Design of an improved multiplex PCR method for diagnosis of enterohaemoraghic E.coli and enteropathogic E.coli pathotypes

Abstract

Aim: We aimed to develop a multiplex PCR assay for specific detection of EPEC and EHEC pathotypes based on specific marker genes.

Background: About 2.5 million infant's morbidity in developing countries occurs by E.coli pathotypes because of diarrhea and intestinal diseases. The traditional phenotypic methods are time consuming and sometimes detection and differentiation of the pathotypes are not done easily. Multiplex PCR technology is used as a sensitive, specific and rapid molecular method for detection of various pathogens.

Patients and methods: PCR reactions were performed with primers which targeted the virulence genes selected for each category (stx 1 , stx 2 genes for EHEC and bfpA for EPEC). For preparation of a positive control, the PCR products were cloned in pTZ57R/T plasmid. The same PCR reactions were done but in presence of genomes of various negative control bacteria for evaluation of test specificity.

Results: As expected, gel agarose electrophoresis of PCR products of the stx 1 , stx 2 and bfpA, showed 329bp, 586bp and459bp bands respectively. Result of amplification using negative control genomes as template was negative.

Conclusion: The multiplex PCR assay followed by capillary electrophoresis presented in the present paper provides a simple, reliable, and rapid procedure that in a single reaction identifies the four main pathotypes of E. coli. This assay will replace the previous molecular genetics methods used in our laboratory and work as an important supplement to the more time consuming phenotypic assays.

Keywords: Diarrheagenic; Escherichia coli; Multiplex PCR assay.

Figure 1

Both uniplex and multiplex PCR amplification of stx ₁ (329bp), stx ₂ (586bp) and bfpA (459bp) genes