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


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.


CONCLUSIONS
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.
Islamic Azad University, Qom Branch, Qom, Iran.
Mohammad
M
Soleimani
Islamic Azad University, Qom Branch, Qom, Iran.
Abbas
A
Morovvati
Islamic Azad University, Qom Branch, Qom, Iran.
Seyede Zohreh
SZ
Hosseini
Islamic Azad University, Qom Branch, Qom, Iran.
Mohammad Reza
MR
Zolfaghari
Diarrheagenic; Escherichia coli; Multiplex PCR assay