| Summary: | 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 <i>Salmonella</i> serovar strains, and the amplified products were sequenced. The prevalent <i>Salmonella enterica</i> subspecies enterica serovars, including Typhimurium; Enteritidis, Agona, enterica, Typhi, and Abony, were amplified and sequenced. The amplified sequences of six <i>Salmonella</i> 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 (<i>SBG</i>, <i>dedA</i>, <i>yacG</i>, <i>mrcB</i>, <i>mesJ</i>, <i>metN</i>, <i>rihA/B</i>, <i>modA</i>, <i>hutG</i>, <i>yehX</i>, <i>ybiY</i>, <i>moeB</i>, and <i>sopA</i>), were developed for PCR confirmation of target <i>Salmonella</i> serovar strains. Finally, the SNPs in four genes, including <i>fliA</i> gene (<i>S.</i> Enteritidis), <i>modA</i> (<i>S</i>. Agona and <i>S</i>. <i>enterica</i>), <i>sopA</i> (<i>S.</i> Abony), and <i>mrcB</i> (<i>S.</i> Typhimurium and <i>S</i>. Typhi), were used for detection markers of six target <i>Salmonella</i> serotypes. We developed an m-PCR primer set in which <i>Salmonella</i> serovars were detected in a single reaction. Nevertheless, m-PCR was validated with 21 <i>Salmonella</i> isolates (at least one isolate was taken from one positive animal fecal, and <i>n</i> = 6 reference <i>Salmonella</i> strains) and non-<i>Salmonella</i> bacteria isolates. The SNP-based m-PCR method would identify prevalent <i>Salmonella</i> serotypes, minimize the infection, and control outbreaks.
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