Abstract：Objective To establish a rapid GeXP-based multiplex reverse transcription-PCR assay (GeXP assay) for simultaneous detection of 5 subtypes of diarrheogenic Escherichia coli. Methods Specific primers were designed according to reserved sequences of 12 virulence genes in enterotoxigenic E. coli (ETEC), enterinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (ETEC), and PCR amplification was performed with a single pair of primers to validate the specificity of PCR assay with a single template and a single pair of primers. The specificity of the GeXP assay was evaluated with the genomic DNA of 5 subtypes of diarrheogenic E. coli as the template in a mixture of 12 pairs of primers, and the sensitivity of the GeXP assay was evaluated with the mixed suspensions of 5 subtypes of diarrheogenic E. coli at concentrations of 106, 105, 104 and 103 CFU/mL as the template. Foods purchased from supermarkets and agricultural retail markets were prepared into 34 spiked samples, and 5 subtypes of diarrheogenic E. coli were detected using the GeXP assay and compared with the fluorescent real-time multiple PCR assay. Results The sizes of sth, pic, bfpB, astA, lt, escV, aggR, stx1, uidA, invE, stx2 and stp genes amplification products were consistent with expected sizes using a single template and a single pair of primers, with a fluorescent signal intensity of more than 25 000 A.U. The sizes of the GeXP assay amplification products of 12 virulence genes in 5 subtypes of diarrheogenic E. coli were consistent with expected sizes, with a high specificity. If the concentration of the mixed suspensions of 5 subtypes of diarrheogenic E. coli was 103 CFU/mL, the GeXP assay was effective for simultaneous detection of 12 virulence genes, with a high fluorescent signal intensity, consistent repeated detection results and a less than 10% coefficient of variation. The GeXP assay detected 3 ETEC isolates, 12 EAEC isolates, one EIEC isolate, one EPEC isolate and one EHEC isolate among the 34 spiked samples, which was in agreement with the detection of 5 subtypes of diarrheogenic E. coli with commercial fluorescent real-time multiple PCR assay kits. Conclusions A GeXP assay has been successfully established for simultaneous detection of 12 virulence genes in diarrheogenic E. coli, which is effective for clinical differential diagnosis and epidemiological surveys of diarrheogenic E. coli.
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