Construction of a Chimeric Plasmid Vector and its Investigation for Usage as an Internal Control for Detection of Shiga Toxin-Producing Escherichia coli by Polymerase Chain Reaction and Real-Time PCR

Background: Shiga-toxigenic Escherichia coli (STEC) is an important foodborne pathogen and considered a worldwide problem. The infection can cause severe illnesses, including hemolytic uremic syndrome. Traditional methods for STEC detection are currently not done in clinical laboratories due to the cost and related long turnaround time, which necessitates the development of quick and reliable alternative detection methods. Objectives: In this study, the application of PCR and real-time PCR methods for rapid detection of STEC in DNA extracts of stool samples was investigated. Methods: A synthetic control DNA vector, containing target genes of stx1, stx2, EhxA, and eae, was designed to evaluate the validity of these methods. Moreover, efficiency of primers targeting the genes, limit of detection, repeatability, sensitivity, and specificity of real-time PCR assay was compared with PCR results. Results: Repeatability of real-time PCR assay showed percent coefficient of variation of 3.3% - 16.1% and sensitivity of 66% to 100%. Efficiency of all the primers was 100% with a detection limit of 103 copies of DNA per reaction for both of the two assays. Comparison of the results showed that real-time PCR was more sensitive than conventional PCR. The chimeric vector interfered with template DNA when it was used as an internal control in PCR and real-time PCR assays; however, its application as external control was approved for detection of all targeted genes in STEC. Conclusions: These results confirmed application of the chimeric vector as an external control in detection of STEC by both PCR and real-time PCR assays, but its usage as an internal control was not approved in this study.