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Detection of 5 CFU/g of Escherichia coli O157:H7 on lettuce using activated charcoal and real-time PCR without enrichment.
Food Microbiol. 2011 May; 28(3):562-7.FM

Abstract

A sample treatment method which separates Escherichia coli O157:H7 from lettuce and removes PCR inhibitors allowing 5 CFU/g of target cells to be detected using real-time PCR is described. Lettuce leaves inoculated with E. coli O157:H7 were rinsed with 0.025% sodium dodecyl sulfate (SDS). In this study, there were two major factors that strongly affected the recovery of E. coli O157:H7 during sample preparation, the amount of bentonite coated activated charcoal used to remove PCR inhibitors and the agitated contact time of the samples with the coated charcoal. When 3.0 g of activated carbon coated with bentonite were mixed with target cell suspensions (30 ml) derived from 50 g of lettuce, a high recovery of E. coli O157:H7 (93%) was obtained. Sample agitation with bentonite coated activated charcoal for 15 min resulted in 95% recovery of E. coli O157:H7. When a commercial DNA purification resin was used for detection of E. coli O157:H7 without the use of the bentonite treated charcoal, the real-time PCR (Rti-PCR) failed to detect 1 × 10(2) CFU/g. In contrast, with the use of use of bentonite coated activated charcoal and a commercial DNA purifying resin together, Rti-PCR was able to detect 5 CFU of E. coli O157:H7/g of lettuce which was equivalent to 2.8 CFU/Rti-PCR. Such a successful detection level was the result of the bentonite coated activated charcoal's ability to absorb the PCR inhibitors released from seeded lettuce during detachment. A standard curve was generated by plotting the Ct values against the log of CFU of target bacterial cells. A linear range of DNA amplification was exhibited from 5.0 × 10(0) to 1.0 × 10(4) CFU/g by using Rti-PCR.

Authors+Show Affiliations

Department of Human Ecology, College of Agriculture & Related Sciences, Delaware State University, Dover, DE 19901, USA.No affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

21356465

Citation

Lee, Jung-Lim, and Robert E. Levin. "Detection of 5 CFU/g of Escherichia Coli O157:H7 On Lettuce Using Activated Charcoal and Real-time PCR Without Enrichment." Food Microbiology, vol. 28, no. 3, 2011, pp. 562-7.
Lee JL, Levin RE. Detection of 5 CFU/g of Escherichia coli O157:H7 on lettuce using activated charcoal and real-time PCR without enrichment. Food Microbiol. 2011;28(3):562-7.
Lee, J. L., & Levin, R. E. (2011). Detection of 5 CFU/g of Escherichia coli O157:H7 on lettuce using activated charcoal and real-time PCR without enrichment. Food Microbiology, 28(3), 562-7. https://doi.org/10.1016/j.fm.2010.11.007
Lee JL, Levin RE. Detection of 5 CFU/g of Escherichia Coli O157:H7 On Lettuce Using Activated Charcoal and Real-time PCR Without Enrichment. Food Microbiol. 2011;28(3):562-7. PubMed PMID: 21356465.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Detection of 5 CFU/g of Escherichia coli O157:H7 on lettuce using activated charcoal and real-time PCR without enrichment. AU - Lee,Jung-Lim, AU - Levin,Robert E, Y1 - 2010/11/22/ PY - 2010/07/19/received PY - 2010/11/09/revised PY - 2010/11/12/accepted PY - 2011/3/2/entrez PY - 2011/3/2/pubmed PY - 2011/4/13/medline SP - 562 EP - 7 JF - Food microbiology JO - Food Microbiol VL - 28 IS - 3 N2 - A sample treatment method which separates Escherichia coli O157:H7 from lettuce and removes PCR inhibitors allowing 5 CFU/g of target cells to be detected using real-time PCR is described. Lettuce leaves inoculated with E. coli O157:H7 were rinsed with 0.025% sodium dodecyl sulfate (SDS). In this study, there were two major factors that strongly affected the recovery of E. coli O157:H7 during sample preparation, the amount of bentonite coated activated charcoal used to remove PCR inhibitors and the agitated contact time of the samples with the coated charcoal. When 3.0 g of activated carbon coated with bentonite were mixed with target cell suspensions (30 ml) derived from 50 g of lettuce, a high recovery of E. coli O157:H7 (93%) was obtained. Sample agitation with bentonite coated activated charcoal for 15 min resulted in 95% recovery of E. coli O157:H7. When a commercial DNA purification resin was used for detection of E. coli O157:H7 without the use of the bentonite treated charcoal, the real-time PCR (Rti-PCR) failed to detect 1 × 10(2) CFU/g. In contrast, with the use of use of bentonite coated activated charcoal and a commercial DNA purifying resin together, Rti-PCR was able to detect 5 CFU of E. coli O157:H7/g of lettuce which was equivalent to 2.8 CFU/Rti-PCR. Such a successful detection level was the result of the bentonite coated activated charcoal's ability to absorb the PCR inhibitors released from seeded lettuce during detachment. A standard curve was generated by plotting the Ct values against the log of CFU of target bacterial cells. A linear range of DNA amplification was exhibited from 5.0 × 10(0) to 1.0 × 10(4) CFU/g by using Rti-PCR. SN - 1095-9998 UR - https://www.unboundmedicine.com/medline/citation/21356465/Detection_of_5_CFU/g_of_Escherichia_coli_O157:H7_on_lettuce_using_activated_charcoal_and_real_time_PCR_without_enrichment_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0740-0020(10)00289-3 DB - PRIME DP - Unbound Medicine ER -