Development and Effective Utilization of a Rapid Multiplex Real-Time PCR of Diarrheagenic Escherichia coli for Food Poisoning Cases

Diarrheagenic (DEC) causes diarrheal symptoms in humans. The comprehensive detection of DEC from feces using SYBR Green real-time PCR assay requires multiple runs. Moreover, PCR screening can have discrepancies related to the conformance between the results from PCR screening and culturing. We aimed to develop a real-time PCR for the comprehensive testing of DEC for diagnostic support that can be used in any general laboratory and proposed its effective utilization. We tested specificity for the designed primer sets using 100 strains. Moreover, screening and isolation of DEC were performed using the proposed multiplex real-time PCR system for 308 fecal samples collected from 37 food poisoning incidences that occurred in Gifu Prefecture, Japan from 2017 to 2019. Furthermore, the factor of discrepant results between PCR screening and culturing was analyzed by quantifying the number of DEC cell and whole cell using real-time PCR for 47 PCR screening-positive fecal samples. The results obtained from the developed multiplex real-time PCR system were in 99% concordance with those from the conventional techniques. A total of 49 fecal samples were detected with virulence genes for the screening. Of the samples which were positive with virulence genes by PCR screening, 38.3% could not be detected from the strain for bacterial culture. We found that the culturing positive samples were significantly high in numbers for the DEC cells, but no significant difference was noted in the whole cells with culturing negative samples. The multiplex real-time PCR developed in this study was found to be rapid and practical for DEC testing. The PCR screening for DEC using this method can provide rapid information toward the diagnostic support of DEC infection.