细粒棘球绦虫TaqMan实时荧光定量PCR检测方法的建立及应用

目的 为建立高效、特异的细粒棘球绦虫(Echinococcus granulosus,Eg)检测方法。方法 本研究设计了一组基于Eg线粒体基因的特异性引物和TaqMan探针,通过反应体系及条件的优化,建立了Eg荧光定量PCR检测方法。结果 该方法线性关系好,灵敏度高,在10⁹~10²拷贝/μL相关系数达0.998,灵敏度达10²拷贝/L;在特异性试验中,与其他病原虫株无交叉反应,特异性较好;重复性试验变异系数均低于3%。应用该方法对50份临床样品进行检测,发现6份为Eg感染阳性。结论 由此可见,建立的荧光定量PCR方法可快速、灵敏、特异地检测细粒棘球绦虫,具有一定的实际应用价值。

Establishment and application of TaqMan real-time PCR assays for detection of Echinococcus granulosus

To develop an efficient and specific detection method for Echinococcus granulosus (Eg), we designed a set of specific primers and TaqMan probes based on the Eg mitochondrial gene. Through optimization of the reaction system and conditions, the real-time PCR assay was established. The assay showed a good linear relationship at 10⁹-10² copies/μL, the correlation coefficient reached 0.998, and the assay sensitivity reached 10² copies/μL. In the specificity test, no cross reaction with other pathogenic strains was observed, and the specificity was better. The intra- and inter-assay coefficients of variation were less than 3%. A total of 50 clinical samples were detected with the established assay, and six samples were positive for Eg infection. The results indicated that the fluorescence quantitative PCR method can detect Echinococcus granulosus in a rapid, sensitive and specific manner, and thus has practical application value.