ENRICHMENT METHODS FOR THE DETECTION OF PATHOGENS AND OTHER MICROBES

• US 20090269760A1
• Filed: 01/27/2009
• Published: 10/29/2009
• Est. Priority Date: 05/16/2003
• Status: Active Grant

• Current Assignee: Institute for Environmental Health, Inc.
• Original Assignee: Institute for Environmental Health, Inc.
• Inventors: Samadpour, Mansour
• Granted Patent: US 9,845,486 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence

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 pathogen or other microbe, comprising:

obtaining a test, the sample being solid or semi-solid;

diluting the sample with liquid enrichment medium at a ratio of sample to diluent substantially less than 1;

10 (wt./vol.);

incubating the diluted sample at an optimal temperature for the particular pathogen or other microbe to be detected in an incubator; and

determining, by assaying the diluted incubated test sample, or a portion thereof, with an assay suitable to detect the particular pathogen or other microbe, whether the sample is contaminated with the particular pathogen or other 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;

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;

0.5 (wt./vol.) or lesser dilution.

4. The method of claim 1, wherein diluting is at a ratio of sample to diluent between about 1:

0.1 to about 1;

0.1 (wt./vol.) or lesser dilutions.

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

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

C.

9. The method of claim 1, wherein 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.

10. The method of claim 1, wherein 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, parasitic pathogens;

indicator organisms including heterotrophes, generic E. coli, total and fecal coliforms and enterococcus;

spoilage organisms including Pseudomonas;

and combinations thereof.

11. The method of claim 10, wherein the pathogen or microbe is Escherichia coli O157:

H7 (E. coli O157;

H7, or H negative), Salmonella, Listeria, EHEC, and Campylobacter.

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

13. 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, water samples, wastewater samples, bio-solid samples, air samples, samples taken by spore traps, settled dust, impingers, or filtration, and combinations thereof.

22. 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.

24. 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 target organism, and combinations thereof.

26. 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 pathogen or other microbe, whether the sample is contaminated with the particular pathogen or other microbe, is at the second location.

27. The method of claim 26, 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 target organism, and combinations thereof.

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

32. 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.

6. (canceled)

8. (canceled)

14. A method for remote detection of a pathogen or other microbe:

obtaining a liquid sample;

diluting the sample with liquid enrichment medium at a ratio of sample to diluent substantially less than 1;

10, or not diluting the sample;

incubating the sample at an optimal temperature for the particular pathogen or other microbe to be detected in an incubator;

determining, by assaying the diluted incubated test sample, or a portion thereof, with an assay suitable to detect the particular pathogen or other microbe, whether the sample is contaminated with the particular pathogen or other microbe.

15. The method of claim 14, wherein the optimal temperature is within the optimal growth temperature range of the particular pathogen or other microbe in the liquid enrichment medium.

17. The method of claim 15, wherein the optimal temperature range is from about 25 to about 45°

C.

19. The method of claim 14, wherein 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.

20. The method of claim 14, wherein 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, parasitic pathogens;

indicator organisms including heterotrophes, generic E. coli, total and fecal coliforms and enterococcus;

spoilage organisms including Pseudomonas; and

combinations thereof.

21. The method of claim 20, wherein the pathogen or microbe is Escherichia coli O157:

H7 (E. coli O157;

or H negative), Salmonella, Listeria, EHEC, and Campylobacter.

23. The method of claim 14, 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.

25. The method of claim 14, 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 target organism, and combinations thereof.

29. The method of claim 14, 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 pathogen or other microbe, whether the sample is contaminated with the particular pathogen or other microbe, is at the second location.

30. The method of claim 29, 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 target organism, and combinations thereof.

31. the method of claim 29, wherein the second location is an in-house or local lab, or a remote location.

33. The method of claim 14, wherein diluting is at a ratio of sample to diluent between about 1:

0.1 to about 1;

5 (wt./vol.) or lesser dilution.

34. The method of claim 14, wherein diluting is at a ratio of sample to diluent between about 1:

0.1 to about 1;

2 (wt./vol.) or lesser dilution.

35. The method of claim 14, 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.

36. The method of claim 14, wherein diluting is at a ratio of sample to diluent between about 1:

0.1 to about 1;

0.1 (wt./vol.) or lesser dilution.

16. (canceled)

18. (canceled)