WO2020106897A1

WO2020106897A1 - Methods and materials for detecting salmonella in beef

Info

Publication number: WO2020106897A1
Application number: PCT/US2019/062493
Authority: WO
Inventors: Alex Brandt; Randal GARRETT; Aaron PLEITNER; Wayne Muraoka MURAOKA; Katlyn HOLZER
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-11-20
Filing date: 2019-11-20
Publication date: 2020-05-28
Anticipated expiration: 2021-05-20

Abstract

Materials and methods for detecting Salmonella in beef are provided herein.

Description

METHODS AND MATERIALS FOR DETECTING SALMONELLA IN BEEF

TECHNICAL FIELD

This document relates to materials and methods for detecting Salmonella in food, and more particularly to materials and methods for detecting Salmonella in a

concentration of at least a starting threshold concentration or a starting threshold abundance in beef.

BACKGROUND

Salmonella is a genus of bacteria that can cause food-borne illnesses. Salmonella can be present on food, for example, eggs, poultry, meat (e.g., beef, pork), fruits, vegetables, and nuts. Salmonella can cause salmonellosis, characterized by diarrhea, fever, abdominal cramps, and vomiting.

SUMMARY

This document is based, at least in part, on methods and material for detecting Salmonella in beef.

In one aspect, this document features a method of determining whether

Salmonella is present at a starting abundance of at least about 10 CFU/g in a sample of beef. The method comprises, or consists essentially of or consists of, providing a sample of beef, combining the sample of beef with an enrichment media to form a culture, incubating the culture for a period of time (e.g., from about 3.5 hours to about 4.5 hours, from about 3.75 hours to about 4.25 hours, or for about 4 hours) at about 41 to 43°C, performing an assay on a portion of the culture, wherein the assay detects the presence or absence of Salmonella , and either determining that Salmonella was present at a starting abundance of at least about 10 CFU/g in the sample of beef if the assay detects the presence of Salmonella or determining that Salmonella was not present at a starting abundance of at least about 10 CFU/g in a sample of beef if the assay does not detect the presence of Salmonella. In some cases, enrichment media can include sodium phosphate. In some cases, the assay can be a nucleic acid amplification assay. In some cases, the assay can be a real-time (RT) PCR assay. In some cases, the method can further include homogenizing the sample of beef, the culture, or both. In some cases, the sample of beef can be a N60 Plus beef trim sample.

In another aspect, this document features a method of determining whether Salmonella is present in at least about a starting threshold abundance in a sample of beef. The method comprises, or consists essentially of or consists of, choosing an assay that detects the presence or absence of Salmonella , determining a set of sample preparation conditions, a set of growth conditions, and a set of assay conditions that result in the detection of Salmonella in at least about a starting threshold abundance using the chosen assay, preparing a sample of beef using the determined sample preparation conditions to form a culture, incubating the culture using the determined growth conditions, performing the assay on a portion of the culture using the determined assay conditions, and either determining that Salmonella was present in at least about the starting threshold abundance in the sample of beef if the assay detects the presence of Salmonella or determining that Salmonella was not present in at least about the starting threshold abundance in a sample of beef if the assay does not detect the presence of Salmonella. In some cases, the sample preparation conditions can include combining a sample of beef with an enrichment media to form a culture. In some cases, the enrichment media can include sodium phosphate. In some cases, the assay can be a nucleic acid amplification assay. In some cases, the assay can be a real-time (RT) PCR assay. In some cases, the sample preparation conditions can include homogenizing the sample of beef, the culture, or both. In some cases, the sample of beef can be an N60 Plus beef trim sample. In some cases, the starting threshold abundance can be at least about 10 CFU/g.

In another aspect, this document features a method of determining whether Salmonella is present at a starting abundance of at least about 10 CFU/g in a sampling device (e.g., a beef sampling device). The method comprises, or consists essentially of or consists of, providing a sampling device (e.g., beef sampling device), combining the sampling device (e.g., beef sampling device) with an enrichment media to form a culture, incubating the culture for a period of time (e.g., from about 3.5 hours to about 4.5 hours, from about 3.75 hours to about 4.25 hours, or for about 4 hours) at about 41 to 43°C, performing an assay on a portion of the culture, wherein the assay detects the presence or absence of Salmonella , and either determining that Salmonella was present at a starting abundance of at least about 10 CFU/g in the sampling device (e.g., beef sampling device) if the assay detects the presence of Salmonella or determining that Salmonella was not present at a starting abundance of at least about 10 CFU/g in the sampling device (e.g., beef sampling device) if the assay does not detect the presence of Salmonella. In some cases, enrichment media can include sodium phosphate. In some cases, the assay can be a nucleic acid amplification assay. In some cases, the assay can be a real-time (RT) PCR assay. In some cases, the sampling device can be a beef sampling device in the form of a swab, sheet, or cloth for contacting and testing beef trim.

In another aspect, this document features a method of determining whether Salmonella is present in at least about a starting threshold abundance in a sampling device (e.g., a beef sampling device). The method comprises, or consists essentially of or consists of, choosing an assay that detects the presence or absence of Salmonella , determining a set of sample preparation conditions, a set of growth conditions, and a set of assay conditions that result in the detection of Salmonella in at least about a starting threshold abundance using the chosen assay, obtaining a sampling device (e.g., a beef sampling device) and using the sampling device and the determined sample preparation conditions to form a culture, incubating the culture using the determined growth conditions, performing the assay on a portion of the culture using the determined assay conditions, and either determining that Salmonella was present in at least about the starting threshold abundance in the sampling device (e.g., beef sampling device) if the assay detects the presence of Salmonella or determining that Salmonella was not present in at least about the starting threshold abundance in the sampling device (e.g., beef sampling device) if the assay does not detect the presence of Salmonella. In some cases, the sample preparation conditions can include combining the sampling device (e.g., beef sampling device) with an enrichment media to form a culture. In some cases, the enrichment media can include sodium phosphate. In some cases, the assay can be a nucleic acid amplification assay. In some cases, the assay can be a real-time (RT) PCR assay. In some cases, the sampling device can be a beef sampling device in the form of a swab, sheet, or cloth for contacting and testing beef trim. In some cases, the starting threshold abundance can be at least about 10 CFU/g.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although methods and materials similar or equivalent to those described herein can be used to practice aspects of the disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the disclosure are set forth in the description below. Other features, objects, and advantages of the disclosure will be apparent from the claims.

DETAILED DESCRIPTION

This document provides materials and methods related to the detection of Salmonella (e.g., any bacteria belonging to the Salmonella genus) in a sample of beef or in a sampling device (e.g., a beef sampling device). This document also provides methods and materials related to detecting Salmonella in an amount of at least a threshold abundance (e.g., in CFU/g of sample). For example, this document provides methods and materials related to performing an assay to detect Salmonella in a sample of beef or in a sampling device (e.g., a beef sampling device). In some embodiments, this document provides methods and materials related to performing a nucleic acid amplification assay to detect Salmonella in a sample of beef or in a sampling device (e.g., a beef sampling device). In some cases, the techniques provided herein can be used in a reference laboratory to detect Salmonella in a sample of beef (or in a sampling device such as a beef sampling device) to determine whether a beef sample (or a sampling device such as a beef sampling device) contains Salmonella in an amount of at least a threshold abundance. In one aspect, this document provides a limits testing approach to detecting Salmonella in a sample of beef or in a sampling device (e.g., a beef sampling device). In a limits testing approach, a qualitative assay can be used in a semi-quantitative manner to detect levels of the assay’s target bacterium (e.g., Salmonella ) by pulling aliquots of an enriched culture for testing at a time point that is earlier than the normal time point for testing. In some cases, a limits testing approach can be used to detect high levels of the target bacterium. A high level of a target bacterium can, in some cases, be defined as the concentration in the food at which the bacterium can produce a significant public health concern. In some embodiments, a high level of a bacterium can be measured in colony forming units (CFU)/g of sample. For example, a high level of a bacterium can be at least about 1 CFU/g (e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 14, 15, 16, 17, 19, 19, 20, 25, 30, 35, 40, 45, or 50 CFU/g). In some embodiments, a high level of

Salmonella can be at least about 10 CFU/g.

Without being bound by any particular theory, it is believed that every qualitative assay has a limit of detection, which corresponds to a particular concentration (e.g., CFU/mL) or abundance (e.g., CFU/g) of the target bacterium being present in a sample (e.g., a portion of a culture). During incubation of a culture (e.g., a culture formed by combination of a sample of beef and an enrichment media, or a culture formed by combination of a sampling device and an enrichment media), bacteria will multiply and eventually reach the limit of detection for the assay. If the starting concentration of the bacteria is higher, the bacteria will multiply and reach the limit of detection for the assay at a faster rate. If the starting concentration of the bacteria is lower, the bacteria will multiply and reach the limit of detection for the assay at a slower rate. Thus, with a limits testing approach, determining whether a sample of beef (or a sampling device such as a beef sampling device) had a starting threshold concentration (e.g., in CFU/mL) or starting threshold abundance (e.g., in CFU/g) of a bacterium can be achieved by knowing the conditions at which the limit of detection for the assay will be reached to produce a positive result for that particular starting threshold concentration or starting threshold abundance. In some embodiments, the conditions can include a time point during incubation (also called a target time). Thus, in some cases, once this time point is determined, an aliquot of the culture can be pulled for testing at that particular time point of incubation in order to only capture samples that meet or exceed the starting threshold concentration or the starting threshold abundance of the target bacterium. If an aliquot is taken before the predetermined target time, samples that meet or exceed the starting threshold starting concentration or starting threshold abundance may not be detected. If an aliquot is taken too late, samples that are below the target starting threshold concentration or the starting threshold abundance may be also detected.

Some non-limiting examples of conditions that could affect a limit of detection include sample preparation conditions (e.g., sample size, homogenization of the sample, enrichment media composition, and amount of enrichment media used), culture growth (e.g., incubation time, incubation temperature, and aeration or shaking of the culture), and assay conditions (e.g., type of assay and temperature of assay).

In some embodiments, one or more of the conditions at which the limit of detection for the assay will be reached can overlap with conditions from another testing protocol. Such an overlap, in some cases, can make testing more efficient. For example, at least one (e.g., at least 2, 3, 4, 5, 6 7, 8, 9, or 10) conditions can be the same as conditions from another testing protocol. As another example, one or more (e.g., 2, 3, 4,

5, or more, or all) of the sample preparation conditions can be the same as sample preparation conditions for another testing protocol. As another example, one or more (e.g., 2, 3, 4, 5, or more, or all) of the culture growth conditions can be the same as the culture growth conditions from another testing protocol. As yet another example, one or more (e.g., 2, 3, 4, 5, or more, or all) of the assay conditions can be the same as the assay conditions from another testing protocol. For example, in some embodiments, the conditions that could affect a limit of detection can overlap with the testing conditions for another bacterium (e.g., E. coli (e.g., 017:H7)).

A sample of beef can be any appropriate sample of beef. A sample of beef can be a beef cut (e.g., a beef cut of any muscle or quality), ground beef (e.g., ground beef of any fat percentage or quality), or a beef-containing food product. For example, a sample of beef can be a N60 Plus beef trim sample. In some cases, a sample of beef is refrigerated (e.g., between about 2 and 6 °C) before use. A sample of beef can be any appropriate size. For example, a sample of beef can be from about 50 g to about 500 g (e.g., from about 50 g to about 100 g, from about 50 g to about 150 g, from about 50 g to about 200 g, from about 50 g to about 250 g, from about 50 g to about 300 g, from about 50 g to about 400g, from about 50 g to about 500 g, from about 100 g to about 150g, from about 100 g to about 200 g, from about 100 g to about 250 g, from about 100 g to about 300 g, from about 100 g to about 400 g, from about 100 g to about 500 g, from about 150 g to about 200 g, from about 150 g to about 250 g, from about 150 g to about 300 g, from about 150 g to about 400 g, from about 150 g to about 500 g, from about 200 g to about 250 g, from about 200 g to about 300 g, from about 200 g to about 400 g, from about 200 g to about 500 g, from about 250 g to about 300 g, from about 250 g to about 400 g, from about 250 g to about 500 g, from about 300 g to about 400 g, from about 300 g to about 500g, or from about 400 g to about 500 g). In some examples, a sample of beef can be from about 150 g to about 250 g.

A sampling device can be any appropriate sampling device. For example, a beef sampling device that was used to sample beef or a surface that was used to prepare beef (e.g., a surface of a beef processing line) can be used as described herein. A sampling device can be in the form of a swab, sheet, or cloth. For example, a beef sampling device can be a swab that was used to wipe a sample of beef or that was used to wipe a surface that was used to prepare beef (e.g., a beef processing line). An example of a sampling device that can be used as described herein includes, without limitation, the MicroTally™ sampling device (manual sampling swabs and continuous sampling sheets provided by Fremonta Corp.).

Sample preparation methods can include any appropriate sample preparation methods. For example, a sample of beef can be cut, chopped, ground, or homogenized. In some cases, cutting, chopping, grinding, or homogenizing can occur at low temperature (e.g., at or below about 4°C, at or below about 0°C, or at or below about -78°C). In some examples, a sample of beef can be homogenized after being combined with an enrichment media. For example, homogenization can be carried out using a Bag Mixer (Intercience Laboratories, Inc., Woburn, MA). An enrichment media can include any appropriate components, and an enrichment media can be at any appropriate pH. In some cases, an enrichment media can include water and one or more of: sodium phosphate, disodium phosphate, potassium phosphate, dipotassium phosphate, sodium chloride, potassium chloride, magnesium chloride, magnesium sulfate, sodium acetate, hydrochloric acid, sodium hydroxide, potassium hydroxide, glucose, lactose, a tryptone, yeast extract, phytone, or sodium hydrogen selenite. In some cases, an enrichment media can include water, tryptone, phytone, sodium chloride, dipotassium phosphate, and glucose. In some examples, an enrichment media can be tryptic soy broth (TSB). In some cases, an enrichment media can be BAX® System MP media (e.g., part no. MED2003 from Hygenia). In some cases, an enrichment media can be at a pH of about 3.8 to about 9.5 (e.g., about 3.8 to about 4.5, about 4.0 to about 4.5, about 4.5 to about 5.0, about 5.0 to about 5.5, about 5.5 to about 6.0, about 6.0 to about 6.5, about 6.5 to about 7.0, about 7.0 to about 7.5, about 7.5 to about 8.0, about 8.0 to about 8.5, about 8.5 to about 9.0, or about 9.0 to about 9.5). Combining an enrichment media and a sample of beef (or a sampling device such as a beef sampling device) can form a culture. In some cases, an enrichment media can be combined with a sample of beef (or a sampling device such as a beef sampling device) at an elevated temperature (e.g., any appropriate temperature). In some examples, an enrichment media can be combined with a sample of beef (or a sampling device such as a beef sampling device) at about 45°C. An enrichment media can be combined with a sample of beef (or a sampling device such as a beef sampling device) in any appropriate amount or ratio. For example, an enrichment media can be combined with a sample of beef in a ratio of about 1 part by weight of the sample of beef to about 1 to about 10 parts (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts) by weight of the enrichment media. In some cases, an enrichment media can be combined with a sample of beef in a ratio of about 1 part by weight of the sample of beef to about 4 parts by weight of the enrichment media.

Culture growth conditions can include any appropriate culture growth conditions. For example, culture growth conditions can include any appropriate temperature. In some cases, the temperature of culture growth can be from about 35°C to about 45°C (e.g., from about 35°C to about 38°C, from about 38°C to about 41°C, from about 41°C to about 43°C, from about 43°C to about 45°C, or from about 42°C to about 45°C). In some cases, a culture can be grown between from about 41°C and about 43°C. For example, culture growth conditions can include any appropriate aeration process at any appropriate frequency. For example, a culture can shaken, rotated, or bubbled. Aeration can occur continuously (e.g., shaking at any appropriate rpm), regularly (e.g., every 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, or 12 hours), or irregularly. In some cases, a culture can be shaken every hour. In some cases, a culture can be shaken every hour, starting at the second hour. A culture can be grown for any appropriate period of time. For example, a culture can be grown for about 1 hour to about 5 days (e.g., from about 1 hour to about 2 hours, from about 1 hour to about 3 hours, from about 1 hour to about 4 hours, from about 1 hour to about 5 hours, from about 1 hour to about 6 hours, from about 1 hour to about 12 hours, from about 1 hour to about 1 day, from about 1 hour to about 3 days, from about 1 hour to about 5 days, from about 2 hours to about 3 hours, from about 2 hours to about 4 hours, from about 2 hours to about 5 hours, from about 2 hours to about 6 hours, from about 2 hours to about 12 hours, from about 2 hours to about 1 day, from about 2 hours to about 3 days, from about 2 hours to about 5 days, from about 3 hours to about 4 hours, from about 3 hours to about 5 hours, from about 3 hours to about 6 hours, from about 3 hours to about 12 hours, from about 3 hours to about 1 day, from about 3 hours to about 3 days, from about 3 hours to about 5 days, from about 4 hours to about 5 hours, from about 4 hours to about 6 hours, from about 4 hours to about 12 hours, from about 4 hours to about 1 day, from about 4 hours to about 3 days, from about 4 hours to about 5 days, from about 5 hours to about 6 hours, from about 5 hours to about 12 hours, from about 5 hours to about 1 day, from about 5 hours to about 3 days, from about 5 hours to about 5 days, from about 6 hours to about 12 hours, from about 6 hours to about 1 day, from about 6 hours to about 3 days, from about 6 hours to about 5 days, from about 12 hours to about 1 day, from about 12 hours to about 3 days, from about 12 hours to about 5 days, from about 1 day to about 3 days, from about 1 day to about 5 days, from about 3 days to about 5 days, for about 1 hour, for about 2 hours, for about 3 hours, for about 4 hours, for about 5 hours, for about 6 hours, for about 12 hours, for about 1 day, for about 3 days, or for about 5 days). In some cases, a culture can be grown for about 4 hours. A portion of a culture (e.g., an aliquot) used for an assay can be any appropriate volume. For example, portion of a culture used for an assay can be from about 1 pL to about 500 pL (e.g., from about 1 pL to about 5 pL, from about 1 pL to about 10 pL, from about 1 pL to about 50 pL, from about 1 pL to about 100 pL, from about 1 pL to about 250 pL, about 5 pL to about 10 pL, from about 5 pL to about 50 pL, from about 5 pL to about 100 pL, from about 5 pL to about 250 pL, from about 5 pL to about 500 pL, from about 10 pL to about 50 pL, from about 10 pL to about 100 pL, from about 10 pL to about 250 pL, from about 10 pL to about 500 pL, from about 50 pL to about 100 pL, from about 50 pL to about 250 pL, from about 50 pL to about 500 pL, from about 100 pL to about 250 pL, from about 100 pL to about 500 pL, from about 250 pL to about 500 pL, about 1 pL, about 5 pL, about 10 pL, about 50 pL, about 100 pL, about 250 pL, or about 500 pL). For example, a portion of a culture can be about 5 pL. In some cases, the volume of a portion of a culture can be dependent on the assay used.

Assay conditions can be any appropriate assay conditions. One assay condition can be the choice of assay. An assay can be any appropriate assay. For example, an assay can utilize antibodies (e.g., an enzyme-linked immunosorbent (ELISA) assay, or a blotting assay), flow cytometry, nucleic acid hybridization, nucleic acid amplification, or mass spectrometry. In some cases, an assay can use nucleic acid amplification. In some cases, an assay can be a polymerase chain reaction (PCR) assay. In some cases, an assay can be a real-time (RT) PCR assay. Primers can be designed for a PCR assay using any appropriate method. For example, primers can be designed to amplify nucleic acids that are unique to Salmonella. Any appropriate nucleic acids that are unique to Salmonella can be used. For example, primers can be designed to amplify one or more of: invA,prt (paratose synthase), tyv (tyvelose epimerase), Sdf-1 {Salmonella- differentiating fragment), stn {Salmonella enterotoxin gene), Fur-regulated gene, a histidine transport operon, the junction between SipB and SipC, 16S rDNA, a Salmonella pathogenicity island I virulence gene, or sdiA (a signal receptor of the LuxR family). In some cases, an assay can be the BAX^® System Real-Time PCR Assay for Salmonella (Hygenia, product code KIT2006). In some cases, an assay can be the BAX^® System Standard PCR Assays for Salmonella (Hygenia, product code KIT2012). In some cases, an assay can be the BAX® System Standard PCR Assays for Salmonella 2 (Hygenia, product code

KIT2011). The limit of detection for any of these assays can be determined by any appropriate method. Other assay conditions, such as assay temperature, can be dependent on the choice of assay, and can be any appropriate conditions.

A starting threshold concentration or a starting threshold abundance of

Salmonella can be any appropriate starting threshold concentration or starting threshold abundance of Salmonella. For example, a starting threshold concentration of Salmonella can be at least about 1 CFU/mL (e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 14, 15, 16, 17, 19, 19, 20, 25, 30, 35, 40, 45, or 50 CFU/mL). For example, a starting threshold abundance of Salmonella can be at least about 1 CFU/g (e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 14, 15, 16, 17, 19, 19, 20, 25, 30, 35, 40, 45, or 50 CFU/g). In some examples, a starting threshold abundance of Salmonella can be at least about 10 CFU/g.

To determine the conditions at which the limit of detection for the assay will be reached, any appropriate methodology can be used. For example, a starting threshold concentration or a starting threshold abundance can be determined, and one or more of: the sample preparation conditions, the culture growth conditions, and the assay conditions (e.g., the sample preparation conditions alone, the culture growth conditions alone, the assay conditions alone, the sample preparation conditions and the culture growth conditions, the sample preparation and the assay conditions, or the culture growth conditions and the assay conditions), can be fixed, and the other conditions can be adjusted. In some cases, a starting threshold concentration or a starting threshold abundance can be determined. In some cases, a starting threshold concentration or a starting threshold abundance can be determined, and the sample preparation conditions and the assay conditions can be fixed. In such a case, the culture growth conditions can be adjusted to determine the conditions at which the limit of detection for the assay will be reached. In some such cases, all of the culture growth conditions except for culture time can be fixed, and the culture time can be adjusted to determine the conditions at which the limit of detection for the assay will be reached. The culture time at which the limit of detection for the assay will be reached when other conditions are fixed can be determined using any appropriate method. For example, a number of samples of beef (or sampling devices such as beef sampling devices) can each be inoculated with a starting threshold abundance of Salmonella , and aliquots of the culture (e.g., produced by any appropriate sample preparation methods and culture growth conditions, such as those described herein) can be pulled at regular intervals. For example, a regular interval can be every 10 minutes, every 30 minutes, every hour, every 2 hours, every 3 hours, every 4 hours, or every 6 hours. In some cases, the regular interval is every hour. At each interval, the aliquot can be assayed (e.g., using any appropriate assay conditions, such as those described herein) to determine if the limit of detection is reached. A culture time at which some (e.g., 50%, 60%, 70%, 80%, 90%, or 100%) of the number of samples reach the limit of detection can be the culture time at which the limit of detection for the assay is reached. In some cases, a culture time in which all of the samples reach the limit of detection can be the culture time at which the limit of detection for the assay is reached. In some cases, the same conditions (e.g., including the culture time, as determined above) can be used to determine if a sample of beef (or a sampling device such as a beef sampling device) has a starting concentration or starting abundance of Salmonella that is at least the starting threshold concentration or the starting threshold abundance of Salmonella.

EXAMPLES

Example 1

Preparation of Salmonella Inoculum

The following Salmonella strain was used for this study: Salmonella enterica subsp. enterica serotype Typhimurium ATCC 14028.

An individual culture of the Salmonella strain was prepared by streaking a loopful of culture from the -80°C freezer stock of that strain onto Tryptic Soy Agar (TSA;

Becton, Dickinson, and Company, Franklin Lakes, NJ) and incubating for 21 ± 3 hours at 35 ± 2°C. One isolated colony from the TSA plate was then transferred into a 10 mL tube of Tryptic Soy Broth (TSB; Becton, Dickinson, and Company), which was then incubated at 35 ± 2°C for > 12 hours to achieve stationary phase growth.

The > 12-hour TSB culture was then serially diluted in 9.0 mL blanks of

Butterfield’s Phosphate Buffer (BPB; made in-house) and spread-plated to triplicate TSA plates to determine the“PRE” concentration of Salmonella (CFU/mL) in the TSB culture. The TSB culture was then placed into refrigerated retain (4 ± 2°C) to halt growth, and TSA plates were then incubated 35 ± 2°C for > 12 hours before being counted. The > 12 hour time point was the minimum incubation time point at which Salmonella colonies were visible on the TSA plates. This time point was chosen for enumeration in order to reduce the amount of time that the TSB culture had to remain in refrigerated retain (4 ± 2°C) before being used. The“PRE” concentration (CFU/mL) of Salmonella in the TSB culture was then calculated from the TSA plate counts and their serial dilutions, and is reported in Table 1.

On the day of the experiment described in Examples 2 and 3, the TSB culture was removed from refrigerated retain (4 ± 2°C) and was again serially diluted in 9.0 mL blanks of BPB. Based on the“PRE” concentration (CFU/mL) of Salmonella in the TSB culture that was determined on > 12 hour TSA plates as described above, the volume of the 10 ⁵ dilution of the TSB culture that needed to be added to each N60 Plus beef trim sample was calculated and added to each sample as described below in Example 2. These serial dilutions were also spread-plated to triplicate TSA plates to determine the“POST” concentration of Salmonella (CFU/mL) in the TSB culture. This“POST” concentration of Salmonella (CFU/mL) in the TSB culture was used to verify that the amount of cells added to each sample on the day of the experiment described in Examples 2 and 3 (which was based on the“PRE” concentration) was accurately estimated based on the“PRE” concentration. This“POST” concentration of Salmonella (CFU/ml) is also reported in Table 1. Table 1 :“PRE” and“POST” Concentrations of the Salmonella TSB Culture

Example 2

Preparation and Inoculation of N60 Plus Beef Trim Samples

N60 Plus beef trim samples were obtained and kept under refrigerated storage (4 ± 2°C) until being used for experimentation.

Each of 30 N60 Plus beef trim samples were weighed. The weight of each sample is provided in Table 2. These weights were then used along with the Salmonella“PRE” concentration in Table 1 to determine the volume of the 10 ⁵ dilution of the Salmonella TSB culture to add to each sample in order to achieve a target inoculation rate of 10 CFU/g; the volume added to each sample is also provided in Table 2. The final CFU/g calculated for each sample based on the“POST” concentration from Table 1, the volume of the 10 ⁵ dilution of the Salmonella TSB culture to add to each sample, and the weight of the sample is also provided in Table 2. Likewise, because the enrichment media was be added to each sample at a rate of 4X the weight of the sample, the volume of enrichment media to add was also calculated, and is provided in Table 2.

Table 2: N60 Plus Beef Trim Sample Weights, Volumes of Inoculum Added, Volumes of Enrichment Added, and Final CFU/g Concentrations of Salmonella

Once all of these parameters were calculated, the aliquot of the 10 ⁵ dilution of the

Salmonella TSB culture that needed to be added to each sample was added. The samples were then enriched, sampled, and tested as described in Example 3. Example 3

Enrichment, Sampling, and Testing of N60 Plus Beef Trim Samples

Once each N60 Plus beef trim sample was inoculated with the appropriate volume of the 10 ⁵ dilution of the Salmonella TSB culture, as described in Example 2, it was enriched with the volume of pre-warmed (45°C) BAX® System MP Media (BAX MP; Hygiena, LLC) that was reported in Table 2. The samples were then homogenized for 2 min in a Bag Mixer (Interscience Laboratories, Inc., Woburn, MA), or equivalent. The samples were then placed into 42 ± 1°C incubation.

At 2 hours (h), 3 h, 4 h, 5 h, and 6 h of incubation, the sample bags were shaken and 5 pL aliquots were removed from the enrichment of each sample. These aliquots were then placed into 200 pL aliquots of previously-prepared lysis buffer from the BAX® System Real-Time PCR Assay for Salmonella (SAL BAX RT). Samples were not removed from 42 ± 1°C incubation during removal of the aliquots. A walk-in incubator was used for incubation, and aliquots were removed from samples inside of the walk-in incubator. The rationale behind this was not to allow for a temperature drop at each time point, which would affect the rate of incubation. Aliquots were also collected as expeditiously as possible.

After each 5 pL aliquot was collected and placed into a 200 pL aliquot of previously-prepared BAX SAL RT Assay lysis buffer, the tube was capped and was placed onto a 37°C heating block for 20 minutes. After this, the tube was transferred to a 95°C heating block for 10 minutes. This was followed by chilling the samples in refrigerated storage (4 ± 2°C) overnight.

Once lysates were created for all samples and the lysates were ready to be run on the SAL BAX RT Assay, a 30 pL aliquot was removed from each lysis tube and was placed into a SAL BAX RT Assay PCR tube that was situated in a cooling block (2-8°C). The tubes were PCR tubes were then capped with flat optical caps, and were allowed to sit undisturbed for 10 minutes in order to allow for full hydration of the PCR pellet. The tubes were then loaded into BAX® System Q7 (Hygiena, LLC) instrument and the SAL BAX RT program was run in order to obtain SAL BAX RT Assay PCR results. The detailed SAL BAX RT Assay PCR results for each of the N60 Plus samples at the 2 h, 3 h, 4 h, 5 h, and 6 h time points are provided below in Table 3 (“=” is a negative result;“+” is a positive result). A summary of these results is provided in Table

4.

Table 3: Detailed SAL BAX RT Assay PCR Results for N60 Plus Beef Trim Samples at Each Time Point of Incubation

Table 4

Summarized SAL BAX RT Assay PCR Results for N60 Plus Beef Trim Samples at

Each Time Point of Incubation

As noted above in Table 2, a starting concentration of 10 CFU/g of Salmonella was successfully obtained for each N60 Plus beef trim sample.

Based on the PCR results from the SAL BAX RT Assay at each of the incubation time points (Table 3 and Table 4), the 4 h time point was the first time point at which the N60 Plus beef trim samples contaminated with > 10 CFU/g of Salmonella were 100% positive for Salmonella. Prior to this, at the 3 h time point, only 23.3% of the samples contaminated with 10 CFU/g were positive. Thus, 3 h was deemed an unacceptable incubation time point to pull 5 pL aliquots for testing since a majority of the samples with 10 CFU/g Salmonella were identified as negative for Salmonella. Beyond the 4 h time point, the 5 h and 6 h time points also produced 100% positive Salmonella results. However, since the targeted starting concentration was > 10 CFU/g, the 5 h and 6 h time points also were deemed unacceptable time points to pull 5 pL aliquots for testing since samples that contained < 10 CFU/g Salmonella could theoretically also produce positive results at these time points.

OTHER EMBODIMENTS

It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of determining whether Salmonella is present in a starting abundance of at least about 10 CFU/g in a sample of beef comprising:

(a) combining a sample of beef with an enrichment media to form a culture; (b) incubating the culture for about 4 hours at about 41 to 43 °C;

(c) performing an assay on a portion of the culture, wherein the assay detects the presence or absence of Salmonella ; and

(d) determining (i) that Salmonella was present in a starting abundance of at least about 10 CFU/g in the sample of beef if the assay detects the presence of Salmonella or (ii) that Salmonella was not present in a starting abundance of at least about 10 CFU/g in the sample of beef if the assay does not detect the presence of Salmonella.

2. The method of claim 1, wherein the enrichment media comprises sodium phosphate.

3. The method of any one of claims 1-2, wherein the assay is a nucleic acid amplification assay.

4. The method of claim 3, wherein the assay is a real-time (RT) PCR assay.

5. The method of any one of claims 1-4, wherein the method further comprises homogenizing the sample of beef or the culture.

6. The method of any one of claims 1-5, wherein the sample of beef is an N60 Plus beef trim sample.

7. A method of determining whether Salmonella is present in a starting

concentration of at least about a starting threshold abundance in a sample of beef comprising:

(a) choosing an assay that detects the presence or absence of Salmonella ;

(b) determining a set of sample preparation conditions, a set of growth conditions, and a set of assay conditions that result in the detection of Salmonella in a starting concentration of at least about a starting threshold abundance using the assay in (a);

(c) preparing a sample of beef using the sample preparation conditions determined in step (b) to form a culture;

(d) incubating the culture using the growth conditions determined in step

(b);

(e) performing the assay on a portion of the culture using the assay conditions in step (b); and

(f) determining (i) that Salmonella was present in at least about the starting threshold abundance in the sample of beef if the assay detects the presence of Salmonella or (ii) that Salmonella was not present in at least about the starting threshold abundance in a sample of beef if the assay does not detect the presence of Salmonella.

8. The method of claim 7, wherein the sample preparation conditions include combining a sample of beef with an enrichment media to form a culture.

9. The method of claim 8, wherein the enrichment media comprises sodium phosphate.

10. The method of any one of claims 7-9, wherein the assay is a nucleic acid amplification assay.

11. The method of claim 10, wherein the assay is a real-time (RT) PCR assay.

12. The method of any one of claims 7-11, wherein the sample preparation conditions include homogenizing the sample of beef or the culture.

13. The method of any one of claims 7-12, wherein the sample of beef is an N60 Plus beef trim sample.

14. The method of any one of claims 7-13, wherein the starting threshold abundance is at least about 10 CFU/g.

15. A method of determining whether Salmonella is present in a starting abundance of at least about 10 CFU/g in a sampling device comprising:

(a) combining a sampling device with an enrichment media to form a culture; wherein said sampling device is a sampling device that was contacted with material to be tested;

(b) incubating the culture for about 4 hours at about 41 to 43 °C;

(d) determining (i) that Salmonella was present in a starting abundance of at least about 10 CFU/g in the sampling device if the assay detects the presence of Salmonella or (ii) that Salmonella was not present in a starting abundance of at least about 10 CFU/g in the sampling device if the assay does not detect the presence of Salmonella.

16. The method of claim 15, wherein the enrichment media comprises sodium phosphate.

17. The method of any one of claims 15-16, wherein the assay is a nucleic acid amplification assay.

18. The method of claim 17, wherein the assay is a real-time (RT) PCR assay.

19. The method of any one of claims 15-18, wherein the sampling device is a swab, sheet, or cloth.

20. The method of any one of claims 15-19, wherein the material to be tested was beef or a preparation surface used to prepare beef.

21. A method of determining whether Salmonella is present in a starting

concentration of at least about a starting threshold abundance in a sampling device comprising:

(a) choosing an assay that detects the presence or absence of Salmonella ;

(c) obtaining a sampling device that was contacted with material to be tested;

(d) using the sampling device and the sample preparation conditions determined in step (b) to form a culture;

(e) incubating the culture using the growth conditions determined in step

(b);

(f) performing the assay on a portion of the culture using the assay conditions in step (b); and

(g) determining (i) that Salmonella was present in at least about the starting threshold abundance in the sampling device if the assay detects the presence of Salmonella or (ii) that Salmonella was not present in at least about the starting threshold abundance in the sampling device if the assay does not detect the presence of Salmonella.

22. The method of claim 21, wherein the sample preparation conditions include combining a sample of beef with an enrichment media to form a culture.

23. The method of claim 22, wherein the enrichment media comprises sodium phosphate.

24. The method of any one of claims 21-23, wherein the assay is a nucleic acid amplification assay.

25. The method of claim 24, wherein the assay is a real-time (RT) PCR assay.

26. The method of any one of claims 21-25, wherein the sampling device is a swab, sheet, or cloth.

27. The method of any one of claims 21-26, wherein the material to be tested was beef or a preparation surface used to prepare beef.

28. The method of any one of claims 21-27, wherein the starting threshold abundance is at least about 10 CFU/g.