猪链球菌9型荧光定量PCR检测方法的建立及应用

Establishment and application of Real-time TaqMan-quantitative PCR assay for detection of Streptococcus suis serotype 9

【目的】建立猪链球菌9型(SS9)的快速诊断和定量分析方法。【方法】根据GenBank已登录的SS9cps9H基因保守部分序列设计合成引物和TaqMan荧光探针,建立了SS9的TaqMan荧光定量PCR检测方法(Taq-Man FQ-PCR),并进行了敏感性、特异性和重复性试验;利用所建立的检测方法对河南省11例疑似猪链球菌临床样品进行了应用检测,并与常规PCR方法进行了对比。【结果】成功建立了SS9的FQ-PCR检测方法和定量标准曲线,FQ-PCR方法的检测灵敏度可达1.0拷贝/μL,特异性高且重复性良好;利用该方法对11份临床疑似猪链球菌感染组织病料进行的应用检测表明,其中有3份样品为阳性,与常规PCR方法检测的阳性符合率为100%。【结论】成功建立的SS9 FQ-PCR诊断方法,可用于临床SS9型病菌感染的快速诊断及样品中细菌含量的定量分析。

[Objective] The study is to establish a rapid detection and quantitative analysis diagnosis method for Streptococcus suis Serotype 9 （SS9） clinic infection. [Method] The TaqMan Fluorescent probes and primers were designed and synthesized according to the conserved district of cps9H gene of SS9 available in GenBank, and then reaction parameters were optimized and standard carve was established to develop a real-time TaqMan-fluoreseent-quantitative PCR assay （FQ-PCR） of Streptococcus suis Serotype 9. The sensitivity,specificity and repetition assay of FQ-PCR were tested, and 11 suspected tissue samples from pigs on farms in Henan Province were detected by using the established FQ-PCR assay compared with the routine PCR method. [Result] The FQ-PCR method and quantitative standard carve were successfully established. The developed FQ-PCR assay,which exhibited high specificity and good reproducible,could detect 1.0 eopy/μL of plasmid DNA and its sensitivity was 100 times higher than that of the routine PCR. The FQ-PCR was used to detect the DNA of 11 tonsil tissue samples taken from suspicious S. suis infected pigs and 3 samples were displayed positive,which was completely consistent with the results of the routine PCR. [Conclusion] A SS9 FQ-PCR method was successfully established, which could be used to diagnose rapidly and analyse quantitatively clinic SS9 infection samples.