Abstract
An accurate diagnostic method for Salmonella serovars is fundamental to preventing the spread of associated diseases. A diagnostic polymerase chain reaction (PCR)-based method has proven to be an effective tool for detecting pathogenic bacteria. However, the gene markers currently used in real-time PCR to detect Salmonella serovars have low specificity and are developed for only a few serovars. Therefore, in this study, we explored the novel unique gene markers for 60 serovars that share similar antigenic formulas and show high prevalence using pangenome analysis and developed a real-time PCR to detect them. Before exploring gene markers, the 535 Salmonella genomes were evaluated, and some genomes had serovars different from the designated serovar information. Based on these analyses, serovar-specific gene markers were explored. These markers were identified as genes present in all strains of target serovar genomes but absent in strains of other serovar genomes. Serovar-specific primer pairs were designed from the gene markers, and a real-time PCR method that can distinguish between 60 of the most common Salmonella serovars in a single 96-well plate assay was developed. As a result, real-time PCR showed 100% specificity for 199 Salmonella and 29 non-Salmonella strains. Subsequently, the method developed was applied successfully to both strains with identified serovars and an unknown strain, demonstrating that real-time PCR can accurately detect serovars of strains compared with traditional serotyping methods, such as antisera agglutination. Therefore, our method enables rapid and economical Salmonella serotyping compared with the traditional serotyping method.
Highlights
The genus Salmonella, the causative agent of foodborne salmonellosis, can infect both animals and humans, leading to public health problems and economic loss (Gand M. et al, 2020a)
The purpose of this study is to evaluate Salmonella genomes by in silico serotyping, to select novel serovar-specific gene markers based on pangenome analysis, and to develop a real-time polymerase chain reaction (PCR) method that can distinguish between 60 of the most common Salmonella serovars in a single 96-well plate by detecting unique serovar-specific gene markers
SeqSero is a web-based serotyping tool that can predict many Salmonella serovars using whole-genome sequence data based on a database of Salmonella serovar determinants (Ibrahim and Morin, 2018)
Summary
The genus Salmonella, the causative agent of foodborne salmonellosis, can infect both animals and humans, leading to public health problems and economic loss (Gand M. et al, 2020a). Real-Time PCR for Salmonella Serotyping into more than 2,600 serovars following the White–Kauffman– Le Minor scheme, using antigenic agglutination reactions to three cell-surface antigens of somatic O, and flagellar H antigens denoted as H1 and H2 (Grimont and Weill, 2007; Yachison et al, 2017; Zhang et al, 2019; Gand M. et al, 2020a). As a reliable surveillance protocol is critical for detecting outbreaks or preventing their spread, using a differential serotyping method that identifies serogroups and serovars of Salmonella isolates from causative agents is important (Kasturi, 2020). PCR is reasonably rapid and inexpensive compared with conventional serotyping methods, the limiting factors of this assay are that molecular serotyping does not diagnose numerous serovars and focuses mainly on the most common serovars, such as Salmonella Typhimurium and Enteritidis (Ibrahim and Morin, 2018)
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