Application of real-time fluorescence quantitative polymerase chain reaction for rapid identification and detection of Salmonella Anatum

ObjectiveTo establish a quantitative real time PCR (qPCR) for rapid identification and detection of Salmonella Anatum to prevent bacterial serotype misdiagnosis and improve outbreak response.

MethodsA total of 60 strains of S. Anatum and 238 reference strains of common enteric pathogens were selected randomly. Primer sets targeting the specific gene AW58_15605 of S. Anatum were designed and a qPCR assay based on DNA molecules was established. The specificity, sensitivity and detection limit of the qPCR assay were evaluated by using pure strains, simulated stool samples and clinical stool samples from diarrheal patients.

ResultsThe qPCR amplification results indicated that the 60 S. Anatum strains and 50 clinical S. Anatum culture positive stool samples were all positive for S. Anatum specific gene AW58_15605, but negative for the specific gens of other Salmonella serotypes and enteric pathogens. The detection limit for purified S. Anatum DNA was 8 copies/reaction. After enrichment the detection limit of the qPCR assay significantly decreased to 59.10 cfu/g with extracted DNA as template for the simulated human stool samples, which was significantly lower than those of pre-enrichment and isolation method.

ConclusionIn this study, a qPCR assay based on a specific gene AW58_15605 was established to identify S. Anatum with high specificity and high sensitivity, and the assay can be used for rapid screening and identification of S. Anatum and diagnosis of related diarrheal diseases.