Genome-Wide Searching Single Nucleotide-Polymorphisms (SNPs) and SNPs-Targeting a Multiplex Primer for Identification of Common Salmonella Serotypes.

A rapid and high-quality single-nucleotide polymorphisms (SNPs)-based method was developed to improve detection and reduce salmonellosis burden. In this study, whole-genome sequence (WGS) was used to investigate SNPs, the most common genetic marker for identifying bacteria. SNP-sites encompassing 15 sets of primers (666-863 bp) were selected and used to amplify the target Salmonella serovar strains, and the amplified products were sequenced. The prevalent Salmonella enterica subspecies enterica serovars, including Typhimurium; Enteritidis, Agona, enterica, Typhi, and Abony, were amplified and sequenced. The amplified sequences of six Salmonella serovars with 15 sets of SNP-sites encompassing primers were aligned, explored SNPs, and SNPs-carrying primers (23 sets) were designed to develop a multiplex PCR marker (m-PCR). Each primer exists in at least two SNPs bases at the 3' end of each primer, such as one was wild, and another was a mismatched base by transition or transversion mutation. Thus, twenty-three sets of SNP primers (242-670 bp), including 13 genes ( SBG , dedA , yacG , mrcB , mesJ , metN , rihA/B , modA , hutG , yehX , ybiY , moeB , and sopA ), were developed for PCR confirmation of target Salmonella serovar strains. Finally, the SNPs in four genes, including fliA gene ( S. Enteritidis), modA ( S . Agona and S . enterica ), sopA ( S. Abony), and mrcB ( S. Typhimurium and S . Typhi), were used for detection markers of six target Salmonella serotypes. We developed an m-PCR primer set in which Salmonella serovars were detected in a single reaction. Nevertheless, m-PCR was validated with 21 Salmonella isolates (at least one isolate was taken from one positive animal fecal, and n = 6 reference Salmonella strains) and non- Salmonella bacteria isolates. The SNP-based m-PCR method would identify prevalent Salmonella serotypes, minimize the infection, and control outbreaks.