Modular compositing-multiple lot screening protocols for detection of pathogens, microbial contaminants and/or constituents

• US 7,534,584 B2
• Filed: 08/08/2005
• Issued: 05/19/2009
• Est. Priority Date: 08/06/2004
• Status: Active Grant

• Current Assignee: Institute for Environmental Health, Inc.
• Original Assignee: Institute for Environmental Health, Inc.
• Inventors: Samadpour, Mansour
• Primary Examiner(s): Weber; Jon P
• Assistant Examiner(s): Srivastava; Kailash C
• Application Number:
  US11/199,871
• Publication Number:
  US 20060134726A1
• Time in Patent Office: 1,380 Days
• Field of Search: 435/32, 435/34, 435/36, 435/38, 435/39, 435/40, 435/849
• US Class Current: 435/34
• 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 1/10 Enterobacteria
• CPC Class Codes: C12Q 1/18 Testing for antimicrobial activity of a material
• CPC Class Codes: C12Q 1/24 Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
• CPC Class Codes: G01N 1/28 Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Qmounting specimens on microscopic slides G02B21/34; means for supporting the objects or the materials to be analysed in electron microscopes H01J37/20 ; laboratory gas handling apparatus B01L5/00
• CPC Class Codes: Y10S 435/849 Escherichia coli

Abstract

Particular aspects provide a method of sampling, testing and validating test lots (e.g., single-unit production lots), comprising: assembling a plurality of product portions from each of a plurality of test lots and combining the collected product portions to provide a corresponding set of test lot samples (wherein each test lot sample is attributed to a particular corresponding test lot); enriching the set of test lot samples; removing equal portions of each enriched sample, and combining the removed portions to provide a modular composite sample; and testing of the modular composite sample for the target agent/organism, wherein where such testing is positive, individual test lots may nonetheless yet be validated by further testing of a respective enriched test lot sample and obtaining a negative test result. The methods have broad utility for monitoring all sort of test lots (e.g., environmental lots, production lots, pharmaceutical lots, etc.) and for efficiently affecting informed, targeted remedial measures.

Claims

1. A method of sampling, testing and validating test lots for microbial contamination, comprising:

a) collecting a plurality of portions from each of a plurality of test lots, the test lots each comprising an assemblage of one or more specimens, wherein each test lot is separately sampled by taking said plurality of portions thereof;

b) combining the collected plurality of portions corresponding to each of the separate test lots to provide a corresponding set of separate test lot samples, wherein each separate test lot sample is attributed to a particular corresponding separate test lot;

c) incubating the set of separate test lot samples under conditions suitable to allow levels of a target microbial agent or organism that is present in one or more of the separate test lot samples to reach detectable levels and become uniform, or substantially uniform, throughout the respective one or more separate test lot samples, to provide a set of separate test lot samples enriched for the target microbial agent or organism;

d) removing portions of each enriched separate test lot sample, and combining the removed portions to provide a modular composite sample; and

e) testing of the composite sample, using a suitable detection assay, for the target microbial agent or organism, wherein when such testing is negative all of said separate test lot samples are validated, and wherein when such testing is positive, individual separate test lots are validated by further testing of a portion of the respective enriched separate test lot sample using the same or a more sensitive protocol and obtaining test result for each test lot.

2. The method of claim 1, wherein the test lot is a single-unit production lot, comprised of operationally-linked product specimens.

3. The method of claim 1, further comprising incubating the modular composite sample of d), prior to testing in e).

4. The method of claim 1, wherein incubating each separate test lot sample in c) comprises incubating each separate test lot sample with enrichment medium suitable to enrich for the target microbial agent or organism, and wherein removing in d) comprises removing a portion of the enrichment medium from each enriched separate test lot sample and combining the removed portions to provide a modular composite sample.

5. The method of claim 1, wherein testing of the modular composite sample, using a suitable detection assay further comprises a concentration step to concentrate the microbial agent or organism.

6. The method of claim 1, wherein testing in e) of the modular composite sample is positive, and wherein the individual separate test lot samples are further tested, such further testing comprises the more sensitive protocol.

7. The method of claim 1, further comprising, forming an additional modular composite sample from the set of enriched separate test lot samples of c) and testing the additional modular composite sample, using the same or a different suitable assay, to confirm the testing result.

8. The method of claim 1, further comprising, forming an additional modular composite sample from an additional enriched separate test lot sample along with any enriched separate test lot samples of c) that test negative in e), and testing the additional modular composite sample, using the same or a different suitable assay.

9. The method of claim 1, further comprising, where testing of the modular composite sample in e) is negative, forming an additional modular composite sample from the set of enriched separate test lot samples of c), and testing the additional modular composite sample, using a different suitable assay, to confirm the negative testing status.

10. The method of claim 1, wherein the test lot is selected from the lot group consisting of a single combo, single bin, single pallet, and a production assemblage.

11. The method of claim 1, wherein the number of separate test lots is from 3 to 10.

12. The method of claim 1, further comprising, where testing of the modular composite sample is positive, determining which individual separate test lots are positive, application of remedial measures to said separate positive test lots, and validating by further testing of a portion of the respective remediated separate test lots and obtaining a negative test result.

13. The method of claim 1, wherein, in a), separately collecting said plurality of portions from each of said plurality of separate test lots comprises separately collecting from each separate test lot a number of portions from 5 to 100.

14. The method of claim 1, wherein, in a), separately collecting said plurality of portions from each of said plurality of separate test lots comprises separately collecting from each separate test lot 30 to 60 portions.

15. The method of claim 1, wherein, in a), separately collecting said plurality of portions from each of said plurality of separate test lots comprises separately collecting 30 portions from each separate test lot.

16. The method of claim 1, wherein, in a), separately collecting said plurality of portions from each of said plurality of separate test lots comprises separately collecting from each separate test lot about 60 portions.

18. The method of claim 1, wherein removing portions in step d) is removing equal portions, aseptically, so as to preclude contamination of the enriched separate test lot samples.

19. The method of claim 1, wherein the target microbial agent or organism comprises at least one selected from the group consisting of Listeria, Salmonella and Escherichia coli.

20. The method of claim 1, wherein the target microbial agent or organism comprises at least one selected from the group consisting of Listeria spp., Listeria monocytogenes, Salmonella spp., Escherichia coli O157:

H7, coliforms and fecal coliforms.

21. The method of claim 1, wherein the target microbial agent or organism comprises a spoilage organism.

22. The method of claim 1, wherein removing portions of each enriched separate test lot sample comprises removing equal volume portions of each enriched separate test lot sample.

23. The method of claim 1, wherein testing of the modular composite sample, using a suitable detection assay comprises use of at least one of a DNA-based method, an immunochemistry-based method, and a biosensor-based method.

24. The method of claim 1, wherein the separate test lots are production lots of a meat or poultry product, comprising an assemblage of one or more meat or poultry specimens, respectively.

25. The method of claim 1, wherein the separate test lots are separate production lots of a fish or seafood product, comprising an assemblage of one or more fish or seafood specimens, respectively.

26. The method of claim 1, wherein the separate test lots are separate production lots of a ready-to-eat product.

27. The method of claim 2, wherein product specimens are synonymous with product portions.

28. The method of claim 4, wherein the medium comprises a selective agent that will inhibit a competing non-target microbial agent or organism.

29. The method of claim 5 wherein the concentration step comprises at least one of immunomagnetic bead separation, and affinity chromatography.

30. The method of claim 6, wherein the more sensitive protocol further comprises a concentration step to concentrate the microbial agent or organism, in combination with the same test protocol that is used to test the modular composite sample.

32. The method of claim 24, wherein the meat or poultry specimens are meat trim pieces or poultry trim pieces, respectively.

33. The method of claim 24, wherein the meat is that of beef, pork, sheep or bison.

17. A method of sampling, testing and validating test lots for microbial contamination, comprising:

a) collecting a plurality of portions from each of a plurality of single-unit production lots, the single-unit production lots each comprising an assemblage of one or more production specimens, wherein each single-unit production lot is separately sampled by taking said plurality of portions thereof;

b) combining the collected plurality of product portions corresponding to each of the separate single-unit production lots to provide a corresponding set of separate single-unit production lot samples, wherein each separate single-unit production lot sample is attributed to a particular corresponding separate single-unit production lot;

c) incubating the set of separate single-unit production lot samples under conditions suitable to allow levels of a target microbial agent or organism that is present in one or more of the separate single-unit production lot samples to reach detectable levels and become uniform, or substantially uniform, throughout the respective one or more separate single-unit production lot samples, to provide a set of separate single-unit production lot samples enriched for the target microbial agent or organism;

d) removing portions of each enriched separate single-unit production lot sample, and combining the removed portions to provide a modular composite sample; and

e) testing of the composite sample, using a suitable detection assay, for the target microbial agent or organism, wherein when such testing is negative all of said separate single-unit production lot samples are validated, and wherein when such testing is positive, individual separate single-unit production lots are validated by further testing of a portion of the respective enriched separate single-unit production lot sample using the same or a more sensitive protocol and obtaining test result for each single-unit production lot.

31. The method of claim 17, wherein removing portions in step d) is removing equal portions, aseptically, so as to preclude contamination of the enriched separate single-unit production lot samples.