沙门氏菌血清型快速PCR鉴定方法的建立

Development of Rapid PCR Determination Method of Salmonella Serovars

通过传统玻片凝集试验对78株沙门氏菌进行血清型检测,结果将这些沙门氏菌分成以鼠伤寒沙门氏菌和肠炎沙门氏菌为主的21种血清型,其中沙门氏菌SJTUF10023证实为自凝菌,无法鉴定血清型。利用MLST可以较精确地预测、区分不同血清型菌株,聚类的各个分支与血清型基本呈对应关系(96.2%,75/78),其中有3株沙门氏菌(1株阿贡纳、1株阿伯丁和1株斯坦利)聚类结果与血清型不一致。根据O抗原(rfb基因簇)、H1抗原(fli C基因)和H_2抗原(flj B基因)分别设计引物进行PCR反应,并与测序相结合,对78株沙门氏菌进行血清型测定,结果仅1株伤寒沙门氏菌的H_2抗原出现假阳性结果,准确度达98.7%(77/78),且编号为SJTUF10023的自凝菌被鉴定为鼠伤寒沙门氏菌,说明该方法能够准确、快速检测沙门氏菌血清型,有望在沙门氏菌血清型鉴定中成为替代传统血清分型的方法。

In this study,78 Salmonella strains were detected into 21 serovars by using traditional slide agglutination test,mainly representing S.Typhimurium and S.Enteritidis.A straincoded as SJTUF10023 was proved to be self-curing,which led to the failure of serovar deternination by slide agglutination test.MLST was used to predict different serovars,and it turned out that each clustering branch was substantially corresponded with one single serovar （96.2%,75/78）.However,three exceptions were found in one S.Agona,one S.Aberdeen and one S.Stanley.According to the molecular basis of serovars,O antigen （rfb gene cluster）,H1 antigen （fliC gene） and H2 antigen （filjB gene）,PCR reactions were designed and combined with DNA sequencing to serotyping the 78 Salmonella strains.The accuracy was as high as 98.7% （77/78） with the only one exception that happened to the false positive result of S.TyphiH2 antigen;moreover,the self-curing Salmonella SJTUF10023 was identified as S.Typhimurium.The results demonstrated that the PCR determination method established in this study can accurately and rapidly detect Salmonella serovars,which is promising to be an alternative to traditional serotyping methods of Salmonella.