Scientific Publications by FDA Staff

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.