Development of a Duplex TaqMan Real-Time Polymerase Chain Reaction for Accurate Identification and Quantification of Salmonella Enteritidis from Laboratory Samples and Contaminated Chicken Eggs.

Salmonella   enteritidis is a major causative agent of foodborne illnesses worldwide. As the traditional serotyping and quantification methods are labor-intensive, time-consuming, and expensive, faster and more convenient molecular diagnostic methods are needed. In this study, we developed and validated a rapid duplex TaqMan real-time polymerase chain reaction (PCR) for the accurate identification and quantification of S. enteritidis . The primers and TaqMan probes were designed based on the S. enteritidis -specific gene lygD and the Salmonella genus-specific gene invA . The melt curve and gel electrophoresis analysis showed that the designed primers had potent specificity for the amplification of lygD and invA . The duplex real-time PCR specifically identified S. enteritidis from a panel of 40 Salmonella strains that represented 29 serovars and 12 non- Salmonella organisms. The duplex real-time PCR assay detected four copies of S. enteritidis DNA per reaction. The intra- and inter- assays indicated a high degree of reproducibility. The real-time PCR could accurately detect and quantify S. enteritidis in chicken organs after Salmonella infection. Furthermore, the assay identified 100% of the S. enteritidis and Salmonella genus isolates from chicken egg samples with superior sensitivity after 6 h of pre-enrichment compared to the traditional culture method. Additionally, the most-probable-number (MPN) combined with qPCR and a shortened incubation time (MPN-qPCR-SIT) method was developed for the population determination of S. enteritidis and compared with various enumeration methods. Thus, we have established and validated a new duplex real-time PCR assay and MPN-qPCR-SIT method for the accurate detection and quantification of S. enteritidis , which could contribute to meeting the need for fast detection and identification in prevention and control measures for food safety.