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

Abstract

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.

Prevalence of Salmonella spp. and Escherichia coli in the feces of free-roaming wildlife throughout South Korea

“…Higher bootstrap values seem to be more accurate in the phylogenetic tree obtained using MEGA software. The partial mrcB sequences of ten haplotypes found in South Korean Salmonella isolates (n = 21 Salmonella strains) are marked by a star in Fig 1 , which can be accessed via GenBank with accession numbers OM793284–OM793304 [ 67 ]. The MJN indicated the haplotypes (n = 21, denoted as Hap-1–Hap-21) in animal hosts ( Fig 5A ) and places ( Fig 5B ) in the respective MJN clade, and phylogenetic analysis and gene sequences were collected from South Korean wild animals (n = 21), NCBI acquired (n = 27), and reference Salmonella (n = 6).…”

“…E . coli O157:H7 was identified using a single nucleotide polymorphism (SNP)-based marker [ 67 ] The STEC-positive colonies were amplified and sequenced with the developed SNP-encompassing-based forward primer ( ) and reverse primer ( ) according to our previous study [ 68 ]. The following sequence accession numbers were submitted to GenBank (ON205852–ON205944), and detailed sample information is provided in S3 Table .…”

“…All Salmonella sequences were compared with those in the NCBI BLAST database (maximum score, coverage, identity, and E-value; checked with the naked eye). The Salmonella serovar was considered to align the reference serovar sequences (six reference sequences) with the topmost matched Salmonella strains (highest similarity, coverage, and E-value) and SNP-based multiplex marker [ 67 ].…”

“…The STEC colonies on CT-SMAC agar were molecularly identified by PCR amplification of Shiga toxin genes (stx1 or stx2) using the stx1 or stx2 primer set [66]. E. coli O157:H7 was identified using a single nucleotide polymorphism (SNP)-based marker [67] The STEC-positive colonies were amplified and sequenced with the developed SNP-encompassing-based forward primer (TGACTCGCCGTATGTGCC) and reverse primer (TGCCCGGCAGATAAGTGC) according to our previous study [68]. The following sequence accession numbers were submitted to GenBank (ON205852-ON205944), and detailed sample information is provided in S3 Table . Salmonella colonies on the SS, HE, and XLT4 plates were identified by amplifying the invA or iroB genes, which are shared by all Salmonella species, using PCR with the invA or iroB primer sets [69,70].…”

Development of Molecular-Based Species Identification and Optimization of Reaction Conditions for Molecular Diagnosis of Three Major Asian Planthoppers (Hemiptera: Delphacidae)

“…Multiplex PCR has many applications, including insect diagnosis [ 2 ], pathogen detection [ 51 ], and forensic testing [ 52 ]. Its simplicity of use and low setup cost has opened up new opportunities for its development.…”