (MPN) technique is used to estimate the concentration of viable microorganisms in a sample by means of performing replicate dilutions of a sample in an appropriate broth. The sample to be enumerated in the MPN technique is diluted to obtain at least one negative result (absence of growth). An MPN index number is based on the statistical probability of the coincidence of microorganisms in each sample replicate. The MPN method is particularly useful for determination of low concentrations of Salmonella in food matrices. For MPN method, three or five replicates are tested from three of 10-fold serial dilutions. All samples are then tested by using the traditional culture method including primary enrichment, secondary enrichment and streaking onto selective / differential agar plates. The positive and negative combination results in relation to the dilutions generate a MPN value in the original sample. The MPN method is tedious and takes three to five days to obtain the results.

[0004] There remains a need for improved rapid methods of pathogen quantification toimprove food safety. 59402683.2Attorney Docket No.: 234994-563211 SUMMARY OF THE INVENTION

[0005] The present invention provides a method for quantifying the presence of a pathogenin a sample.

[0006] In one aspect, provided herein is a method for quantifying the presence of apathogen in a sample comprising: providing an initial sample comprising a pathogen; growing the pathogen in the initial sample in an enrichment medium to produce an enriched sample; concentrating the enriched sample to produce a concentrated sample; determining the quantity of the pathogen in the concentrated sample via a pathogen measurement assay; and quantifying the presence of the pathogen in the initial sample by a statistical method that outputs quantification of the pathogen in the initial sample based on the quantity of the pathogen in the concentrated sample.

[0007] Various embodiments of these and other aspects may include one or more of thefeatures as described in the detailed description below. DRAWINGS

[0008] FIG. 1 is a fitted line plot generated in Example 3 to compare the enumeration ofSalmonella in the chicken rinse between 6 h enrichment method and the traditional MPN method.

[0009] FIG. 2A is a published workflow for BAX® System SalQuant™.

[0010] FIG. 2B is a published workflow for GENE-UP® Quant Salmonella.

[0011] FIGS. 3A and 3B are linear regression plots used to compute the lot-specificcalibration factors for two different lots of Neogen MDS V2 Salmonella (Cat# MDA2SAL96).

[0012] FIG. 4A is a fitted line plot for a further regression analysis showing that lot ID is asignificant (p-value<0.05) contributor to the measurement in Example 7.

[0013] FIG. 4B is a fitted line plot showing fit statistics are superior for the calibrated lotcase according to Example 7. DETAILED DESCRIPTION

[0014] In the following description, numerous specific details are given to provide athorough understanding of the embodiments. The embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other 2 59402683.2Attorney Docket No.: 234994-563211 instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the embodiments.

[0015] Reference throughout this specification to "one embodiment," "an embodiment," or"embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0016] Unless indicated otherwise, when a range of any type is disclosed or claimed, it isintended to disclose or claim individually each possible number that such a range could reasonably encompass, including any sub-ranges encompassed therein. Moreover, when a range of values is disclosed or claimed, which Applicants intend to reflect individually each possible number that such a range could reasonably encompass, Applicants also intend for the disclosure of a range to reflect, and be interchangeable with, disclosing any and all sub- ranges and combinations of sub-ranges encompassed therein.

[0017] The terms "comprises" and variations thereof do not have a limiting meaning wherethese terms appear in the description and claims.

[0018] As used herein, "a," "an," "the," "at least one," and "one or more" are usedinterchangeably. Thus, for example, a microorganism can be interpreted to mean "one or more" microorganisms.

[0019] The term "and / or" means one or all of the listed elements or a combination of anytwo or more of the listed elements.

[0020] The present invention provides a method for quantifying the presence of a pathogenin a sample. The present invention allows for the ability to quantitate low levels of a pathogen in a short time. For example, the use of enrichment and concentrating steps according to the present methods improve the ability to quantitate pathogens in a sample with low pathogen levels. In addition, the use of certain enrichment media as described herein increases the growth rate, leading to faster quantitative detection. The present methods may have a low limit of quantification, for example as low as 1 CFU / mL or 1 CFU / g.

[0021] In one aspect, provided herein is a method for quantifying the presence of apathogen in a sample comprising: providing an initial sample comprising a pathogen; growing the pathogen in the initial sample in an enrichment medium to produce an enriched sample; 3 59402683.2Attorney Docket No.: 234994-563211 concentrating the enriched sample to produce a concentrated sample; determining the quantity of the pathogen in the concentrated sample via a pathogen measurement assay; and quantifying the presence of the pathogen in the initial sample by a statistical method that outputs quantification of the pathogen in the initial sample based on the quantity of the pathogen in the concentrated sample. Samples and Pathogens

[0022] Providing a sample to be tested (or “initial sample”) may comprise providing asample that is suspected of containing a target pathogen or target microorganism. Non- limiting examples of suitable samples include environmental samples (e.g., surface swabs / sponges, soil, sediments, fomites), food samples (e.g., raw materials, in-process samples, and finished-product samples), beverages, clinical / veterinary samples (e.g., blood, serum, plasma, urine, sputum, tissue, mucous, feces, wound exudate, pus, cerebrospinal fluid), and water (e.g., surface water, potable water, process water).

[0023] In some embodiments, the sample (initial sample) is a food sample. In someembodiments, the food sample is a meat sample or a poultry sample.

[0024] In other embodiments, the sample (initial sample) is an environmental sample.

[0025] In some embodiments, the initial sample has a volume of from about 10 mL toabout 100 mL, e.g., about 15 mL to about 50 mL, or about 20 mL to about 40 mL, or about 30 mL.

[0026] In some embodiments, the initial food sample has a size of from about 25 g to about375 g, e.g., about 25g to about 125 g or about 325 g or up to about 375g.

[0027] The sample to be tested contains or is suspected to contain a pathogen or othermicroorganism. Microorganisms of interest include prokaryotic and eukaryotic organisms, such as gram-positive bacteria, gram-negative bacteria, fungi, mycoplasma, and yeast. Relevant organisms include members of the family Enterobacteriaceae, or the family Micrococcaceae or the genera Staphylococcus spp., Streptococcus spp., Pseudomonas spp., Enterococcus spp., Salmonella spp., Legionella spp., Shigella spp. Yersinia spp., Enterobacter spp., Escherichia spp., Bacillus spp., Listeria spp., Vibrio spp., Corynebacteria spp. as well as, Aspergillus spp., Fusarium spp., and Candida spp. Particularly virulent organisms include Staphylococcus aureus ( ) S. epidermidis, Streptococcus pneumoniae, S. agalactiae, S. pyogenes, Enterococcus faecalis, Bacillus anthracis, Pseudomonas aeruginosa, Escherichia coli, Aspergillus niger, A. fumigatus, A. clavatus, Fusarium solani, F. oxysporum, F. chlamydosporum, Listeria monocytogenes, Listeria ivanovii, Vibrio cholera, V 4 59402683.2Attorney Docket No.: 234994-563211 parahemolyticus, Salmonella cholerasuis, S. typhi, S. typhimurium, Candida albicans, C. glabrata, C. krusei, and Cronobacter sakazakii.

[0028] In some embodiments, the pathogen is a bacterium. In some embodiments, thebacterium is a gram-negative bacterium. In some embodiments, the gram-negative bacterium is Salmonella. In some embodiments, the bacterium is a gram-positive bacterium. In some embodiments, the gram-positive bacterium is Listeria. Enrichment

[0029] The sample is enriched to increase the concentration of pathogen in the sample toallow for quantitative detection. This is particularly useful in samples with low concentration of pathogen, e.g., less than 10 CFU / ml, less than 5 CFU / ml, and even as little as 1 CFU / ml or less than 10 CFU / g, less than 5 CFU / g or as little as 1 CFU / g.

[0030] The sample is enriched in an enrichment medium or broth. The pathogen is grownby use of the enrichment medium and growth conditions, e.g., incubation temperatures.

[0031] By utilizing growth in enrichment media, the concentration of pathogen can beincreased to improve detection and quantitation in a quantitative assay. But compared to other methods, the growth in the enrichment medium can be performed in a shorter amount of time, e.g., within a single work shift. Use of particular enrichment media as described herein may also allow for reduced growth times.

[0032] In some embodiments, the pathogen in the initial sample is grown in the enrichmentmedium for about 2 to about 8 hours. In some embodiments, the pathogen in the initial sample is grown in the enrichment medium for about 4 hours to about 8 hours. In some embodiments, the pathogen in the initial sample is grown in the enrichment medium for about 5 hours to about 7 hours. In some embodiments, the sample is not allowed to reach stationary growth phase.

[0033] In some embodiments, the volume of enrichment media used is to grow thepathogen in the initial sample, is from about 10 mL to about 100 mL of enrichment media, e.g., about 15 mL to about 50 mL, or about 20 mL to about 40 mL, or about 30 mL of enrichment media.

[0034] In some embodiments, growth of the pathogen in the enrichment medium includesincubating the pathogen (e.g., bacteria) in the enrichment medium. In some embodiments, the incubation temperature is from about 34°C to about 43 °C, e.g., about 35 °C or about 42°C.

[0035] In some embodiments, growing the pathogen in the initial sample in an enrichmentmedium, concentrating the enriched sample, determining the quantity of the pathogen in the concentrated sample, and quantifying the presence of the pathogen in the initial sample is 5 59402683.2Attorney Docket No.: 234994-563211 completed in a total time of from about 2 to about 12 hours, or from about 6 to about 10 hours, or from about 6 to about 8 hours. For comparison, the MPN method can detect low concentration of pathogens (e.g., as low as 1 CFU / ml), but takes three to five days to obtain results. Enrichment Medium

[0036] The present methods utilize an enrichment medium to grow the pathogen in thesample, i.e., to increase the concentration of pathogen in the sample by reproduction. The enrichment medium supports such growth and accelerates growth compared to other media.

[0037] In some embodiments, the enrichment medium (e.g., enrichment broth) comprisespyruvic acid, magnesium, yeast extract, a selectivity agent that inhibits the growth of gram- positive bacteria, and water.

[0038] In some embodiments, the enrichment medium (e.g., enrichment broth) comprisessodium pyruvate, magnesium sulfate, yeast extract, a selectivity agent that inhibits the growth of gram-positive bacteria, and water.

[0039] In other embodiments, the enrichment medium can be one that isolates gram-positive bacteria or fungi (e.g., yeasts or molds) using their respective sets of selective agents (e.g., nalidixic acid, polymyxin B, colistin sulfate, potassium tellurite, chloramphenicol, chlortetracycline, or other antimicrobial agent).

[0040] The combined use of pyruvic acid (e.g., sodium pyruvate), magnesium (e.g.,magnesium sulfate), and yeast extract reduces growth times compared to other enrichment media, such as buffered peptone water (BPW). The use of magnesium sulfate in combination with yeast extract provides the unexpected benefit of reduced growth time while also reducing the cost of the enrichment media due to the lower cost of magnesium sulfate compared to yeast extract.

[0041] The enrichment medium may include a selectivity agent that inhibits the growth ofgram-positive bacteria. This selectivity is beneficial for faster growth of gram-negative bacteria, such as Salmonella, E. coli, or Cronobacter. For example, when selectivity agents are not present, growth rate may be slowed due to nutrient exclusion as a result of proliferation of competitive bacteria.

[0042] In some embodiments, the selectivity agent is selected from the group consisting ofantibiotics, detergents, and combinations thereof. In some embodiments, the selectivity agent is selected from the group consisting of vancomycin or a salt thereof, sodium dodecylbenzenesulfonate (SDBS), novobiocin or a salt thereof, cefsulodin or a salt thereof, sulfadiazine or a salt thereof, bile salts, sodium lauryl sulfate (SLS), sodium dodecylbenzesulfonate (SDBS), Tergitol 4, and combinations thereof. 6 59402683.2Attorney Docket No.: 234994-563211

[0043] In some embodiments, the selectivity agent is selected from the group consisting ofvancomycin or a salt thereof, SDBS, and combinations thereof. In some embodiments, the selectivity agent is a combination of vancomycin or a salt thereof and SDBS. In some embodiments, the selectivity agent is vancomycin or a salt thereof. In some embodiments, the selectivity agent is vancomycin. In some embodiments, the selectivity agent is SDBS. In some embodiments, SDBS is the only selectivity agent. Vancomycin and / or SDBS inhibit gram-positive bacteria without harming injured pathogen cells. This also contributes to rapid growth rate of bacteria using the enrichment medium of the present invention.

[0044] Pyruvic acid may be in the form of a salt. For example, the pyruvic acid salt may besodium pyruvate, potassium pyruvate, or a combination thereof. In some embodiments, the pyruvic acid salt is sodium pyruvate. In solution, the pyruvic acid and the sodium cation may be dissociated, such that the sodium pyruvate is in the form of separate sodium cations and pyruvic acid anions. Likewise, potassium pyruvate may be in the form of separate potassium cations and pyruvic acid anions.

[0045] In some embodiments, the sodium pyruvate is present in the enrichment medium ina concentration of from about 0.1 to about 5 grams per liter, e.g., from about 0.2 to about 3 grams per liter, from about 0.5 to about 2 grams per liter, or from about 1 to about 1.5 grams per liter. Sodium pyruvate was found to significantly increase growth rate of bacteria in the enrichment medium of the present invention.

[0046] In some embodiments, pyruvic acid is present in the enrichment medium in aconcentration of from about 0.08 to about 4 grams per liter, e.g., from about 0.16 to about 2.4 grams per liter, from about 0.4 to about 1.6 grams per liter, or from about 0.8 to about 1.2 grams per liter.

[0047] Magnesium may be in the form of a salt. For example, the magnesium may bemagnesium sulfate, magnesium chloride, or a combination thereof. In some embodiments, the magnesium salt is magnesium sulfate. In some embodiments, the magnesium sulfate in the enrichment medium is anhydrous magnesium sulfate.

[0048] In some embodiments, the concentration of yeast extract in the enrichment mediumis from about 1 to about 9 grams per liter (e.g., from about 2.5 to about 7.5 grams per liter or from about 4 to about 6 grams per liter) and the concentration of magnesium sulfate is from about 0.5 to about 10 millimolar (e.g., from about 1 to about 5 millimolar or from about 2 to about 4 millimolar). As noted above, the combined use of pyruvic acid (e.g., sodium pyruvate), magnesium (e.g., magnesium sulfate), and yeast extract is a surprising synergistic combination that reduces growth times compared to other enrichment media, such as buffered 7 59402683.2Attorney Docket No.: 234994-563211 peptone water (BPW). The concentrations of sodium pyruvate, magnesium sulfate, and yeast extract in the enrichment medium also contribute to the reduced growth times.

[0049] In some embodiments, the concentration of yeast extract in the enrichment mediumis from about 1 to about 9 grams per liter (e.g., from about 2.5 to about 7.5 grams per liter or from about 4 to about 6 grams per liter) and the concentration of magnesium is from about 0.5 to about 10 millimolar (e.g., from about 1 to about 5 millimolar or from about 2 to about 4 millimolar).

[0050] In some embodiments, the enrichment medium further comprises potassiumchloride. In some embodiments, the enrichment medium further comprises sodium chloride. In some embodiments, the sodium chloride is in a concentration of less than or equal to about 3 grams per liter, e.g., from about 1.5 to about 2.5 grams per liter. Sodium chloride levels are normally 5 grams per liter in traditional media like buffered peptone water. The enrichment medium of the present invention may have reduced levels of sodium chloride, such as less than 5 grams per liter, less than about 3 grams per liter, or from about 1.5 to about 2.5 grams per liter. In some embodiments, the enrichment medium comprises from about 1 gram per liter to about 5 grams per liter of potassium chloride, e.g., from about 1.5 grams per liter to about 3 grams per liter.

[0051] In some embodiments, the enrichment medium is substantially free of sodiumphosphate. In some embodiments, the enrichment medium further comprises potassium phosphate (e.g., monobasic and / or dibasic). In some embodiments, higher phosphate levels are utilized in the enrichment medium of the present invention, which reduces shifts in pH.

[0052] In some embodiments, the enrichment medium further comprises phenol red. Thepresence of phenol red may aid as a pH indicator in case the sample enrichment starts off with an unusually acidic starting pH. In such an instance, NaOH may be added to balance pH to neutral. In some embodiments, the pH of the enrichment medium is from about 6.5 to about 7.5, e.g., from about 6.8 to about 7.2, or about 7.

[0053] In some embodiments, the enrichment medium further comprises glucose. In someembodiments, the enrichment medium comprises dextrose. In some embodiments, the enrichment medium comprises from about 1 gram per liter to about 5 grams per liter of dextrose, e.g., from about 1 gram per liter to about 3 grams per liter.

[0054] In some embodiments, the enrichment medium further comprises casein peptone. Insome embodiments, the concentration of casein peptone in the enrichment medium is reduced compared to traditional enrichment medium. For example, the concentration of casein peptone in the enrichment medium may be less than about 10 grams per liter, less than about 7.5 grams per liter, from about 2 grams per liter to about 10 grams per liter, from about 2.5 8 59402683.2Attorney Docket No.: 234994-563211 grams per liter to about 7.5 grams per liter, or from about 4 grams per liter to about 6 grams per liter.

[0055] In some embodiments, the enrichment medium further comprises trisodium citratedihydrate. In some embodiments, the concentration of trisodium citrate dihydrate in the enrichment medium is from about 0.1 to about 2 grams per liter, e.g. from about 0.2 to about 1 grams per liter.

[0056] In some embodiments, the enrichment medium further comprises polysorbate 80(Tween 80).

[0057] In some embodiments, the enrichment medium is substantially free of chicken fat,thioglycolate, and / or mannitol. In some embodiments, the enrichment medium is substantially free of iron. In some embodiments, the enrichment medium is substantially free of casamino acids and / or lactose.

[0058] The enrichment medium may be irradiated, autoclavable, and / or ready-to-use.

[0059] In some embodiments, the enrichment medium comprises:from about 0.2 to about 3 grams per liter of sodium pyruvate; from about 2.5 to about 7.5 grams per liter of yeast extract; from about 1 to about 5 millimolar of magnesium sulfate; and vancomycin, SDBS, or a combination thereof.

[0060] In some embodiments, the enrichment medium comprises:from about 0.2 to about 3 grams per liter of sodium pyruvate; from about 2.5 to about 7.5 grams per liter of yeast extract; from about 1 to about 5 millimolar of magnesium sulfate; vancomycin, SDBS, or a combination thereof; casein peptone; sodium chloride; and potassium phosphate.

[0061] In other embodiments, the enrichment medium comprises from about 0.5 to about 2grams per liter of sodium pyruvate; from about 2.5 to about 7.5 grams per liter of yeast extract; from about 1 to about 5 millimolar of magnesium sulfate; and water. In some embodiments, the concentration of yeast extract is from about 4 to about 6 grams per liter, and the concentration of magnesium sulfate is from about 2 to about 4 millimolar. In some embodiments, the enrichment medium further comprises potassium chloride and less than or equal to about 3 grams per liter of sodium chloride.

[0062] In yet other embodiments, the enrichment medium comprises from about 0.4 to about1.6 grams per liter of pyruvic acid; from about 2.5 to about 7.5 grams per liter of yeast 9 59402683.2Attorney Docket No.: 234994-563211 extract; from about 1 to about 5 millimolar of magnesium; and water. In some embodiments, the concentration of yeast extract is from about 4 to about 6 grams per liter, and the concentration of magnesium is from about 2 to about 4 millimolar. In some embodiments, the enrichment medium further comprises potassium chloride and less than or equal to about 3 grams per liter of sodium chloride.

[0063] In some embodiments, the enrichment medium is autoclaved prior to use for growinga pathogen. In some embodiments, the enrichment medium is autoclaved at a temperature of less than or equal to 120 °C, e.g., less than or equal to 115 °C, less than or equal to about 110 °C, from about 100 to about 120 °C, from about 105 to about 115°C, or about 110 °C. The enrichment medium may include vancomycin in the medium. Autoclaving at lower temperature allows for the inclusion of vancomycin in the medium without the need to add vancomycin after autoclaving, as would be required at higher temperatures. This allows vancomycin to be included in an autoclavable and / or ready to use product. Concentrating

[0064] The speed and consistency of detection time is improved by concentrating thesample. Various methods for concentrating a sample are known in the art and may be employed in the present methods, including centrifugation, beads (e.g., magnetic beads), filtration, IMS, or column techniques).

[0065] In some embodiments, concentrating the enriched sample comprises centrifugingthe sample. In some embodiments, centrifuging the sample comprises producing a centrifuged sample comprising a pellet and a supernatant liquid, and removing the supernatant liquid and resuspending the pellet. In some embodiments, concentrating is performed via a single centrifugation step, i.e., without additional centrifugation steps.

[0066] In some embodiments, the centrifugation speed is from about 4,000 rcf (relativecentrifugal force) to about 10,000 rcf. In some embodiments, the centrifugation time is about 3 to about 10 minutes. In some embodiments, the centrifugation speed is from about 4,000 to about 6,000 rcf, e.g., for a thick matrix sample such as ground meat. In some embodiments, the speed of centrifugation is about 5,000 rcf, and centrifugation time is about 5 minutes.

[0067] In some embodiments, the centrifuge is a microcentrifuge or mini centrifuge,designed for centrifugation of small sample volumes, e.g., about 0.5 to about 2 mL (e.g., 1.5 mL). In some embodiments, a small aliquot from the enriched sample is used for concentrating. For example, from about 0.5 to about 5 mL, e.g., from about 1 to about 2 mL, e.g., about 1.5 mL of the enriched sample may be used for concentrating to produce the concentrated sample. 10 59402683.2Attorney Docket No.: 234994-563211

[0068] In some embodiments, the extent of concentrating is to produce a sample havingfrom about 5 times to about 20 times the concentration of pathogen per unit volume. For example, the concentrating may produce a concentrated sample having 10 times the concentration of the pathogen as compared to prior to concentrating the sample.

[0069] In other embodiments, concentrating the sample comprises using beads toconcentrate the sample. In some embodiments, the beads are magnetic beads.

[0070] In some embodiments, centrifugation and beads are used in combination toconcentrate the sample. Pathogen Measurement Assay

[0071] A pathogen measurement assay is used to quantify the pathogen in the concentratedsample.

[0072] The concentrated sample may be lysed prior to the pathogen measurement assay (oras part of the assay) in order to prepare the concentrated sample for detection by the assay. Lysis of pathogen cells in the sample can be performed by any suitable methods known in the art such as chemical lysis, thermal lysis, or mechanical lysis.

[0073] A sample volume of the concentrated sample may be aliquoted for lysis. Thevolume may be from about 10 uL to about 1 mL, e.g., from about 20 uL to about 100 uL, or about 30 uL to about 70 uL, or about 50 uL. Lysis solution may be added to the aliquot of the concentrated sample, e.g., 10 uL to about 1 mL, or from about 20 uL to about 100 uL, or about 30 uL to about 70 uL, or about 50 uL of lysis solution. A sample volume of the lysed sample may be aliquoted for use in the pathogen measurement assay, e.g., from about 5 uL to about 100 uL, from about 10 uL to about 40 uL, or about 20 uL.

[0074] In some embodiments, the pathogen measurement assay is a loop-mediatedisothermal amplification (LAMP) assay. For example, the Neogen Molecular Detection System (including MDA2SAL96 for Salmonella) uses a LAMP assay.

[0075] In some embodiments, the pathogen measurement assay is a quantitative real-timepolymerase chain reaction (qPCR) assay. In other embodiments, the pathogen measurement assay is a nicking-enzyme amplification reaction (NEAR) assay, a helicase-dependent amplification (HDA) assay, a nucleic acid sequence-based amplification (NASBA) assay, or a transcription-mediated amplification (TMA) assay.

[0076] Suitable variations of such assays may be used. Variations on a traditional LAMPassay that may be used may include colorimetric LAMP (cLAMP) assays, in which pH changes driven by the accumulation of protons during LAMP can be visualized via observation of color changes of a pH-sensitive colorimetric dye that occur with nucleic acid amplification. Other such variations may include turbidity-LAMP assays, in which formation 11 59402683.2Attorney Docket No.: 234994-563211 of magnesium pyrophosphate during LAMP results in turbidity that increases in correlation with nucleic acid yield and that can be quantified in real-time. Materials and methods used in such variations on traditional LAMP assays, and / or on PCR assays, may be understood by those of skill in the art and thus are not described in detail here. Statistical Methods

[0077] The amount of pathogen that grows during enrichment and is concentrated by theconcentrating method is correlated via a statistical method to the amount of pathogen initially in the sample.

[0078] In the present methods, the initial number of target organisms may be calculatedbased on the detection value (in minutes), e.g., using an algorithm.

[0079] In some embodiments of the present methods, the statistical method is a regressionmethod, e.g., linear regression.

[0080] The statistical method may be based on calibration data collected from samples witha known initial quantity of a pathogen. In some embodiments, the calibration data is used to generate a calibration curve or calibration line. In some embodiments, the statistical method is based on the relationship between the detection time, and the amount of the target DNA / RNA.

[0081] Lot-specific calibration

[0082] To increase accuracy, the calibration data may be generated with materials from thesame lot as the materials used in the initial sample and / or enrichment medium. The ability to control for noise in the reagents used in the present quantitative methods is also advantageous. All assays have inherent variability due to the manufacturing process, such that not every lot yields the same response. In some embodiments, each lot of reagents is calibrated against known concentration standards. The information from this calibration may be distributed to customers via a cloud system. In some embodiments, molecular reagents prepared such that under normal shipping and storage conditions over time do not appreciably change their ‘detection value’ when challenged by a defined level of stimulus (i.e. copy number). In some embodiments, a “calibration” or reference line is generated at the time the molecular reagents are made. Such a calibration correlates the arbitrary ‘detection value’ to a true, physical value (gene copy number, CFU, etc.)

[0083] In some embodiments, the calibration or reference line is distributed to the endusers of the test. Such information can then be used by the method at any time in the shelf- life window of the product. 12 59402683.2Attorney Docket No.: 234994-563211

[0084] EXAMPLES

[0085] The molecular quantification methods of the following examples are based on therelationship between the detection time, and the amount of the target DNA / RNA. Increasing detection time is correlated with decreasing copy numbers. Variations increase when the target copy number is low which results in the uncertainty of quantification accuracy. A short time enrichment in a superior growth media allows the fast recovery of stressed cells and the rapid growth of the target microorganism. Including a concentrating step decreases the variation of the detection time and increases the accuracy for quantification.

[0086] Example 1

[0087] Inoculum Preparation

[0088] A single colony of Salmonella Infantis was grown in tryptic soy broth (TSB) at35 °C for 18-22 hours. The overnight culture of Salmonella was serially diluted in phosphate buffered saline (PBS) and plated on tryptic soy agar (TSA) to determine the titer.

[0089] Chicken Rinse Inoculation and Holding

[0090] Chicken broiler carcass was rinsed with 400 ml of neutralizing buffered peptonewater (nBPW). Discarded the chicken. Divided the chicken rinse into 30 ml aliquot samples.

[0091] Inoculated samples with diluted Salmonella at 1.4 CFU / mL. The samples were heldat 4 °C overnight before enrichment to simulate the cold storage organism stress.

[0092] Enrichment

[0093] Thirty milliliters (30 mL) of pre-warmed (at 50 °C) Example 5 Broth or bufferedpeptone water (BPW) were added to each chicken carcass rinse sample. The samples were incubated at 42 °C for 6 hours.

[0094] Sample Analysis with MDA2SAL96

[0095] After 6 h enrichment, the sample was analyzed with or without concentrating usingthe Neogen® Molecular Detection Assay 2, Salmonella (MDA2SAL96) (Neogen Corporation, Lansing, Michigan). For samples that were tested without concentrating, 20 uL of enrichment broth was added to lysis cluster tubes. For concentrating the sample, 1.5 mL of enrichment broth was added to the microcentrifuge tube, centrifuged for 5 mins at 5000 rcf, and supernatant was discarded. After resuspending the resultant pellet with 100 uL of BPW; 20 uL was withdrawn to the lysis cluster tubes.

[0096] Lysis was performed by incubating the cluster tubes for 15 minutes at 100 °C, andthen cooling the tubes at 4 °C for 5 – 10 minutes. Twenty microliters (20 uL) of lysate was transferred to MDA2SAL96 reaction tubes. Reaction tubes were loaded into the Molecular Detection Instrument, and the MDA 2 - Salmonella assay was run using the software instructions. 13 59402683.2Attorney Docket No.: 234994-563211

[0097] Culture confirmation

[0098] Continued incubation of the enrichment samples for 24 hours (primary enrichment).Streaked the primary enrichment broth on Salmonella differential / selective media. Incubated the plates at 35°C for 24 hours. Checked the plates for typical Salmonella colonies.

[0099] In addition, transferred 100 uL of primary enrichment to RV (Rappaport-Vassiliadis) media (10 mL) for secondary enrichment. Incubated the tubes at 42 °C for 24 hours.

[0100] Streaked the secondary enrichment broth on Salmonella differential / selective media.Incubated the plates at 35°C for 24 hours. Checked the plates for typical Salmonella colonies.

[0101] The data collected is shown in Table 1, below. Example 5 Broth showed better andfaster growth for Salmonella in the chicken carcass rinse than traditional BPW after 6 h enrichment. Incorporating a concentrating step by centrifugation, decreased the variations of detection times.

[0102] Table 1MDA2SAL96 Culture14 59402683.2Attorney Docket No.: 234994-563211

[0103] Example 2

[0104] Inoculum Preparation

[0105] A single colony of Salmonella Typhimurium was grown in TSB at 35 °C for 18-22hours. The overnight culture of Salmonella was serially diluted in PBS and plated on TSA to determine the titer.

[0106] Chicken Rinse Inoculation and Holding

[0107] Chicken broiler carcass was rinsed with 400 ml of nBPW. Discarded the chicken.Divided the chicken rinse into 30 ml aliquot samples.

[0108] Inoculated samples with diluted Salmonella at 0.74 CFU / mL. The samples wereheld at 4 °C over the weekend before enrichment.

[0109] Enrichment

[0110] Thirty milliliters (30 mL) of pre-warmed (at 50 °C) Example 5 Broth were added toeach chicken carcass rinse sample. The samples were incubated at 42°C for 6 hours.

[0111] Sample Analysis with MDA2SAL96

[0112] After 6h enrichment, the sample was analyzed with or without concentrating usingthe Neogen® Molecular Detection Assay 2, Salmonella (MDA2SAL96) (Neogen Corporation, Lansing, Michigan). For samples that were tested without concentrating, 20 uL of enrichment broth was added to lysis cluster tubes. For concentrating the samples, 1.5 mL of enrichment broth was transferred to the microcentrifuge tube, 30 uL of Dynabeads® Anti- Salmonella beads were added, and samples were centrifuged to concentrate the sample. The beads were resuspended in 150 uL 0.1% Peptone water, and 20 uL were withdrawn to the lysis cluster tubes.

[0113] Lysis was performed by incubating the cluster tubes for 15 minutes at 100 °C, andthen cooling the tubes at 4 °C for 5 – 10 minutes. Twenty microliters (20 uL) of lysate was transferred to MDA2SAL96 reaction tubes. Reaction tubes for each sample (A, B, C, D, E, F) were loaded into the Molecular Detection Instrument, and the MDA 2 - Salmonella assay was run according to manufacturer’s instructions.

[0114] The results are shown in Table 2, below.15 59402683.2Attorney Docket No.: 234994-563211

[0115] Table 2n

[0116] Incorporating a concentrating step using Dynabeads® Anti-Salmonella beadsdecrease the variations of detection times, which will generate a more accurate result for quantification.

[0117] Example 3

[0118] Inoculum Preparation

[0119] A single colony of Salmonella Typhimurium was grown in TSB at 35 °C for 18-22hours. The overnight culture of Salmonella was serially diluted in PBS and plated on TSA to determine the titer.

[0120] Chicken Carcass Inoculation and Holding

[0121] Two whole chickens were purchased from the grocery store. The chicken carcasseswere spiked with Salmonella Typhimurium inside and outside of the bird, one bird at 300 – 400 CFU, the other bird at about 3000 – 4000 CFU. After inoculation, each bird was rinsed with 400 ml of nBPW. Discarded the chicken. Divided chicken rinse into 30ml aliquot samples. The samples were held at 4 °C over the weekend before enrichment.

[0122] Enrichment

[0123] Thirty milliliters (30 mL) of pre-warmed (at 50 °C) Example 5 Broth were added toeach chicken carcass rinse sample. The samples were incubated at 42°C for 6 hours.

[0124] Sample Analysis with MDA2SAL96 or Traditional MPN method16 59402683.2Attorney Docket No.: 234994-563211

[0125] MDA2SAL96 method: After 6h enrichment, transferred 1.5 mL of enrichment brothto the microcentrifuge tube, centrifuged for 5 mins at 5000 rcf, and discarded the supernatant. After resuspending the resultant pellet with 100 uL of Example 5 Broth, withdrew 50 uL to the lysis cluster tubes.

[0126] Lysis was performed by incubating the cluster tubes for 15 minutes at 100° C, andthen cooling the tubes at 4° C for 5 – 10 minutes. Twenty microliters (20 uL) of lysate was transferred to MDA2SAL96 reaction tubes. Loaded the reaction tubes into the Molecular Detection Instrument, and ran the MDA 2 - Salmonella assay according to assay instructions.

[0127] MPN method: three replicates at each three dilutions were used for MPN. Thesample was enriched in BPW (primary enrichment) at 35 °C for 24h. Transferred 100 uL from BPW enrichment to 10 mL RV tube (secondary enrichment), incubated at 42 °C for 24h. Streaked 10 uL RV enrichment broth on XLD plates, incubated the plates at 35C for 24h. Examined the plates for typical Salmonella colonies, recorded the combination of positive tubes, obtained MPN results from MPN table or MPN calculator.

[0128] Fitted line plot (FIG. 1) was generated to compare the enumeration of Salmonella inthe chicken rinse between 6 h enrichment method and the traditional MPN method.

[0129] The obtained detection times were used to estimate the Log10 CFU / mL ofSalmonella in the chicken rinse sample. The scatter plot was generated to compare the estimate Log10 CFU / mL of Salmonella to the Log MPN / mL in the sample. The R2 is 0.88, Standard Error is 0.25. The accuracy of the present method for calculating the concentration of Salmonella in the sample is very close to MPN method. Utilization of MDS Detection values for estimating Log10 CFU / mL of Salmonella in whole carcass rinsates provides faster time to results, and requires less labor per sample compared to current standard MPN methodology.

[0130] Example 4

[0131] Example 4A: A first exemplary workflow for a quantitative detection method isshown below. 30 mL Poultry Rinse 30 mL Example 5 Broth Incubate at 42 °C for 6 h n 59402683.2Attorney Docket No.: 234994-563211

[0132] Example 4B: A second exemplary workflow for a quantitative detection method isshown below. e

[0133] Example

[0134] An exemplary enrichment medium (“Example 5 Broth”) is shown in the tablebelow. Enrichment Medium 59402683.2Attorney Docket No.: 234994-563211

[0135] Example 6

[0136] CFU concentrations were calculated based on published workflows for BAX®System SalQuant ™ and GENE-UP® Quant Salmonella. Analogous concentrations were calculated based on an exemplary workflow according to the present invention. The calculations are shown in the table below. cfu o ia no ic ss y b e 627to ti e .1https: / / www.hygiena.com / news / everything-you-need-know-about-fsis-regulatory-framework- poultry (accessed March 25, 2024) and are shown in FIG.2A and FIG.2B.

[0138] Example 7

[0139] As known in the molecular field, qPCR is a standard technique that can quantifythe amount of DNA added to a qPCR reaction (https: / / doi.org / 10.3389 / fmicb.2017.00108). However, it is acknowledged that for qPCR to be a true quantitative technique, controls must be run. For small batches of tests the use of controls can double the total cost per test. Furthermore, in manufacturing any diagnostic reagent, variations occur when making or manufacturing each batch of reagent.

[0140] To improve overall test accuracy, we developed a system to control lot-to-lotnoise of reagents, the system comprising (1) a molecular kit where the ‘detection- 19 59402683.2Attorney Docket No.: 234994-563211 value’ of the kit does not change over time when challenged by a defined amount of target pathogenic gene, (2) a means of running controls after production of a given lot of reagent (i.e., a known level of challenge is correlated to a ‘detection- value’ of the assay) and (c) an electronic means (e.g., cloud SW, QR code) to distribute the lot specific calibration information to end-users.

[0141] To exemplify the approach, first the kit-lots were calibrated. To accomplish this,two lots of Neogen MDS V2 Salmonella (Cat# MDA2SAL96) were obtained. A reference DNA material spanning the assay target DNA region was created and copy number were calculated using UV measurement of purified DNA preparation.

[0142] Using the MDS kit, 20 uL of buffered peptone water was added to multiple lysistubes and heat processed according to the kit IFU. While avoiding any precipitate from the BPW, the liquid contents of the tubes were pooled. Using the pooled lysate the challenge reference material was diluted in separate tubes such that there was an expected 500,000; 50,000; 5,000; and 500 copies per 20 uL of pooled lysate (otherwise known as Log10(Reaction Copy) at values of 5.7, 4.7, 3.7, 2.7. From each challenge level, 20 uL aliquots were added to eight separate MDS Salmonella reagent tubes containing lyophilized reagent. Each reagent tube was then capped and vortexed to mix the liquid contents with the lyophilized reagent then spun down using a butterfly rotor until the liquid was collected at the bottom of the tubes. The prepared reagent tubes were run on MDS instruments and software per the IFU.

[0143] After the instrument completed, the results were processed to extract the‘detection-value.’ In this case the time to peak maximum as described in figure 2A of (https: / / journals.plos.org / plosone / article?id=10.1371 / journal.pone.0014155) was used. These values were used in a linear regression to compute the lot- specific calibration factors for the two different lots, which are shown in FIGS.3A and 3B. For Fig.3A (Lot 1), linear regression yielded y = -0.3606x + 12.598 and R² = 0.9455. For Fig.3B (Lot 2), linear regression yielded y = -0.4208x + 13.794 and R² = 0.9671.

[0144] These two MDS lots were then used to test chicken rinses prepared as described inExamples 1-3. The lot-specific calibration information was electronically mailed to a central account and then used in statistical analysis. The calibration data using these two lots were used to examine test data obtained 1 week to 3 months after the calibrations were performed. In this case, a further regression analysis 20 59402683.2Attorney Docket No.: 234994-563211 (FIG.4A and the table below) shows that the lot ID is a significant (p-value<0.05) contributor to the measurement.

[0145] Subsequently, the fit statistics for the calibrated value were determined (FIG. 4B).

[0146] As demonstrated, the fit statistics are superior for the calibrated lot case.Case R^2 S (RMSE)No Kit-Lot Calibration 81% 0.36With Kit-Lot Calibration 87% 0.30sshown in the table below. 21 59402683.2

Claims

Attorney Docket No.: 234994-563211 CLAIMS WHAT IS CLAIMED IS:

1. A method for quantifying the presence of a pathogen in a sample comprising: providing an initial sample comprising a pathogen; growing the pathogen in the initial sample in an enrichment medium to produce an enriched sample; concentrating the enriched sample to produce a concentrated sample; determining the quantity of the pathogen in the concentrated sample via a pathogen measurement assay; and quantifying the presence of the pathogen in the initial sample by a statistical method that outputs quantification of the pathogen in the initial sample based on the quantity of the pathogen in the concentrated sample.

2. The method of claim 1, wherein the sample is a food sample.

3. The method of claim 1, wherein the sample is an environmental sample.

4. The method of any preceding claim, wherein the pathogen is a bacterium.

5. The method of claim 4, wherein the bacteria is a gram-negative bacterium.

6. The method of claim 5, wherein the gram-negative bacteria is Salmonella.

7. The method of claim 4, wherein the pathogen is a gram-positive bacteria, such as Listeria.

8. The method of any preceding claim, wherein the enrichment medium comprises pyruvic acid, magnesium, yeast extract, a selectivity agent that inhibits the growth of gram- positive bacteria, and water.

9. The method of any preceding claim, wherein the enrichment medium comprises sodium pyruvate, magnesium sulfate, yeast extract, a selectivity agent that inhibits the growth of gram-positive bacteria, and water. 22 59402683.2Attorney Docket No.: 234994-563211 10. The method of any preceding claim, wherein the pathogen in the initial sample is grown in the enrichment medium for from about 2 to about 8 hours.

11. The method of claim 10, wherein the pathogen in the initial sample is grown in the enrichment medium for from about 4 hours to about 8 hours.

12. The method of claim 11, wherein the pathogen in the initial sample is grown in the enrichment medium for from about 5 hours to about 7 hours.

13. The method of any preceding claim, wherein concentrating the enriched sample comprises centrifuging the sample.

14. The method of claim 13, wherein centrifuging the sample comprises producing a centrifuged sample comprising a pellet and a supernatant liquid, and removing the supernatant liquid and resuspending the pellet.

15. The method of any preceding claim, wherein concentrating the sample comprises using beads to concentrate the sample.

16. The method of any preceding claim, wherein the pathogen measurement assay is a loop-mediated isothermal amplification (LAMP) assay.

17. The method of any preceding claim, wherein the pathogen measurement assay is a quantitative real-time polymerase chain reaction (qPCR) assay.

18. The method of any preceding claim, wherein the statistical method is a regression method.

19. The method of any preceding claim, wherein the statistical method is a linear regression method.

20. The method of any preceding claim, wherein the statistical method is based on calibration data collected from samples with a known initial quantity of a pathogen. 23 59402683.2Attorney Docket No.: 234994-563211 21. The method of claim 20, wherein the calibration data is used to generate a calibration curve or calibration line.

22. The method of claim 20 or 21, wherein the calibration data is generated with materials from the same lot as the materials used in the initial sample and / or enrichment medium.

23. The method of any preceding claim, wherein the growing, concentrating, determining, and quantifying steps are completed in a total time of from about 2 to about 12 hours.

24. The method of claim 23, wherein the total time is from about 6 to about 10 hours.

25. The method of claim 24, wherein the total time is from about 6 to about 8 hours.

26. The method of any preceding claim, further comprising lysing pathogen cells in the concentrated sample. 24 59402683.2

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