title: A pentaplex PCR assay for the detection and differentiation of Shigella species.
abstract: The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis of Shigella spp. 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 identify invC for Shigella genus, rfc for S. flexneri, wbgZ for S. sonnei, and rfpB for S. dysenteriae, as well as one internal control (ompA) gene, was developed in a single reaction to detect and differentiate Shigella spp. Validation with 120 Shigella strains and 37 non-Shigella strains yielded 100% specificity. The sensitivity of the PCR was 100 pg of genomic DNA, 5.4 × 10(4) 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-Shigella strains 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 the Shigella genus and the three Shigella species responsible for the majority of shigellosis cases.
authors.affiliation: Department of Medical Microbiology & Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia.
authors.forename: Suvash Chandra
authors.initials: SC
authors.lastname: Ojha
authors.forename: Chan
authors.initials: C
authors.lastname: Yean Yean
authors.forename: Asma
authors.initials: A
authors.lastname: Ismail
authors.forename: Kirnpal-Kaur Banga
authors.initials: KK
authors.lastname: Singh
mesh_terms: D001431:Bacteriological Techniques; D004269:DNA, Bacterial / Q000032:analysis; D003967:Diarrhea / Q000382:microbiology; D005243:Feces / Q000382:microbiology; D006090:Gram-Negative Bacteria / Q000302:isolation & purification; D006801:Humans; D008296:Malaysia; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012760:Shigella / Q000302:isolation & purification*; D013045:Species Specificity; D013234:Stem Cells
chemical_list: D004269:DNA, Bacterial