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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 To explore the real-time fluorescent PCR for Salmonella species identification. Methods According to the consensus sequence of Salmonella choleraesuis (SC) and Salmonella paratyphi C (SP) (Gen Bank: AE017220.1), Salmonella Typhi (ST; Gen Bank: The primers of SCP, STP, SGP and Taq Man were designed according to the specific sequence of Salmonella Gallinarum (NC_016832.1) and Salmonella Gallinarum (SG; Gen Bank: HQ703462) TET, a multi-fluorescence PCR detection method based on Taq Man probe was established. At the same time, SPP primers and probes were designed according to SC and SP differential sequences (Gen Bank: CP000857), ROX was labeled at the 5 ’end of the probe, distinguishing between SC and SP. Results The results showed that 25 SC and 22 SP were amplified by SCP. 31 ST and 34 SG were amplified by STP and SGP, respectively. However, the amplification results of other serotypes of Salmonella and non-Salmonella were negative. SPP amplified 22 SP, 25 SC negative results. Amplification system evaluation results, SCP, STP, SGP and SPP amplification efficiency were 80% to 100%, the linear correlation coefficient r2 ≥ 0.944. Conclusion The established method is specific and sensitive and can be used for the identification of SC, SP, ST and SG.