A Pentaplex PCR Assay for the Detection and Differentiation ofShigellaSpecies

Abstract

The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis ofShigellaspp. is laborious and time consuming and has low sensitivity. Hence, in the present study, a molecular-based diagnostic assay which amplifies simultaneously four specific genes to identifyinvCforShigellagenus,rfcforS. flexneri,wbgZforS. sonnei, andrfpBforS. dysenteriae, as well as one internal control (ompA) gene, was developed in a single reaction to detect and differentiateShigellaspp. Validation with 120Shigellastrains and 37 non-Shigellastrains yielded 100% specificity. The sensitivity of the PCR was 100 pg of genomic DNA, 5.4 × 104 CFU/ml, or approximately 120 CFU per reaction mixture of bacteria. The sensitivity of the pentaplex PCR assay was further improved following preincubation of the stool samples in Gram-negative broth. A preliminary study with 30 diarrhoeal specimens resulted in no cross-reaction with other non-Shigellastrains tested. We conclude that the developed pentaplex PCR assay is robust and can provide information about the four target genes that are essential for the identification of theShigellagenus and the threeShigellaspecies responsible for the majority of shigellosis cases.