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.

一种准确的诊断方法 沙门氏菌血清型是预防相关疾病传播的基础。基于诊断性聚合酶链反应 (PCR) 的方法已被证明是检测病原菌的有效工具。然而，目前用于实时 PCR 检测的基因标记沙门氏菌血清型特异性低，仅针对少数血清型开发。因此，在本研究中，我们探索了 60 个具有相似抗原分子式并使用泛基因组分析显示出高流行率的血清型的新型独特基因标记，并开发了实时 PCR 来检测它们。在探索基因标记之前，535沙门氏菌基因组进行了评估，一些基因组的血清型与指定的血清型信息不同。基于这些分析，探索了血清型特异性基因标记。这些标记被鉴定为存在于目标血清型基因组的所有菌株中但在其他血清型基因组的菌株中不存在的基因。血清型特异性引物对是根据基因标记设计的，实时 PCR 方法可以区分 60 个最常见的沙门氏菌开发了单个 96 孔板测定中的血清型。结果，实时 PCR 对 199沙门氏菌 和 29 个非沙门氏菌菌株。随后，所开发的方法成功应用于具有已鉴定血清型的菌株和未知菌株，表明与传统的血清分型方法（如抗血清凝集法）相比，实时 PCR 可以准确检测菌株的血清型。因此，我们的方法可以快速且经济地沙门氏菌 血清分型与传统的血清分型方法相比。