实时荧光PCR技术在沙门菌种间鉴别中的应用

Species differentiation and identification in Salmonella isolates by real-time PCR

目的探讨用实时荧光PCR对沙门菌进行种间鉴别。方法根据猪霍乱沙门菌(Salmonella Choleraesuis,SC)和丙型副伤寒沙门菌(Salmonella paratyphi C,SP)的共有序列(Gen Bank:AE017220.1),伤寒沙门菌(Salmonella Typhi,ST;Gen Bank:NC_016832.1)和鸡伤寒沙门菌(Salmonella Gallinarum,SG;Gen Bank:HQ703462)的特异序列分别设计SCP、STP、SGP引物和Taq Man探针,在探针的5'端分别标记FAM、HEX、TET,建立基于Taq Man探针多重荧光PCR检测方法。同时,根据SC和SP差异序列(Gen Bank:CP000857)设计SPP引物和探针,在探针的5'端标记ROX,区分SC和SP。结果 SCP特异性扩增出25株SC和22株SP,STP和SGP分别扩增出31株ST和34株SG,而其他不同血清型沙门菌和非沙门菌扩增结果阴性。SPP扩增出22株SP,25株SC扩增结果阴性。扩增体系评价结果,SCP、STP、SGP和SPP扩增效率均在80%~100%,线性相关系数r2≥0.994。结论建立的方法特异性强、敏感性高,可用于SC、SP、ST、SG的种间鉴别。

Objective The study makes a research on species differentiation and identification of Salmonella isolates useing real-time PCR. Methods To establish multiplex real-time PCR detection methods,the specific primer pairs SCP,STP,SGP and probes（ The 5＇-end of probes labeled with FAM,HEX and TET respectively） based on consensus sequence（ GenBank： AE017220. 1） of Salmonella Choleraesuis（ SC） and Salmonella paratyphi C（ SP）,specific sequence of Salmonella Typhi（ ST,Gen Bank： NC_016832. 1） and Salmonella Gallinarum（ SG,Gen Bank： HQ703462） were designed; according to the discrimination sequences of SC and SP（ Gen Bank： CP000857） to establish SPP primer and probe; the 5＇-end of probes was labeled with ROX to discriminate SC and SP. Results Test results showed SCP can amply 22 strains of SC and 25 strains of SP;STP and SGP amplied 31 strains of ST and 34 strains of SG respectively,and other serotype salmonella and non-salmonella were negative. SPP amplied 25 strains of SP and 22 strains of SC,which were both negative. The amplification efficiency of SCP,STP,SGP and SPP was within 80%-100%,and the linear correlation coefficient（ r2） was more than 0. 994. Conclusion The established method has high specificity and good sensitivity,and is suitable for species differentiation and identification for SC,SP,ST,SG isolates quickly and accurately.