Application of a multiplex PCR assay for the detection of Shigella, Escherichia coli and Shiga toxin-producing Esch. coli in milk

A multiplex PCR (mPCR) assay using previously known genetic markers of Shigella, Escherichia coli and Shiga-toxic Esch. coli was standardized. uidA gene was targeted for the common detection of Esch. coli and Shigella, whereas ipaH and stx1 genes were used as markers for the detection of Shigella and shiga-toxin producing strains, respectively. The standardized assays detected the target organism specifically and selectively. The mPCR developed by combining all the three reactions generated specific products. The inclusivity and exclusivity tests depicted the precise specificity of the mPCR assay. Results were interpreted on the basis of the pattern of amplicons generated: amplifications of the ipaH and uidA gene fragments indicated the presence of Shigella spp., amplification of uidA alone revealed the presence of Esch. coli and additional presence of verotoxin gene amplicon indicated verotoxinogenic nature of the strain. Specific patterns of bands were obtained when different strains of Esch. coli and Shigella spp. were subjected to this assay. The reactions, individually as well as in the mPCR, could detect approximately 1 cell per 20-μl PCR assay. The protocols were validated by analyzing the coded samples of full fat milk spiked with different pathogens. In naturally contaminated raw milk samples (n=100), Esch. coli were detected in all samples and verotoxinogenic Esch. coli in 15 samples. Shigella, however, was not detected in any of the samples. When DNA purified from the samples found positive for Shiga-toxic Esch. coli was directly used as template for the mPCR, the results showed agreement with the enrichment based detection. The mPCR assay, standardized in this study, may be used for rapid microbiological evaluation of milk samples. Further, the study emphasizes the need for better hygienic conditions in dairies.