Enrichment methods for the detection of pathogens and other microbes

Abstract

The present invention provides novel enrichment, testing and detection methods for detection of pathogens or other microbes in a food, water, wastewater, industrial, pharmaceutical, botanical, environmental samples and other types of samples analyzed by enrichment-detection methods. In preferred aspects, a sample is obtained at a first location and is diluted (e.g., in the case of a solid or semi-solid sample or liquid) at the first location at a ratio of about 1:0 (wt./vol.) to 1:2 (wt./vol.), or greater, preferably at a ratio of about 1:0.1 (wt./vol.) or greater, or more preferably, at a ratio of about 1:2 (wt./vol.) or greater. The diluted sample is incubated at an optimal temperature in an incubator and either tested locally, or sent in a shipping incubator to a second location that is a remote test location. The incubated sample is received and tested at the second location by assaying the sample, or a portion thereof, with an assay suitable to detect the pathogen or other microbe. In alternate embodiments, no dilution at the first location is required, and optionally minimal additions to adjust intrinsic deficiencies may be made, but the sample is nonetheless incubated during transit to the test location.

Claims

1. A method for detection of a particular bacterial pathogen or bacterial microbe, comprising: obtaining a test sample, the sample being solid or semi-solid; diluting the sample with liquid enrichment medium at a ratio of sample to diluent between about 1:0.1 to about 1:5 (wt./vol.) or lesser dilution; incubating, in an incubator, the diluted sample at an optimal temperature for the particular bacterial pathogen or bacterial microbe to be detected for a time period sufficient to allow levels of the particular bacterial pathogen or microbe to reach levels detectable by use of an assay suitable to detect the particular bacterial pathogen or bacterial microbe; and determining, by assaying the diluted incubated test sample, or a portion thereof, with the assay suitable to detect the particular bacterial pathogen or bacterial microbe, whether the sample is contaminated with the particular bacterial pathogen or bacterial microbe.

2. The method of claim 1, wherein diluting is at a ratio of sample to diluent between about 1:0.1 to about 1:0.5 (wt./vol.) or lesser dilution.

3. The method of claim 1, wherein diluting is at a ratio of sample to diluent between about 1:0.1 to about 1:0.3 (wt./vol.) or lesser dilution.

4. The method of claim 1, wherein the optimal temperature is within the optimal growth temperature range of the particular bacterial pathogen or bacterial microbe in the liquid enrichment medium.

5. The method of claim 4, wherein the optimal temperature range is from about 25° C. to about 45° C.

6. The method of claim 1, wherein the assay suitable for detection of the particular bacterial pathogen or bacterial microbe contamination is selected from the assay group consisting of immunoassays, nucleic acid amplification-based assays, PCR-based assays, nucleic acid hybridization-based assays, bio-sensor assays, immunostaining-microscopy-based assays, nucleic acid-array-based assays, DNA chip-based assays, classical microbiology-based assays, and chemical or biochemical assays based on the detection of compounds associated with the particular bacterial pathogen or bacterial microbe, and combinations thereof.

7. The method of claim 1, wherein the particular bacterial pathogen or bacterial microbe is selected from the group consisting of Escherichia coli O157:H7 (E. coli O157:H7), enterohemorrhagic Escherichia coli (EHEC), enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC), enterpathogenic Escherichia coli (EPEC), Salmonella, Listeria, Yersinis, Campylobacter, Clostridial species, Staphylococcus spp.; frank and opportunistic bacteria, indicator organisms including heterotrophes, generic E. Coli, total and fecal coliforms and enterococcus; spoilage organisms including Pseudomonas, and combinations thereof.

8. The method of claim 7, wherein the particular bacterial pathogen or bacterial microbe is Escherichia coli O157:H7 (E. coli O157:H7, or H negative), Salmonella, Listeria, EHEC, and Campylobacter.

9. The method of claim 1, wherein obtaining a sample is obtaining a standard Lot-unit sample.

10. The method of claim 1, wherein the sample is a composite-Lot sample, corresponding to a combination of samples selected from the group consisting of samples or subsamples taken from sublots/lots of raw or processed samples, environmental samples, industrial samples, pharmaceutical samples, bio-solid samples, samples taken by spore traps, settled dust, impingers, or filtration, and combinations thereof.

11. A method for detection of a particular bacterial pathogen or bacterial microbe, comprising: obtaining a liquid sample; diluting the sample with liquid enrichment medium at a ratio of sample to diluent between about 1:0.1 to about 1:5 (vol./vol.) or lesser dilution, or not diluting the sample; incubating, in an incubator, the diluted or the not diluted sample at an optimal temperature for the particular bacterial pathogen or bacterial microbe to be detected for a time period sufficient to allow levels of the particular bacterial pathogen or bacterial microbe to reach levels detectable by use of an assay suitable to detect the particular bacterial pathogen or bacterial microbe; and determining, by assaying the diluted or the not diluted incubated test sample, or a portion thereof, with the assay suitable to detect the particular bacterial pathogen or bacterial microbe, whether the sample is contaminated with the particular bacterial pathogen or bacterial microbe.

12. The method of claim 11, wherein the optimal temperature is within the optimal growth temperature range of the particular bacterial pathogen or bacterial microbe in the liquid enrichment medium.

13. The method of claim 12, wherein the optimal temperature range is from about 25° C. to about 45° C.

14. The method of claim 11, wherein the assay suitable for detection of the particular bacterial pathogen or bacterial microbe contamination is selected from the assay group consisting of immunoassays, nucleic acid amplification-based assays, PCR-based assays, nucleic acid hybridization-based assays, bio-sensor assays, immunostaining-microscopy-based assays, nucleic acid-array-based assays, DNA chip-based assays, classical microbiology-based assays, and chemical or biochemical assays based on the detection of compounds associated with the particular bacterial pathogen or bacterial microbe, and combinations thereof.

15. The method of claim 11, wherein the particular bacterial microbe or bacterial pathogen is selected from the group consisting of Escherichia coli O157:H7 (E. coli O157:H7), enterohemorrhagic Escherichia coli (EHEC), enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC), enterpathogenic Escherichia coli (EPEC), Salmonella, Listeria, Yersinis, Campylobacter, Clostridial species, Staphylococcus spp.; frank and opportunistic bacteria, indicator organisms including heterotrophes, generic E. Coli, total and fecal coliforms and enterococcus; spoilage organisms including Pseudomonas; and combinations thereof.

16. The method of claim 15, wherein the particular bacterial pathogen or bacterial microbe is Escherichia coli O157:H7 (E. coli O157: or H negative), Salmonella, Listeria, EHEC, and Campylobacter.

17. The method of claim 1, where in the sample is selected from the group consisting of beef, pork, sheep, bison, deer, elk, poultry, fish, produce, dairy products, dry goods, raw and processed foods, environmental samples, soil, sludge, surface samples, samples taken by impingers and filtration, pharmaceuticals, and samples analyzed using enrichment-detection protocols.

18. The method of claim 11, wherein the liquid sample is selected from the group consisting of fruit juice, vegetable juice, milk and dairy products, raw and processed liquid foods, environmental samples, water, wastewater, samples taken by impingers and filtration, botanical liquid, industrial liquids, pharmaceutical liquids, and other liquid samples analyzed using enrichment-detection protocols.

19. The method of claim 1, wherein the diluting comprises addition to the sample of a supplement selected from the group consisting of water, sugars, proteins, minerals, organics, vitamins and cofactors, antibiotics, dyes, indicators, buffers, agents to adjust the pH, water activity, nutritional contents, selective pressure to optimize the growth conditions for the particular bacterial pathogen or bacterial microbe, and combinations thereof.

20. The method of claim 11, wherein the diluting comprises addition to the sample of a supplement selected from the group consisting of water, sugars, proteins, minerals, organics, vitamins and cofactors, antibiotics, dyes, indicators, buffers, agents to adjust the pH, water activity, nutritional contents, selective pressure to optimize the growth conditions for the particular bacterial pathogen or bacterial microbe, and combinations thereof.

21. The method of claim 1, wherein the obtaining and diluting of the solid or semi-solid sample with liquid enrichment medium is at a first location, wherein incubating the diluted sample at an optimal temperature in the incubator is during transit of the diluted sample to a second location that is not the site of sample collection and dilution, and wherein determining by assaying the diluted incubated test sample, or a portion thereof, with the assay suitable to detect the particular bacterial pathogen or bacterial microbe, whether the sample is contaminated with the particular bacterial pathogen or bacterial microbe, is at the second location.

22. The method of claim 21, further comprising addition to the sample, prior to or during incubation, of a supplement selected from the group consisting of water, sugars, proteins, minerals, organics, vitamins and cofactors, antibiotics, dyes, indicators, buffers, agents to adjust the pH, water activity, nutritional contents, selective pressure to optimize the growth conditions for the particular bacterial pathogen or bacterial microbe, and combinations thereof.

23. The method of claim 21, wherein the second location is an in-house or local lab, or a remote location.

24. The method of claim 11, wherein the obtaining and diluting of the liquid sample with liquid enrichment medium is at a first location, wherein incubating the diluted sample at an optimal temperature in the incubator is during transit of the diluted sample to a second location that is not the site of sample collection and dilution, and wherein determining by assaying the diluted incubated test sample, or a portion thereof, with the assay suitable to detect the particular bacterial pathogen or bacterial microbe, whether the sample is contaminated with the particular bacterial pathogen or bacterial microbe, is at the second location.

25. The method of claim 24, further comprising addition to the sample, prior to or during incubation, of a supplement selected from the group consisting of water, sugars, proteins, minerals, organics, vitamins and cofactors, antibiotics, dyes, indicators, buffers, agents to adjust the pH, water activity, nutritional contents, selective pressure to optimize the growth conditions for the particular bacterial pathogen or bacterial microbe, and combinations thereof.

26. The method of claim 24, wherein the second location is an in-house or local lab, or a remote location.

27. The method of claim 1, wherein diluting is at a ratio of sample to diluent between about 1:0.1 to about 1:2 (wt./vol.) or lesser dilution.

28. The method of claim 11, wherein diluting is at a ratio of sample to diluent between about 1:0.1 to about 1:2 (vol./vol.) or lesser dilution.

29. The method of claim 11, wherein diluting is at a ratio of sample to diluent between about 1:0.1 to about 1:0.5 (vol./vol.) or lesser dilution.

30. The method of claim 11, wherein diluting is at a ratio of sample to diluent between about 1:0.1 to about 1:0.3 (vol./vol.) or lesser dilution.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1 and 2 show amplification products obtained by multiplex PCR of E. coli O157:H7 inoculated on meat trimmings incubated with a standard method (FIG. 1), or an embodiment of the inventive ‘dry’ method (FIG. 2) for 6 h, and concentrated by Dynabeads®. Numbers 1, 2, 3 were inoculation levels at 2.4×10.sup.1, 0.36×10.sup.1, and 0.036×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(2) FIGS. 3 and 4 show amplification products obtained by multiplex PCR of E. coli O157:H7 inoculated on meat trimmings incubated with a standard method (FIG. 3), or an embodiment of the inventive ‘dry’ method (FIG. 4), for 7 h, and concentrated by Dynabeads®. Numbers 1, 2, 3 were inoculation levels at 2.4×10.sup.1, 0.36×10.sup.1, and 0.036×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(3) FIGS. 5 and 6 show amplification products obtained by multiplex PCR of E. coli O157:H7 inoculated on meat trimmings incubated with a standard method (FIG. 5), or an embodiment of the inventive ‘dry’ method (FIG. 6), for 8 h, and concentrated by Dynabeads®. Numbers 1, 2, 3 were inoculation levels at 2.4×10.sup.1, 0.36×10.sup.1, and 0.036×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(4) FIGS. 7 and 8 show amplification products obtained by multiplex PCR of E. coli O157:H7 inoculated on meat trimmings incubated with a standard method (FIG. 7), or an embodiment of the inventive ‘dry’ method (FIG. 8), for 6 h. Numbers 1, 2, 3 were inoculation levels at 2.4×10.sup.1, 0.36×10.sup.1, and 0.036×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(5) FIGS. 9 and 10 show amplification products obtained by multiplex PCR of E. coli O157:H7 inoculated on meat trimmings incubated with a standard method (FIG. 9), or an embodiment of the inventive ‘dry’ method (FIG. 10), for 7 h. Numbers 1, 2, 3 were inoculation levels at 2.4×10.sup.1, 0.36×10.sup.1, and 0.036×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(6) FIGS. 11 and 12 show amplification products obtained by multiplex PCR of E. coli O157:H7 inoculated on meat trimmings incubated with a standard method (FIG. 11), or an embodiment of the inventive ‘dry’ method (FIG. 12), for 8 h. Numbers 1, 2, 3 were inoculation levels at 2.4×10.sup.1, 0.36×10.sup.1, and 0.036×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(7) FIGS. 13 and 14 show amplification products obtained by multiplex PCR of E. coli O157:H7 inoculated on meat trimmings incubated with a standard method (FIG. 13), or an embodiment of the inventive ‘dry’ method (FIG. 14), for 24 h. Numbers 1, 2, 3 were inoculation levels at 2.4×10.sup.1, 0.36×10.sup.1, and 0.036×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(8) FIGS. 15 and 16 show amplification products obtained by multiplex PCR of Salmonella spp. inoculated on meat trimmings incubated with a standard method (FIG. 15), or an embodiment of the inventive ‘dry’ method (FIG. 16), for 6 h. Numbers 1, 2, 3 were inoculation levels at 1.6×10.sup.1, 0.24×10.sup.1, 0.042×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(9) FIGS. 17 and 18 show amplification products obtained by multiplex PCR of Salmonella spp. inoculated on meat trimmings incubated with a standard method (FIG. 17), or an embodiment of the inventive ‘dry’ method (FIG. 18), for 7 h. Numbers 1, 2, 3 were inoculation levels at 1.6×10.sup.1, 0.24×10.sup.1, 0.042×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(10) FIGS. 19 and 20 show amplification products obtained by multiplex PCR of Salmonella spp. inoculated on meat trimmings incubated with a standard method (FIG. 19), or an embodiment of the inventive ‘dry’ method (FIG. 20), for 8 h. Numbers 1, 2, 3 were inoculation levels at 1.6×10.sup.1, 0.24×10.sup.1, 0.042×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(11) FIGS. 21 and 22 show amplification products obtained by multiplex PCR of Salmonella spp. inoculated on meat trimmings incubated with a standard method (FIG. 21), or an embodiment of the inventive ‘dry’ method (FIG. 22), for 24 h. Numbers 1, 2, 3 were inoculation levels at 1.6×10.sup.1, 0.24×10.sup.1, 0.042×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

DETAILED DESCRIPTION OF THE INVENTION

(12) Prior to the present invention, low-dilution enrichment, transit incubation methods were not known or appreciated in the relevant art. Prior art methods, as summarized herein, under the “Background” section, are based on 1:10 (wt./vol.) dilutions of the sample.

(13) In various aspects, the present invention provides novel methods for remote testing of one or more pathogens or other microbes in food, water, wastewater, sludge, pharmaceutical, industrial samples, and the like. In particular aspects, a dry-enrichment or a semi-dry-enrichment process allows for incubation, during transit to a remote testing location, of the food samples either without (e.g., liquid samples) the addition of enrichment media, or with (e.g., solid or semi-solid samples) addition of only relatively small quantities of media and/or supplements, for testing at the remote location of contaminating pathogens or other microbes.

(14) The present novel embodiments provide accurate, cost effective, and timely sampling and testing methods under circumstances where shipping of the sample to a remote testing location is required or desirable. The present invention, in various aspects, allows, inter alia, for: (i) savings in the cost of enrichment media/buffers; (ii) the ability to ship the samples economically (with up to 90% less weight), and with far less chances of spills (e.g., because of 90% less liquids in enriched samples), in specially designed dry/semi-dry shipping incubators, to remote/distant laboratories; (iii) increased sensitivity in the detection of the target organisms, due to smaller dilution factor; and (iv) the ability to form centralized laboratories (that may be remote from the sampling sites) that use the enrichment/incubation-in shipping concept to provide testing services to distant clients with the ability to report as fast as a local laboratory, and at similar or reduced costs relative to local laboratories.

(15) According to the present invention, the samples are incubated immediately at the desired incubation temperature, and/or are incubated in transit by shipping the samples in a temperature-controlled shipping container or incubator allowing for incubation at appropriate temperatures within optimal ranges depending on the pathogen's optimal growth range, and/or optimal competitive growth range.

(16) According to the present invention, for many types of foods, the food itself can be incubated with no addition of inhibitory or supplementary substances. For example, with foods or environmental samples that are in liquid form (e.g., fruit juices and the like), the samples are enriched directly, with or without a concentration step, with or without the addition of supplementary nutrients, inhibitors, or indicators.

(17) In other aspects, with dry and semi-dry foods, the water and/or nutrient activity is adjusted to allow for the growth of the target organism. Addition of supplements depends on the target organism and the nature of the samples which are to be tested. Inventive supplements include water, sugars, proteins, minerals, organics, vitamins and cofactors, antibiotics, dyes, indicators, etc.

(18) The protocols as described allow for the enrichment of pathogens, or target organisms with minimal increase in volume/weight of the samples. For samples that are to be shipped out, the shipping time can be used to enrich the sample without substantial increase in the weight and volume of the product, also minimizing the probability of spillage and/or cross-contamination. The sensitivity of the method is increased due to the elimination of dilution factor; the sample size has not increased substantially in terms of its volume or weight.

(19) Samples testable according to the present invention include, but are not limited to beef, pork, sheep, bison, deer, elk, poultry (e.g., chicken and turkey) and fish, produce, juices, dairy products, dry goods (cereals, etc), and all manners of raw and processed foods, environmental samples (water, wastewater, soil, surface samples, samples taken by impingers and filteration, etc), pharmaceuticals, and other types of samples that are to be analyzed using enrichment-detection protocols.

(20) Definitions

(21) “Carcass” refers to the body of the livestock after harvest, de-hiding (or, as the case may be, de-feathering, de-skinning, de-scaling), and evisceration. Preferred embodiments relate to beef carcasses, but the present invention encompasses carcasses of pigs, sheep, deer, bison, elk, poultry (e.g., turkey, chicken) and other animals (e.g., fish) that are killed and processed into products that may contain pathogens and/or other microbes of interest.

(22) Carcass “splitting” refers to splitting of the carcass into portions, including into two half-carcasses or half-carcass portions. In preferred embodiments, splitting refers to splitting into two half-carcass portions.

(23) “Fabrication” refers to the process of cutting-up half-carcass into marketable cuts (e.g., primals, sub-primals, trim).

(24) “Trim” refers to small pieces of meat and fat which are excised during the fabrication process in order to produce primal and subprimal pieces and marketable cuts.

(25) “Trim testing” refers to the process of testing trim, or raw materials which are to be used for ground meat production for microbial/pathogen content.

(26) A “combo” or “combo-bin” refers to the trim packaging unit. Alternatively raw materials to be tested can be packaged into boxes, bags or other appropriate containers, which can be placed, for example, on pallets.

(27) A “Lot-unit” or “five-combo-lot unit” refers to a composite unit, comprised of five combos (combo-bins). In prior art sampling plans, the Lot-unit represents raw the material (composite trim) upon which sampling, testing and acceptance or rejection is based.

(28) A prior art “Bin-sample” refers to a sample (typically about 75 g), comprised of 1-12 randomly-selected pieces from a single combo-bin. Typically, in prior art sampling plans, the standard manner of collection is to randomly pick five pieces as a comb-bin is being filled.

(29) A prior art “Lot-unit sample” refers to a 375 g composite sample, comprised of seventy-five (75) gram samples from each combo-bin of the Lot-unit. In a variation of the typical prior art protocol, the random piece samples from each combo-bin (the Bin-samples) are subjected to grinding, then composite Lot-samples are made from the respective ground Bin-samples.

(30) “Sampling” or “obtaining samples” refers to obtaining, in a form suitable for pathogen/microbe testing purposes, a sample of the pathogens and/or other microbes of interest present on or within various samples. Samples testable according to the present invention include, but are not limited to beef, pork, sheep, bison, deer, elk, poultry (e.g., chicken and turkey) and fish, produce, juices, dairy products, dry goods (cereals, etc), and all manners of raw and processed foods, environmental samples (water, wastewater, soil, surface samples, samples taken by impingers and filteration, etc), pharmaceuticals, and other types of samples that are to be analyzed using enrichment-detection protocols. Samples, include environmental samples, water, wastewater, samples taken by impingers and filtration, botanical liquid, industrial liquids, pharmaceutical liquids, and other liquid samples analyzed using enrichment-detection protocols.

(31) For meat products, a sample may correspond to the surface of one or more test locations of the carcass or sub-carcass portion. Any sampling method is encompassed, provided that it is suitable to acquire (or include), at least to some extent, the surface pathogens/microbes. Preferably, the sampling method is by excision, or by blotting, swabbing, sponging, and the like (see under “Samples and Sample Locations, herein below).

(32) Preferred Embodiments

(33) Aspects of the present invention provide a method for remote detection of a pathogen or other microbe in a sample comprising: obtaining a test sample at a first location, the sample being solid or semi-solid; diluting, at the first location, the sample with enrichment medium at a ratio of about 1:2.0 (wt./vol.) or greater; incubating the diluted sample at an optimal temperature in an incubator during transit of the diluted sample to a second location that is a remote test location; and determining, at the remote test location, by assaying the diluted incubated test sample, or a portion thereof, with an assay suitable to detect the pathogen or other microbe, whether the sample is contaminated.

(34) Preferably, the sample is selected from the group consisting of beef, pork, sheep, bison, deer, elk, poultry, fish, produce, dairy products, dry goods, raw and processed foods, environmental samples, soil, surface samples, samples taken by impingers and filtration, etc), pharmaceuticals, and samples analyzed using enrichment-detection protocols.

(35) Preferably, diluting is at a ratio of 1:1 (wt./vol.) or greater. More preferably, diluting is at a ratio of 1:0.5 (wt./vol.) or greater. Alternatively, diluting is at a ratio of 1:0.3 (wt./vol.) or greater, or at a ratio of 1:01 (wt./vol.) or greater (i.e., non-diluted).

(36) Preferably, the optimal temperature is within, or substantially within, the optimal growth temperature range of the pathogen or other microbe in the particular enrichment medium. Preferably, the optimal temperature is within, or substantially within, a temperature range that affords an optimal competitive growth advantage, relative to other microbes present in the sample. Preferably, the optimal temperature range is from about 25 to 45° C. Preferably, the optimal competitive growth temperature range is from about 30 to about 45° C.

(37) Preferably, the assay suitable for detection of pathogenic or microbial contamination is selected from the assay group consisting of immunoassays, nucleic acid amplification-based assays, PCR-based assays, nucleic acid hybridization-based assays, bio-sensor assays, immunostaining-microscopy-based assays, nucleic acid-array-based assays, DNA chip-based assays, bacteriophage-detection-based assays, classical microbiology-based assays, and chemical or biochemical assays based on the detection of compounds associated with particular target organisms or groups of target organisms, and combinations thereof.

(38) Preferably, the microbe or pathogen is selected from the group consisting of Escherichia coli O157:H7 (E. coli O157:H7), enterohemorrhagic Escherichia coli (EHEC), enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC), enterpathogenic Escherichia coli (EPEC), Salmonella, Listeria, Yersinis, Campylobacter, Clostridial species, Staphylococcus spp.; frank and opportunistic bacterial, fungal, viral, parsitic pathogens; indicator organisms including heterotrophes, generic E. coli, total and fecal coliforms and enterococcus; spoilage organisms including Pseudomonas; indicator molecules including glial fibillary acid protein (GFAP), transmissable spongiform encephalopathy (TSE) agents (prions), including bovine spongiform encephalopathy (BSE) agents, scrapie, chronic wasting disease; and combinations thereof.

(39) Preferably, the pathogen or microbe is Escherichia coli O157:H7 (E. coli O157:H7).

(40) Preferably, obtaining a sample is obtaining a sample from at least one test location of a carcass or of a split-portion thereof, wherein the test sample is obtained, in the slaughter production process, prior to or during initial chilling of the respective carcass or split-portion thereof. Preferably, the sample is a composite-Lot sample, corresponding to a combination of samples from a plurality of carcasses with a carcass Lot, and wherein the carcass is selected from the carcass group consisting of cattle, sheep, pigs, bison, elk, deer, chicken, turkey, fish and combinations thereof.

(41) In alternative embodiments, supplements are added to the sample, prior to shipping of the sample to the remote testing location. Preferably, supplements are selected from the group consisting of water, sugars, proteins, minerals, organics, vitamins and cofactors, antibiotics, dyes, indicators. Such supplements are well known to those of ordinary skill in the relevant art, and include art-recognized micro- (e.g., Zn, Fe and Mn ) and macro nutrients (e.g., Na, P, K, Ca, and magnesium and sulfur) (see, e.g., U.S. Pat. No. 5,582,627, incorporated by reference herein in its entirety), and/or carbon skeleton sources or other compounds, including but not limited to: sugar—mannose, lactose, dextrose, arythrose, fructose, fucose, galactose, glucose, gulose, maltose, polysaccharide, raffinose, ribose, ribulose, rutinose, saccharose, stachyose, trehalose, xylose, xylulose, adonose, amylose, arabinose, fructose phosphate, fucose-p, galactose-p, glucose-p, lactose-p, maltose-p, mannose-p, ribose-p, ribulose-p, xylose-p, xylulose-p, deoxyribose, corn steep liquor, whey, corn sugar, corn syrup, maple syrup, grape sugar, grape syrup, beet sugar, sorghum molasses, cane molasses, calcium lignosulfonate; sugar alcohol—adonitol, galactitol, glucitol, maltitol, mannitol, mannitol-p, ribitol, sorbitol, sorbitol-p, xylitol; organic acids—glucuronic acid, a-ketoglutaric acid, galactonic acid, glucaric acid, gluconic acid, pyruvic acid, polygalacturonic acid, saccharic acid, citric acid, succinic acid, malic acid, oxaloacetic acid, aspartic acid, phosphoglyceric acid, fulvic acid, ulmic acid, humic acid; nucleotides and bases—adenosine, adenosine-p, adenosine-p-glucose, uridine, uridine-p, uridine-p-glucose, thymine, thymine-p, cytosine, cytosine-p, guanosine, guanosine-p, guanosine-p-glucose, guanine, guanine-p, NADPH, NADH, FMN, FADH; Buffers—phosphate buffer, acetate buffer, AMP buffer, calcium tartrate, glycine buffer, phosphate citrate buffer, and tris buffer; and combinations thereof, are also contemplated (Id).

(42) Optional addition of complexing agents, including but not limited to citric acid; Ca, K, Na and ammonium lignosulfonates, fulvic acid, ulmic acid, humic acid, Katy-J, EDTA, EDDA, EDDHA, HEDTA, CDTA, PTPA, NTA and combination thereof, are also contemplated.

(43) In alternate preferred embodiments, the invention provides a method for remote detection of a pathogen or other microbe in a liquid sample comprising: obtaining a liquid sample at a first location; incubating the sample at an optimal temperature in an incubator during transit of the diluted sample to a second location that is a remote test location; and determining, at the remote test location, by assaying the diluted incubated test sample, or a portion thereof, with an assay suitable to detect the pathogen or other microbe, whether the sample is contaminated.

(44) Preferably, the optimal temperature is within, or substantially within, the optimal growth temperature range of the pathogen or other microbe in the particular enrichment medium. Preferably, the optimal temperature is within, or substantially within, a temperature range that affords an optimal competitive growth advantage, relative to other microbes present in the sample. Preferably, the optimal temperature range is from about 25 to about 45° C. Preferably, the optimal competitive temperature range is from about 30 to about 45° C.

(45) Preferably, detection of pathogenic or microbial contamination is with an assay selected from the assay group consisting of immunoassays, nucleic acid amplification-based assays, PCR-based assays, nucleic acid hybridization-based assays, bio-sensor assays, immunostaining-microscopy-based assays, nucleic acid-array-based assays, DNA chip-based assays, bacteriophage-detection-based assays, classical microbiology-based assays, and chemical or biochemical assays based on the detection of compounds associated with particular target organisms or groups of target organisms, and combinations thereof.

(46) Preferably, the microbe or pathogen is selected from the group consisting of Escherichia coli O157:H7 (E. coli O157:H7), enterohemorrhagic Escherichia coli (EHEC), enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC), enterpathogenic Escherichia coli (EPEC), Salmonella, Listeria, Yersinis, Campylobacter, Clostridial species, Staphylococcus spp.; frank and opportunistic bacterial, fungal, viral, parsitic pathogens; indicator organisms including heterotrophes, generic E. coli, total and fecal coliforms and enterococcus; spoilage organisms including Pseudomonas; indicator molecules including glial fibillary acid protein (GFAP), transmissable spongiform encephalopathy (TSE) agents (prions), including bovine spongiform encephalopathy (BSE) agents, scrapie, chronic wasting disease; and combinations thereof.

(47) Preferably, the pathogen or microbe is Escherichia coli O157:H7 (E. coli O157:H7).

(48) Preferably, the liquid sample is selected from the group consisting of fruit juice, vegetable juice, milk and dairy products, raw and processed liquid foods, environmental samples, water, wastewater, samples taken by impingers and filtration, pharmaceuticals, and other samples analyzed using enrichment-detection protocols.

(49) Alternatively, the methods comprise addition to the sample, prior to transit of the sample to the second location, of a supplement selected from the group consisting of water, sugars, proteins, minerals, organics, vitamins and cofactors, antibiotics, dyes, indicators, buffers and combinations thereof. Exemplary supplements are described elsewhere herein.

(50) Yet further alternate embodiments provide a method for remote detection of a pathogen or other microbe in a solid sample, semi-solid sample or liquid sample comprising: obtaining a test sample at a first location, the sample being solid, semi-solid or liquid; diluting, at the first location, the sample with enrichment medium at a range of ratio of about 1:0.1 to 1:2 (wt./vol.), or not diluting the sample; incubating the enriched sample at an optimal temperature in an incubator for testing in a second location that is an in-house or local lab; and determining, at the in-house or local lab, by assaying the diluted incubated test sample, or a portion thereof, with an assay suitable to detect the pathogen or other microbe, whether the sample is contaminated.

(51) Preferably, the methods further comprise addition to the sample, prior to or during incubation, of a supplement selected from the group consisting of water, sugars, proteins, minerals, organics, vitamins and cofactors, antibiotics, dyes, indicators, buffers, agents to adjust the pH, water activity, nutritional contents, selective pressure to optimize the growth conditions for the target organism, and combinations thereof.

(52) Incubators

(53) Preferred embodiments of the present invention comprise use of an incubator for holding the sample or diluted sample at an optimal temperature, or within an optimal temperature range, during shipment of the sample to the remote testing location. Suitable incubators are known in the art. In a preferred embodiment the incubator comprises an enclosure into which bottles containing sample and enrichment medium/buffer can be placed. Preferably the enclosure is an insulated container with a removable lid. Preferably, a temperature sensor activates ‘on demand’ power to a heating (and/or cooling) element. Preferably, the power supply is a typical 12 v type, able to work in a vehicle (e.g., off the cigarette lighter, or similar connection). In particular embodiments, a fan circulates air around the heating (and/or cooling) element. In preferred embodiments the power source is through a cigarette lighter-type connection within a vehicle. Preferably, the sample bottles are designed to be leak proof.

(54) Samples to be Tested by the Inventive Methods

(55) Samples testable according to the present invention include, but are not limited to beef, pork, sheep, bison, deer, elk, poultry (e.g., chicken and turkey) and fish, produce, juices, dairy products, dry goods (cereals, etc), and all manners of raw and processed foods, environmental samples (water, wastewater, soil, surface samples, samples taken by impingers and filteration, etc), pharmaceuticals, and other types of samples that are to be analyzed using enrichment-detection protocols.

(56) Prior Art Trim Testing Methods in the Slaughter Industry

(57) Prior art pathogen-testing plans are actually either trim-testing plans, final product testing plans, or both, involving random and incomplete sampling at the ‘packing-Lot’ level; that is, testing of trim samples near the end of the production chain (as they enter the bins or after binning, or testing of the ground products by taking samples at given time intervals. A typical trim-testing plan involves analysis of ‘five-combo-lot’ units, and comprises analysis of a single composite-Lot sample of about 375 g, prepared by combining five combo-bin samples (about 75 g each), in each case corresponding to one to five randomly-selected pieces from each combo-bin, such that, on average 5-25 pieces representing the five combo-bins are in the composite sample. The combo-bin is comprised of pieces of a plurality of carcasses, and thus the test results under these prior art systems reflect random and incomplete sampling; that is, only a small fraction of the carcasses are represented in the test results, particularly where, as is true of many such plans, only a sub-fraction of the composite-Lot sample is used for the pathogen-testing assay (e.g., when large pieces of trim are collected). For example, for ground beef production, final product testing comprises taking ground beef samples at given time intervals (e.g., every 10-30 minutes) and compositing a number of samples into one composite sample.

(58) Generally, one of several methods of analysis has been used for pathogen detection after sampling at the packing-Lot level: (1) Immunochemical based detection (e.g., ELISA based immunoassays) following enrichment (e.g., for E. coli O157:H7); (2) Nucleic acid-based (e.g., DNA-based, such as PCR-analysis) detection methods following enrichment (e.g., for E. coli O157:H7), wherein an appropriate medium is inoculated with a composite-Lot sample; and (3) target organisms can be detected by enrichment, followed by immunomagnetic separation followed by plating, immunochemical or DNA based detection. Typically, the levels of sensitivity of most of these methods are set at about 1 colony forming unit (cfu)/25 g of sample.

(59) Typically, to allow time for trim testing results to be obtained, such sampling plans require additional (in addition to initial chilling of the split-carcasses, and the time taken during the processing of the carcasses (fabrication)) ‘holding’ of the trim prior to use. Such holding of the trim will typically add 12 to 24 hrs of extra refrigeration storage time/capacity, and uses up about a day of the product shelf-life.

(60) Trim Testing Methods in the Context of the Present Invention

(61) According to preferred aspects of the present invention, carcasses (e.g., beef, sheep, pigs, deer, elk, bison, poultry, and fish) typically spend up to 24 hours in chilled storage prior to being cut up into, or generating, trim (prior to fabrication), and this is sufficient time to run an adequate detection assay (e.g., presence/absence assays) for microbes or other pathogens. Furthermore, carcass-Lots are identifiable, and carcasses are typically tagged with unique identifiers, and thus, according to the present invention, where a timely and statistically-significant positive result (contaminated carcass) is obtained, the respective contaminated carcass-Lot is precluded from entering the fabrication/production chain. Therefore, cross-contamination is reduced or eliminated, where carcasses carrying contamination are removed from the process stream prior to being cut into, or generating, trim (e.g., the possibility of transferring any contamination to equipment such as knives and conveyor belts is reduced or eliminated).

(62) In a preferred aspect, the inventive method comprises splitting of a carcass-Lot to produce a split-carcass-Lot (Lot of split-portions), and obtaining at least one surface test sample from at least one preferred sample location of one or both split-portions, immediately prior to chilling of the split-portions. In a particularly preferred embodiment, at least three distinct surface samples are taken from each half-carcass. The test samples are combined to form a composite-Lot test sample, and the composite-Lot test sample, or a portion thereof is used for determining whether contamination is present by using an assay suitable to detect the microbe or pathogen of interest. Preferably, a carcass-Lot is a group of 50-100 carcasses. Preferably, at least one half of each carcass is surface-sampled (e.g., by excision, blotting, swabbing or sponging). Preferably, testing is of composite-Lot samples comprising at least 25, at least 50, at least 100, at least 150, at least 200, or at least 300 surface samples (e.g., blotting, swabbing or sponging, or thinly excised sample slices). Preferably, three to four carcass sampling stations (locations) are established. Preferably, the sampling stations correspond to carcass sites that are most likely, according to the present invention, to be contaminated, based on factors regarding the locations and incident rates of a given pathogen such as E. coli O157:H7. Preferably, the test locations are selected in a random or repeated (rotational) order from the group consisting of rump, brisket, back and flank, and combinations thereof. Preferably, from rump, brisket and/or flank.

(63) Type of Pathogens/Microbes to be Detected

(64) In preferred embodiments, the present inventive methods encompass the detection of Escherichia coli O157:H7 (E. coli O157:H7), enterohemorrhagic Escherichia coli (EHEC), enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC), enterpathogenic Escherichia coli (EPEC), Salmonella, Listeria, Yersinis, Campylobacter, Clostridial species, Staphylococcus spp. Additionally, other pathogens and pathogenic agents are encompassed within the present invention, including, but not limited to other frank and opportunistic bacterial, fungal, viral, parsitic pathogens; indicator organisms (total heterotrophes, generic E. coli, total and fecal coliforms, enterococcus, etc); and spoilage organisms (Pseudomonas, etc), and indicator molecules such as glial fibillary acid protein (GFAP), transmissable spongiform encephalopathy (TSE) agents (prions) (e.g., bovine spongiform encephalopathy (BSE) agents, scrapie (sheep), chronic wasting disease (e.g., deer, Elk).

(65) Preferably, the detected microbe is: a pathogen including, but not limited to, Escherichia coli O157:H7 (E. coli O157:H7), Listeria, Salmonella, EHEC, Campylobacter, Staphylococcus, pathogenic Clostridial species, and other frank, or opportunistic pathogens; a spoilage organism including, but not limited to, clostridial and pseudomonas species; or an indicator organism including, but not limited to, generic E. coli, fecal coliforms, total coliforms, etc. More preferably, the pathogen is Escherichia coli O157:H7 (E. coli O157:H7).

(66) Detection Assays

(67) Preferably, the detection assay is selected from the assay group consisting of immunoassays, nucleic acid amplification-based (e.g., PCR-based assays), nucleic acid hybridization-based assays, bio-sensor assays, immunostaining-microscopy based assays, nucleic acid-array-based (e.g., DNA chip-based) assays, bacteriophage detection based assays, classical microbiology based assays, and chemical/biochemical assays based on the detection of compounds associated with particular groups of target organisms, and combinations thereof. Such assays are well known in the relevant art, and a few exemplary assays are as follows:

(68) Reveal® test kits are available from Neogen Corporation, Lansing, Mich., and comprise an ELISA test that combines an immunoassay with chromatography in a lateral flow device (Reveal® test kits are available for E. coli O157:H7, Salmonella, Listeria spp. and Listeria monocytogenes);

(69) VIP® test kits are available from BioControl, Bellevue, Wash., and comprise an ELISA test that is expressed as a lateral flow antibody-chromogen immunoprecipitate assay (VIP® test kits are available for EHEC, Salmonella, Listeria spp. and Listeria monocytogenes);

(70) BAX® Qualicon test kits are available from DuPont, Wilmington, Del., wherein, using the BAX® system, samples are enriched and then lysed to release DNA, which is amplified using PCR techniques, and detected using a fluorescent signal (BAX® test kits are available for E. coli O157:H&, Salmonella, Listeria spp. and Listeria monocytogenes, and enterobacter sakazakii); and

(71) TSE (e.g., in cattle sheep and deer) can be tested by testing nervous system tissue (e.g., brain stem) for the respective agents (PrP) using, for example, ELISA assays, such as the Enfer TSE test, manufactured by Enfer Scientific, Newbridge Ireland, and distributed by Abbott Laboratories (Abbott Park, Ill.; see Abbott Application Note by Klass et al, 2002, “A test for transmissible spongiform encephalopathy,” incorporated herein by reference).

(72) Carcass Samples and Sample Locations

(73) Sampling, as practiced herein, refers to obtaining, in a form suitable for pathogen/microbe testing purposes, a sample of the pathogens and/or other microbes of interest present on the surface of one or more test locations of the carcass or sub-carcass portion. Any sampling method is encompassed, provided that it is suitable to extract (or include), at least to some extent, the surface pathogens/microbes. Preferably, the sampling method is by excision, or by blotting, swabbing, sponging, and the like. Preferably, in order to produce results which allow systematic comparisons with additional monitoring of microbial interventions along process line, samples are collected at the rump, brisket, or flank and combinations thereof (see Elder et al., PNAS 97:2999-3003, 2000; Correlation of enterohemorrhagic Escherichia coli O157 prevalence in feces, hides, and carcasses of beef cattle during processing).

(74) Preferably, obtaining a test sample, or remedial test sample, comprises obtaining one or more surface samples from at least one test location of at least one split-portion, or remedial-split-portion of each carcass. Preferably, the test sample, or remedial test sample, is a surface sample (e.g., swabbed, blotted, sponged, or an excised surface tissue section) corresponding to, or having a surface area of at least 4, at least 6, at least 8, at least 10, at least 12, or at least 16 square inches. More preferably, the test sample, or remedial test sample, is a surface sample corresponding to, or having a dimension of about 16 square inches. Preferably, the test sample, or remedial test sample, comprises three, or at least three, such surface samples, each from a distinct location of the split-portion. Preferably, the test location, or remedial test location, is randomly or rotationally selected from the group consisting of sites most likely to harbor microbial pathogens (e.g., rump, brisket, back and flank, and combinations thereof).

(75) In particularly preferred embodiments, a contaminated split-carcass-Lot is subjected to remedial reconditioning. Preferably, reconditioning comprises sanitizing/pasteurizing by hot-water or steam pasteurization, and/or by using an organic acid spray, (e.g., lactic-acid spray), etc.). Such methods are well known to those of ordinary skill in the relevant art.

(76) The present invention will now be further illustrated by reference to the following EXAMPLES. However, it should be noted that these EXAMPLES, like the embodiments described above, are illustrative and are not to be construed as restricting the enabled scope of the invention in any way.

EXAMPLE I

Enrichment Methods Based on 1:0.5 Dilution Samples to Media Were Found to be Effective

(77) To compare the effectiveness of dry (1:0.5 meat to media) to standard (1:10) enrichment methods on the growth of Escherichia coli O157:H7 and Salmonella spp. on experimentally inoculated meat samples incubated for 6, 7, 8, and 24 h. E. coli and Salmonella spp. were detected with a multiplex polymerase chain reaction (PCR) technique.

(78) Materials and Methods

(79) Inoculum preparation. Colonies of E. coli O157:H7 and Salmonella typhi isolated from clinical samples were grown on MacConkey II agar (BD Diagnostic Systems, Sparks, Md.) and XLD (BD), respectively. Random colonies of E. coli O157 were confirmed with E. coli O157 Latex Test Kit (Oxoid, Hampshire, UK). Each pathogen were independently cultured in E. coli O157 Bax® System media (Dupont Qualicon by Oxoid Ltd., Hampshire, England) at 37° C. for 24 h. Serial dilutions of each inoculum were done separately in 0.1% peptone water (BD). Enumeration was done by plating 1 ml of each dilution on Petrifilm™ Aerobic Count Plates (3M™ Microbiology Products, St. Paul, Minn.). Plates were incubated at 37° C. for up to 48 h. Based on enumeration data, meat trim samples were inoculated with approximately 6.1×10.sup.2, 9.1×10.sup.1, and 0.9×10.sup.1 CFU/ml of E. coli O157:H7, and 4.1×10.sup.2, 6.2×10.sup.1, and 1.0×10.sup.1 CFU/ml of S. typhi.

(80) Meat sample. Prepacked meat trim (beef stew) sample was purchased from a local grocery store in Seattle, Wash. For standard and dry enrichment methods, 25 g and 100 g of duplicate samples were placed in whirl-pack bags, respectively. Meat samples were inoculated with three concentrations of E. coli O157:H7 and S. typhi mentioned earlier at a rate of 1 ml of each pathogen per 25 g of meat sample. This approached resulted in concentrations of 2.4×10.sup.1, 0.36×10.sup.1, and 0.036×10.sup.1 CFU/g of meat for E. coli O157:H7, and 1.67×10.sup.1, 0.24×10.sup.1, 0.042×10.sup.1 CFU/g of meat for S. typhi. Samples were then subjected to a stomacher for 30 s. Standard and dry enrichment samples received 225 ml and 50 ml of pre-warmed (42° C.) E. coli O157 Bax® System media (Dupont), respectively. Samples were again stomached for 30 s. All enrichment bags were placed in an incubator shaker at 42° C. with household (ice) packs between bags. Ice packs were pre-warmed by placing them in a 42° C. incubator two days prior to the experiment.

(81) At 6, 7, 8 and 24 h of incubation, 1 ml of broth of each enrichment bag was taken and placed in 1.5 ml centrifuge tubes. Samples were concentrated by centrifugation for 5 min at low speed. Supernatant was aspirated and pellet was resuspended in 1 ml phosphate buffer saline with 0.05% Tween (PBST).

(82) Prior to E. coli O157:H7 detection by PCR method, an aliquot of sample suspension was concentrated with anti-E. coli O157 Dynabeads® (Dynal Biotech ASA, Oslo, Norway). All sample suspension with and without Dynabeads were detected by PCR.

(83) Background microflora of non-inoculated meat sample was analyzed. Ten grams of meat sample was placed in a stomacher bag and 90 ml of 0.1% peptone water was added to the bag. The meat was subjected to a stomacher for 30 s. Serial dilution was made in 0.1% peptone water and 1 ml was plated on Petrifilm™ Aerobic Count Plates (3M™). Plates were incubated at 37° C. for up to 48 h.

(84) PCR. Multiplex PCR method was used for the detection of E. coli O157:H7 and Salmonella spp. A reaction mixture containing specific regions of the pathogens of interest was used as a positive control. Negative control was distilled water.

(85) E. coli O157: Samples were subjected to a 55 μl mixture of lysis buffer containing MgCl.sub.2 IGEPAL CA-630 detergent, primers, achromopeptidase and Tris buffer. The primer pairs amplified specific fragments of E. coli O157:H7 encoding for O157 antigen (rfb, 985 bp), intemin (eae, 309 bp), and shiga-like toxin 2 (stx 2, 255 bp) and 1 (stx 1,180 bp) genes. Pure Taq Ready-To-Go beads (Amersham Biosciences, Piscataway, N.J.) were used. Taq beads contained Taq polymerase, dNTPs, BSA, and detergent. The reaction conditions were 4° C. for 2 min, 95° C. for 2 min, 95° C. for 2 sec, 65° C. for 30 sec, 72° C. for 20 sec, 33 cycles of 95° C. for 10 sec, followed by 4° C. cooling period.

(86) Salmonella spp.: Samples were subjected to a 55 μl mixture of lysis buffer containing MgCl.sub.2 IGEPAL CA-630 detergent, primers, achromopeptidase and Tris buffer. The primer pairs amplified specific fragments of Salmonella spp. encoding for plasmid DNA (524 bp), Salmonella spp. fragment (300 bp), and chromosome (197 bp) genes. Pure Taq Ready-To-Go beads (Amersham Biosciences) were used. Taq beads contained Taq polymerase, dNTPs, BSA, and detergent. The reaction conditions were 4° C. for 2 min, 95° C. for 2 min, 95° C. for 2 sec, 65° C. for 30 sec, 72° C. for 20 sec, 33 cycles of 95° C. for 10 sec, followed by 4° C. cooling period.

(87) Electrophoresis was performed on 2% (w/v) agarose gel (Fisher Scientific, Fair Lawn, N.J.), stained with ethidium bromide solution and viewed under a UV light source.

(88) Results

(89) Pre-packed meat trim had an average of 3.8 logs CFU/ml of aerobic plate counts. The four (rfb, eae, stx 1, and stx 2) target genes for E. coli O157 were detected by PCR methods with Dynabeads® (FIGS. 1 through 6) and without Dynabeads® (FIGS. 7 through 14) from samples incubated with dry and standard enrichment procedures for 6, 7, 8, and 24 h incubation. Likewise, the three (spvc, sal 2, and inva) target genes for Salmonella spp. were detected by PCR methods (FIGS. 14 through 22) from samples incubated with dry and standard enrichment procedures after 6, 7, 8, and 24 h incubation.

(90) FIG. 1 shows amplification products obtained by multiplex PCR of E. coli O157:H7 inoculated on meat trimmings incubated with standard dilution method (1:10) for 6 h and concentrated by Dynabeads®. Numbers 1, 2, 3 were inoculation levels at 2.4×10.sup.1, 0.36×10.sup.1, and 0.036×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(91) FIG. 2 shows amplification products obtained by multiplex PCR of E. coli O157:H7 inoculated on meat trimmings incubated with a representative dry method (1:0.5) for 6 h and concentrated by Dynabeads®. Numbers 1, 2, 3 were inoculation levels at 2.4×10.sup.1, 0.36×10.sup.1, and 0.036×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(92) FIG. 3 shows amplification products obtained by multiplex PCR of E. coli O157:H7 inoculated on meat trimmings incubated with standard method (1:10) for 7 h and concentrated by Dynabeads®. Numbers 1, 2, 3 were inoculation levels at 2.4×10.sup.1, 0.36×10.sup.1, and 0.036×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(93) FIG. 4 show amplification products obtained by multiplex PCR of E. coli O157:H7 inoculated on meat trimmings incubated with dry method (1:0.5) for 7 h and concentrated by Dynabeads®. Numbers 1, 2, 3 were inoculation levels at 2.4×10.sup.1, 0.36×10.sup.1, and 0.036×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(94) FIG. 5 shows amplification products obtained by multiplex PCR of E. coli O157:H7 inoculated on meat trimmings incubated with standard method (1:10) for 8 h and concentrated by Dynabeads®. Numbers 1, 2, 3 were inoculation levels at 2.4×10.sup.1, 0.36×10.sup.1, and 0.036×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(95) FIG. 6 shows amplification products obtained by multiplex PCR of E. coli O157:H7 inoculated on meat trimmings incubated with dry method (1:0.5) for 8 h and concentrated by Dynabeads®. Numbers 1, 2, 3 were inoculation levels at 2.4×10.sup.1, 0.36×10.sup.1, and 0.036×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(96) FIG. 7 shows amplification products obtained by multiplex PCR of E. coli O157:H7 inoculated on meat trimmings incubated with standard method (1:10) for 6 h. Numbers 1, 2, 3 were inoculation levels at 2.4×10.sup.1, 0.36×10.sup.1, and 0.036×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(97) FIG. 8 shows amplification products obtained by multiplex PCR of E. coli O157:H7 inoculated on meat trimmings incubated with dry method (1:0.5) for 6 h. Numbers 1, 2, 3 were inoculation levels at 2.4×10.sup.1, 0.36×10.sup.1, and 0.036×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(98) FIG. 9 shows amplification products obtained by multiplex PCR of E. coli O157:H7 inoculated on meat trimmings incubated with standard method (1:10) for 7 h. Numbers 1, 2, 3 were inoculation levels at 2.4×10.sup.1, 0.36×10.sup.1, and 0.036×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(99) FIG. 10 amplification products obtained by multiplex PCR of E. coli O157:H7 inoculated on meat trimmings incubated with dry method (1:0.5) for 7 h. Numbers 1, 2, 3 were inoculation levels at 2.4×10.sup.1, 0.36×10.sup.1, and 0.36×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(100) FIG. 11 shows amplification products obtained by multiplex PCR of E. coli O157:H7 inoculated on meat trimmings incubated with standard method (1:10) for 8 h. Numbers 1, 2, 3 were inoculation levels at 2.4×10.sup.1, 0.36×10.sup.1, and 0.36×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(101) FIG. 12 shows amplification products obtained by multiplex PCR of E. coli O157:H7 inoculated on meat trimmings incubated with dry method (1:0.5) for 8 h. Numbers 1, 2, 3 were inoculation levels at 2.4×10.sup.1, 0.36×10.sup.1, and 0.36×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(102) FIG. 13 shows amplification products obtained by multiplex PCR of E. coli O157:H7 inoculated on meat trimmings incubated with standard method (1:10) for 24 h. Numbers 1, 2, 3 were inoculation levels at 2.4×10.sup.1, 0.36×10.sup.1, and 0.36×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(103) FIG. 14 shows amplification products obtained by multiplex PCR of E. coli O157:H7 inoculated on meat trimmings incubated with dry method (1:0.5) for 24 h. Numbers 1, 2, 3 were inoculation levels at 2.4×10.sup.1, 0.36×10.sup.1, and 0.36×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(104) FIG. 15 shows amplification products obtained by multiplex PCR of Salmonella spp. inoculated on meat trimmings incubated with standard method (1:10) for 6 h. Numbers 1, 2, 3 were inoculation levels at 1.6×10.sup.1, 0.24×10.sup.1, 0.42×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(105) FIG. 16 shows amplification products obtained by multiplex PCR of Salmonella spp. inoculated on meat trimmings incubated with dry method (1:0.5) for 6 h. Numbers 1, 2, 3 were inoculation levels at 1.6×10.sup.1, 0.24×10.sup.1, 0.42×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(106) FIG. 17 shows amplification products obtained by multiplex PCR of Salmonella spp. inoculated on meat trimmings incubated with standard method (1:10) or 7 h. Numbers 1, 2, 3 were inoculation levels at 1.6×10.sup.1, 0.24×10.sup.1, 0.42×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(107) FIG. 18 shows amplification products obtained by multiplex PCR of Salmonella spp. inoculated on meat trimmings incubated with dry method (1:0.5) for 7 h. Numbers 1, 2, 3 were inoculation levels at 1.6×10.sup.1, 0.24×10.sup.1, 0.42×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(108) FIG. 19 shows amplification products obtained by multiplex PCR of Salmonella spp. inoculated on meat trimmings incubated with standard method (1:10) for 8 h. Numbers 1, 2, 3 were inoculation levels at 1.6×10.sup.1, 0.24×10.sup.1, 0.42×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(109) FIG. 20 shows amplification products obtained by multiplex PCR of Salmonella spp. inoculated on meat trimmings incubated with dry method (1:0.5) for 8 h. Numbers 1, 2, 3 were inoculation levels at 1.6×10.sup.1, 0.24×10.sup.1, 0.42×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(110) FIG. 21 shows amplification products obtained by multiplex PCR of Salmonella spp. inoculated on meat trimmings incubated with standard method (1:10) for 24 h. Numbers 1, 2, 3 were inoculation levels at 1.6×10.sup.1, 0.24×10.sup.1, 0.42×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(111) FIG. 22 shows amplification products obtained by multiplex PCR of Salmonella spp. inoculated on meat trimmings incubated with dry method (1:0.5) for 24 h. Numbers 1, 2, 3 were inoculation levels at 1.6×10.sup.1, 0.24×10.sup.1, 0.42×10.sup.1 CFU/g of meat, respectively. A and B were duplicate samples.

(112) Collectively, the results show that enrichment of meat trimmings with the inventive ‘dry’ method (e.g., 1:0.5 wt./vol. dilution) gave results comparable to those obtained using standard (USDA) methods employing the art-recognized 1:10 dilution factor. This experiment showed that enrichment of meat trimming with ‘dry’ method (1:0.5 dilution) showed similar results to the standard FDA (1:10 dilution) method. This experiment also showed that the dry enrichment method on samples inoculated with the highest (0.021×10.sup.1 CFU/g) dilution of bacteria was able to produce sufficient signals as shown in the PCR results.

EXAMPLE II

Novel Minimally Dilution Methods Were Shown to be Cost Effective

(113) Costs comparisons of the inventive methods with those of the prior art are as follows:

(114) TSB Medium Comparison:

(115) TSB 1 lb (453.59 g)=>$25-27 (depending on quantity ordered);

(116) Working dilution: 30 g/L;

(117) From 453.59 g, make±15.11 L;

(118) Price per Liter=±$1.65 to $1.78;

(119) Prior art price per sample (225 ml)=(225/1000)*1.65=±$ 0.37 to $0.40; and

(120) Inventive method price per sample (50 ml)=(50/1000)*1.65=+$0.082 to 0.089.

(121) Bax® Medium Comparison:

(122) E. coli O157 Bax® System media (Dupont Qualicon by Oxoid Ltd., Hampshire, England);

(123) 2.5 Kg (2500 g)=>about $300.00;

(124) Working dilution: 36.6 g/L;

(125) From 2,500 g, make±68.3 L;

(126) Price per liter=±$4.39;

(127) Prior art price per sample (225 ml)=(225/1000)*4.39=+$0.98; and

(128) Inventive method price per sample (50 ml)=(50/1000)*4.39+$0.21.