title: Evaluation of a loop-mediated isothermal amplification suite for the rapid, reliable, and robust detection of Shiga toxin-producing Escherichia coli in produce.
abstract: 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 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°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 10(5) to 10(6) CFU per 25 g (i.e., 10(3) to 10(4) CFU per g) in produce, except for strains harboring the stx2c, eae-β, and eae-θ subtypes. After 6 to 8 h of enrichment, the LAMP suite achieved accurate detection of low levels of STEC strains of various stx2 and eae subtypes in lettuce and spinach varieties but not in sprouts. A similar trend of detection was observed for qPCR. The 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.
authors: Division of Animal and Food Microbiology, Office of Research, Center for Veterinary Medicine, U.S. Food and Drug Administration, Laurel, Maryland, USA.
authors: Fei
authors: F
authors: Wang
authors: Qianru
authors: Q
authors: Yang
authors: Yinzhi
authors: Y
authors: Qu
authors: Jianghong
authors: J
authors: Meng
authors: Beilei
authors: B
authors: Ge
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D005516:Food Microbiology / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D054323:Shiga-Toxigenic Escherichia coli / Q000302:isolation & purification*; D014675:Vegetables / Q000382:microbiology*