沙门菌属特异性检测靶点碱基差异位点的识别及其血清型决定力

摘要

以美国国家生物技术信息中心(NCBI)已公布的28株沙门菌(14个血清型)全基因组序列为研究对象,对前期研究获得的7个沙门菌属特异性检测靶点在不同血清型间的单核苷酸多态性进行比较分析,结果表明,S9和S69为碱基差异位点最多的两个靶点,具有沙门菌分子血清分型的潜在能力.随后,通过分析S9和S69在沙门菌不同血清型菌株中的差异位点,分别绘制两个血清分型靶点的差异位点表,从而获得了这两个靶点同时用于14个沙门菌血清型的快速分子血清分型的差异位点组合.在这些组合中,同一血清型具有相同的差异位点,不同血清型可以通过差异位点得以区分.最后,采集食品样品192份,用国标方法获得疑似沙门菌21株;利用血清分子分型靶点S9和S69进行快速分型,并同时用传统玻片凝集反应鉴定方法进行验证,两种方法鉴定结果的符合率为100%.鉴定结果为:21株沙门菌共包括4个不同血清型,其中肠炎沙门菌10株、鼠伤寒沙门菌7株、鸡沙门菌2株、纽波特沙门菌2株.基因组序列分析结果和分离株分型结果均表明,沙门菌特异分子检测靶点S9和S69相结合具备沙门菌的分子血清分型能力,以差异位点表为基础建立的血清分型方法有望替代传统的玻片凝集血清分型这一繁杂步骤,从而降低沙门菌血清鉴定的时间与成本,为聚合酶链式反应技术应用于沙门菌分子血清分型提供新思路.%The single nucleotide polymorphisms(SNPs)in different serotypes of 7 specific targets for Salmonella detection were analyzed based on the whole genome sequences of 28 Salmonella strains(14 serotypes)published on NCBI.The results showed that S9 and S69 had more SNPs than any other target, making them a potential tool for Salmonella serotyping. Subsequently, the distinct nucleotide mutation sites of the two candidate molecular serotyping targets in different serotypes of Salmonella were analyzed.As a result,various combinations of the mutation sites were obtained for rapid molecular serotyping of 14 Salmonella serotypes, and the results showed that identical mutation sites existed in the same serovar, while different mutation sites were found in diverse serovars.Finally,21 suspected Salmonella isolates were obtained by the national standard method from 192 food samples collected from supermarkets and open-air markets in Shanghai. The results of molecular serotyping by targets S9 and S69 were 100% consistent with the results of the traditional slide agglutination test; the Salmonella isolates were assigned to 4 different serovars, 10 strains of Salmonella Enteritidis, 7 strains of Salmonella Typhimurium,2 strains of Salmonella Gallinarum and 2 strains of Salmonella Newport.The results of genome sequence analysis and strain serotyping showed that the combination of Salmonella-specific targets S9 and S69 had the potential for molecular serotyping of Salmonella,which is expected to be a time-saving and cheaper alternative to the traditional slide agglutination test and can provide a new idea for the application of PCR technology in Salmonella molecular serotyping.