Multiplex polymerase chain reaction (PCR) for the detection of diarrheagenic Escherichia coli and Shigella directly from stool

Abstract

Shigella and diarrheagenic Escherichia coli (DEC) are the most common causes of diarrheal diseases in developing countries. A multiplex polymerase chain reaction (mPCR) was developed for identification of DEC infections caused by shiga toxin producing E. coli (STEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli(ETEC), enteroinvasive E. coli (EIEC) and Shigella in feacal samples by simultaneous and specific detection of seven virulence genes (eaeA, stx1, stx2, bfpA, LT, ST and ipaH). The detection limit of the method was 101 to 103 cfu/PCR assay from pure cultures. When the multiplex PCR was tested with DNA purified from artificially spiked stool samples directly from stool or after overnight pre-enrichment in buffered peptone water, the detection limit was106 cfu and 101 - 105 cfu/gm stool, respectively. The developed mPCR is specific, sensitive, easy, rapid and of low cost method for detecting DEC in stool. Key words: Diarrheagenic Escherichia coli, Shigella, shiga toxin producing E. coli(STEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterotoxigenicE. coli (ETEC), multiplex PCR.