Real-Time PCR Method for Detection of Salmonella spp. in Environmental Samples.

The methods currently used for detecting Salmonella in environmental samples require 2 days to produce results and have limited sensitivity. Here, we describe the development and validation of a real-time PCR Salmonella screening method that produces results in 18 to 24 h. Primers and probes specific to the gene invA, group D, and Salmonella enterica serovar Enteritidis organisms were designed and evaluated for inclusivity and exclusivity using a panel of 329 Salmonella isolates representing 126 serovars and 22 non-Salmonella organisms. The invA- and group D-specific sets identified all the isolates accurately. The PCR method had 100% inclusivity and detected 1 to 2 copies of Salmonella DNA per reaction. Primers specific for Salmonella-differentiating fragment 1 (Sdf-1) in conjunction with the group D set had 100% inclusivity for 32 S Enteritidis isolates and 100% exclusivity for the 297 non-Enteritidis Salmonella isolates. Single-laboratory validation performed on 1,741 environmental samples demonstrated that the PCR method detected 55% more positives than the Vitek immunodiagnostic assay system (VIDAS) method. The PCR results correlated well with the culture results, and the method did not report any false-negative results. The receiver operating characteristic (ROC) analysis documented excellent agreement between the results from the culture and PCR methods (area under the curve, 0.90; 95% confidence interval of 0.76 to 1.0) confirming the validity of the PCR method.IMPORTANCE This validated PCR method detects 55% more positives for Salmonella in half the time required for the reference method, VIDAS. The validated PCR method will help to strengthen public health efforts through rapid screening of Salmonella spp. in environmental samples.

A real-time PCR for rapid identification of Salmonella enterica Gaminara serovar.

Salmonella is one of the leading causes of foodborne illnesses in the USA. When a Salmonella outbreak occurs, rapid identification of the causative serovar is important for tracing the source of contamination and for preventing the further spread of the illness. Each serovar is characterized by the presence of a group-specific somatic O-antigen(s) and an assortment of flagellar phase-1 and phase-2 antigens. As the traditional serotyping protocol is time consuming, labor intensive, and expensive, faster and less expensive molecular diagnostic methods are needed. This report outlines the development of a rapid multiplex real-time PCR procedure that facilitates the identification of Salmonella serogroup I and the serovars of the group. Using Salmonella Gaminara serovar (O16:d:1,7) as an example, first the gene(s) responsible for expression of the somatic O antigen, O16, and the nucleotide sequence of the variable-region of genes encoding the flagellar phase-1 (d) and phase-2 (1,7) antigens were identified. Then, a multiplex real-time PCR was designed that incorporated primers and probes specific for the three target genes and confirmed the specificity. The assay had 100% inclusivity for all three gene targets, detecting 2 genomic DNA copies of O16 and 1,7 gene targets and 10 copies of d gene target. Importance: Rapid molecular methods to identify Salmonella serovars should increase the precision of routine surveillance of clinically important serovars and promote public health.