WO2020106897A1 - Procédés et matériaux permettant de détecter la salmonella dans le bœuf - Google Patents

Procédés et matériaux permettant de détecter la salmonella dans le bœuf

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Publication number
WO2020106897A1
WO2020106897A1 PCT/US2019/062493 US2019062493W WO2020106897A1 WO 2020106897 A1 WO2020106897 A1 WO 2020106897A1 US 2019062493 W US2019062493 W US 2019062493W WO 2020106897 A1 WO2020106897 A1 WO 2020106897A1
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WO
WIPO (PCT)
Prior art keywords
assay
salmonella
beef
sample
culture
Prior art date
Application number
PCT/US2019/062493
Other languages
English (en)
Inventor
Alex Brandt
Randal GARRETT
Aaron PLEITNER
Wayne Muraoka MURAOKA
Katlyn HOLZER
Original Assignee
Alex Brandt
Garrett Randal
Pleitner Aaron
Muraoka Wayne Muraoka
Holzer Katlyn
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Alex Brandt, Garrett Randal, Pleitner Aaron, Muraoka Wayne Muraoka, Holzer Katlyn filed Critical Alex Brandt
Publication of WO2020106897A1 publication Critical patent/WO2020106897A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/10—Enterobacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/22—Testing for sterility conditions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/255—Salmonella (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food
    • G01N33/12—Meat; Fish

Definitions

  • This document relates to materials and methods for detecting Salmonella in food, and more particularly to materials and methods for detecting Salmonella in a
  • 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.
  • This document is based, at least in part, on methods and material for detecting Salmonella in beef.
  • 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.
  • enrichment media can include sodium phosphate.
  • the assay can be a nucleic acid amplification assay.
  • the assay can be a real-time (RT) PCR assay.
  • the method can further include homogenizing the sample of beef, the culture, or both.
  • the sample of beef can be a N60 Plus beef trim sample.
  • 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.
  • the sample preparation conditions can include combining a sample of beef with an enrichment media to form a culture.
  • the enrichment media can include sodium phosphate.
  • the assay can be a nucleic acid amplification assay.
  • the assay can be a real-time (RT) PCR assay.
  • the sample preparation conditions can include homogenizing the sample of beef, the culture, or both.
  • the sample of beef can be an N60 Plus beef trim sample.
  • the starting threshold abundance can be at least about 10 CFU/g.
  • 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 C
  • enrichment media can include sodium phosphate.
  • the assay can be a nucleic acid amplification assay.
  • the assay can be a real-time (RT) PCR assay.
  • the sampling device can be a beef sampling device in the form of a swab, sheet, or cloth for contacting and testing beef trim.
  • 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.
  • the sample preparation conditions can include combining the sampling device (e.g., beef sampling device) with an enrichment media to form a culture.
  • the enrichment media can include sodium phosphate.
  • the assay can be a nucleic acid amplification assay.
  • the assay can be a real-time (RT) PCR assay.
  • the sampling device can be a beef sampling device in the form of a swab, sheet, or cloth for contacting and testing beef trim.
  • the starting threshold abundance can be at least about 10 CFU/g.
  • 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).
  • 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).
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • a high level of a bacterium can be measured in colony forming units (CFU)/g of sample.
  • 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.
  • 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).
  • a concentration e.g., CFU/mL
  • abundance e.g., CFU/g
  • 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.
  • 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.
  • the conditions can include a time point during incubation (also called a target time).
  • 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.
  • 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).
  • 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
  • assay conditions e.g., type of assay and temperature of assay.
  • 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,
  • sample preparation conditions can be the same as sample preparation conditions for another testing protocol.
  • 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.
  • 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.
  • 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.
  • a sample of beef can be a N60 Plus beef trim sample.
  • 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.
  • 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
  • a sampling device can be any appropriate sampling device.
  • 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.
  • 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 MicroTallyTM sampling device (manual sampling swabs and continuous sampling sheets provided by Fremonta Corp.).
  • Sample preparation methods can include any appropriate sample preparation methods.
  • a sample of beef can be cut, chopped, ground, or homogenized.
  • 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).
  • a sample of beef can be homogenized after being combined with an enrichment media.
  • 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.
  • 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.
  • an enrichment media can include water, tryptone, phytone, sodium chloride, dipotassium phosphate, and glucose.
  • an enrichment media can be tryptic soy broth (TSB).
  • an enrichment media can be BAX® System MP media (e.g., part no.
  • 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.
  • 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.
  • culture growth conditions can include any appropriate temperature.
  • 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).
  • a culture can be grown between from about 41°C and about 43°C.
  • 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.
  • a culture can be shaken every hour.
  • a culture can be shaken every hour, starting at the second hour.
  • a culture can be grown for any appropriate period of time.
  • 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 5 hours,
  • 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.
  • 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,
  • Assay conditions can be any appropriate assay conditions.
  • One assay condition can be the choice of assay.
  • An assay can be any appropriate assay.
  • 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.
  • an assay can use nucleic acid amplification.
  • an assay can be a polymerase chain reaction (PCR) assay.
  • PCR polymerase chain reaction
  • RT real-time PCR assay. Primers can be designed for a PCR assay using any appropriate method.
  • 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.
  • 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).
  • 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 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.
  • Salmonella can be any appropriate starting threshold concentration or starting threshold abundance of Salmonella.
  • 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).
  • 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).
  • a starting threshold abundance of Salmonella can be at least about 10 CFU/g.
  • 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.
  • a starting threshold concentration or a starting threshold abundance can be determined.
  • a starting threshold concentration or a starting threshold abundance can be determined, and the sample preparation conditions and the assay conditions can be fixed.
  • the culture growth conditions can be adjusted to determine the conditions at which the limit of detection for the assay will be reached.
  • 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.
  • 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.
  • 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.
  • 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.
  • the same conditions 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.
  • 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;
  • TSA Tryptic Soy Broth
  • 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
  • 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.
  • Example 3 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
  • 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.
  • BAX® System MP Media BAX MP; Hygiena, LLC
  • 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.
  • 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.

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Abstract

L'invention concerne des matériaux et des procédés permettant de détecter la Salmonella dans le bœuf.
PCT/US2019/062493 2018-11-20 2019-11-20 Procédés et matériaux permettant de détecter la salmonella dans le bœuf WO2020106897A1 (fr)

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US11319595B2 (en) 2014-04-21 2022-05-03 Natera, Inc. Detecting mutations and ploidy in chromosomal segments

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WO1998020148A1 (fr) * 1996-11-04 1998-05-14 The Regents Of The University Of California Procede de detection de pathogenes dans des aliments
WO2014114688A1 (fr) * 2013-01-22 2014-07-31 Imicroq, S.L. Procédé rapide de détection de pathogène
US20180258467A1 (en) * 2015-04-07 2018-09-13 Polyskope Labs Detection of one or more pathogens

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WO1998020148A1 (fr) * 1996-11-04 1998-05-14 The Regents Of The University Of California Procede de detection de pathogenes dans des aliments
WO2014114688A1 (fr) * 2013-01-22 2014-07-31 Imicroq, S.L. Procédé rapide de détection de pathogène
US20180258467A1 (en) * 2015-04-07 2018-09-13 Polyskope Labs Detection of one or more pathogens

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CORNELIA MEYER ET AL: "Salmonella in Raw Meat and By-Products from Pork and Beef", JOURNAL OF FOOD PROTECTION, vol. 73, no. 10, October 2010 (2010-10-01), pages 1780 - 1784, XP055710813, ISSN: 0362-028X, DOI: 10.4315/0362-028X-73.10.1780 *
PAULA SZYMCZAK ET AL: "Detection of Salmonella enterica in Meat in Less than 5 Hours by a Low-Cost and Noncomplex Sample Preparation Method", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 83, no. 5, 1 March 2016 (2016-03-01), pages 1 - 11, XP055579916, ISSN: 0099-2240, DOI: 10.1128/AEM.02748-16 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11319595B2 (en) 2014-04-21 2022-05-03 Natera, Inc. Detecting mutations and ploidy in chromosomal segments

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