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

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.