Sensitive and rapid detection of Vero toxin-producing Escherichia coli using loop-mediated isothermal amplification

Abstract

A loop-mediated isothermal amplification (LAMP) assay was developed to detect Vero toxin (VT)-producing Escherichia coli rapidly (within 60 min). The 24 strains of VT-producing E. coli were successfully amplified, but 6 strains of non-VT-producing E. coli and 46 bacterial species other than E. coli were not. The sensitivity of the LAMP assay was found to be >0.7 c.f.u. per test using serogroups O157, O26 and O111 of VT-producing E. coli; this sensitivity is greater than that obtained by PCR assay. Furthermore, the LAMP assay was examined for its ability to detect VT-producing E. coli in food because of the difficulty of detection in food samples. The recovery of VT-producing E. coli by LAMP assay from beef and radish sprouts inoculated with the pathogen was high, similar to that obtained using culture methods with direct plating and/or plating after immunomagnetic separation. Although PCR assay was unable to recover VT-producing E. coli from half of the radish samples, LAMP assay was successful in most samples. In addition, VT-producing E. coli was successfully detected in cultures of the beef samples by LAMP assay, but not by the culture method. The LAMP products in naturally contaminated beef samples were analysed to confirm the specific amplification of the VT-encoding gene, and were found to show a specific ladder band pattern on agarose gel after electrophoresis. Additionally the sequences of the LAMP products coincided well with the expected sequences of the VT-encoding gene. These results indicate that the proposed LAMP assay is a rapid, specific and sensitive method of detecting the VT-producing E. coli.

Loop-Mediated Isothermal Amplification Assays for Detecting Shiga Toxin-Producing Escherichia coli in Ground Beef and Human Stools

“…In this study, sequence alignments of several Stx2 and intimin variants were conducted before suitable regions were chosen for LAMP primer design. Consequently, all of the three LAMP assays possessed 100% inclusivity and 100% exclusivity among the 50 STEC and 40 non-STEC strains tested, a specificity similar to that reported previously for LAMP assays targeting stx 1 and stx 2 (20). Noticeably, eae-LAMP showed inferior sensitivity in detecting two strains (STEC O26 97-3250 and a None of the 4-h-enrichment samples tested positive by either LAMP or qPCR.…”

“…With 35 min to 1 h of reaction time, these LAMP assays were capable of detecting between 0.7 and 100 CFU of E. coli per reaction, 10-to 100-fold more sensitive than conventional PCR (20,22,28,37,40,44,47). The three LAMP assays developed here fell within these detection ranges in terms of speed and sensitivity.…”

“…For instance, a recent study reported that 45 to 50% of liver samples inoculated with 1 to 4 CFU/25 g of E. coli O157:H7 strains tested positive by LAMP after overnight enrichment, compared to a 10 to 35% detection rate by culture (34). Two earlier studies by the same group reported that for ground beef samples inoculated with approximately 10 CFU/25 g of E. coli O157 or O26 strains, 100% detection rates were observed after 24 h enrichment (19,20), whereas culture methods detected 100% of ground beef samples spiked with STEC O157 but only 50 to 80% of those spiked with STEC O26 (19). In raw milk, a detection limit of 4.1 ϫ 10 4 CFU/ml of E. coli O157 was reported (40).…”

“…Previously, LAMP assays have been developed for the detection of generic (22) and pathogenic E. coli, including enteroaggregative E. coli (44), enteroinvasive E. coli (37), enterotoxigenic E. coli (43), STEC (20,28,29), and more specifically, E. coli O157:H7 (40,46,47). With 35 min to 1 h of reaction time, these LAMP assays were capable of detecting between 0.7 and 100 CFU of E. coli per reaction, 10-to 100-fold more sensitive than conventional PCR (20,22,28,37,40,44,47).…”

Sensitive and Rapid Detection of Cholera Toxin-Producing Vibrio cholerae Using Loop-Mediated Isothermal Amplification

“…Also, the increase in the turbidity of the reaction mixture according to the production of precipitate correlates with the amount of DNA synthesized [10,11]. Various LAMP assays for the identification of pathogenic organisms have been developed [10][11][12][13]15,16], however, no assay for the detection of CT-producing V. cholerae has been described.…”