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Appl Environ Microbiol 2014 Apr;80(8):2516-25

Evaluation of a Loop-Mediated Isothermal Amplification Suite for the Rapid, Reliable, and Robust Detection of Shiga Toxin-Producing Escherichia coli in Produce.

Wang F, Yang Q, Qu Y, Meng J, Ge B


Shiga toxin-producing Escherichia coli (STEC) strains are a leading cause of produce-associated outbreaks in the United States. Rapid, reliable, and robust detection methods are needed to better ensure produce safety. We recently developed a loop-mediated isothermal amplification (LAMP) suite for STEC detection. In this study, the STEC LAMP suite was comprehensively evaluated against real-time quantitative PCR (qPCR) using a large panel of bacterial strains (n = 156) and in various produce items (several varieties of lettuce, spinach, and sprouts). To simulate real-world contamination events, produce samples were surface-inoculated with a low level (1.2 to 1.8 CFU/25 g) of individual STEC strains belonging to seven serogroups (O26, O45, O103, O111, O121, O145, and O157) and held at 4 degrees C for 48 h before testing. Six DNA extraction methods were also compared using produce enrichment broths. All STEC targets and their subtypes were accurately detected by the LAMP suite. The detection limits were 1 to 20 cells per reaction in pure culture and 105 to 106 CFU per 25 g (i.e., 103 to 104 CFU per g) in produce, except for strains harboring stx2c, eae-beta, and eae-theta subtypes. After 6 to 8 h of enrichment, the LAMP suite achieved accurate detection of low-level STEC strains with various stx2 and eae subtypes in lettuce and spinach varieties but not in sprouts. A similar trend of detection was observed for qPCR. PrepMan Ultra sample preparation reagent yielded the best results among the six DNA extraction methods. This research provided a rapid, reliable, and robust method for detecting STEC in produce during routine sampling and testing. The challenge with sprouts detection by both LAMP and qPCR calls for special attention to further analysis.

Category: Journal Article
PubMed ID: #24509927 DOI: 10.1128/AEM.04203-13
Includes FDA Authors from Scientific Area(s): Animal and Veterinary
Entry Created: 2014-02-11 Entry Last Modified: 2014-05-03