Evaluation of 5' nuclease based assays for the detection of Escherichia coli O157:H7 and Salmonella typhimurium from food, cattle feces and enrichment broth systems.

摘要

Two 5′ nuclease based PCR assays, PCR-LS50B and PCR-7200 (Applied BioSystems, Foster City, CA), were evaluated for their sensitivity to detect E. coli O157:H7 from enrichment broth and meat samples. We found that the PCR-7200 detection system was superior to the PCR-LS-50B detection system. The EHEC enrichment broth (EEB) performed better than modified EC broth (mECB) as an enrichment medium for detection by the 5′ nuclease assay. A cultural, and an immunological assay were compared to the 5′ nuclease (PCR-7700) assay for detection of E. coli O157:H7 from meat samples enriched in mECB. The detection rates (at all spike levels) of the PCR, immunoassay, and USDA cultural methods were 79, 68, and 37%, respectively.; The ability of the 5′ nuclease assay, coupled with an immunomagnetic separation (IMS) step, to detect E. coli O157:H7 from cattle feces was evaluated. The IMS procedure alone detected 104 CFU/mL of enriched feces. When a small aliquot of the IMS sample was post-enriched in EEB for 18 h at 35°C, the detection capability of the IMS-PCR procedure was 102 CFU/mL. A total of 550 cattle feces samples were screened using the cultural and IMS-PCR methods. Excellent repeatability (kappa statistic = 1) was seen for the IMS-PCR method, but there was a poor repeatability for the cultural methods (kappa statistic = −0.74).; The 5′ nuclease based PCR assay detected S. typhimurium at levels of 102–3 CFU/ from pre-enrichment broths (non-selective) and meat samples. The method was less sensitive in detecting Salmonella from selective enrichment broths, especially tetrathionate (TT) broth. A 9 h enrichment time was necessary to detect S. typhimurium from various pre-enrichment broth samples and meat samples enriched in the buffered peptone water (BPW) by the 5′ nuclease method.; When compared to the cultural methods for the detection of E. coli O157:H7 and S. typhimurium, the 5′ nuclease assay is a sensitive and accurate detection method.