多重荧光定量PCR检测鼠感染3种病原体的方法

A multiplex fluorescent quantitative PCR method for detection of three rodent-carrying pathogens

目的建立多重荧光定量PCR快速检测以鼠为宿主伯氏疏螺旋体、弓形虫和恶性疟原虫的方法,对预防3种病原体引发的疫情具有重要意义。方法通过设计特异性引物和探针,扩增伯氏疏螺旋体23S rRNA基因,弓形虫的B1基因和恶性疟原虫的SSU基因,采用倍比梯度稀释法检测该体系的灵敏度,以另外8种以鼠为宿主的致病菌评价检测体系的特异性;建立了同时感染3种病原体的鼠全血模拟样本检测试验,以验证方法的适用性。结果建立自鼠血液模拟样本中同时检测伯氏疏螺旋体、弓形虫和恶性疟原虫的多重荧光定量PCR方法,检测3种病原体的灵敏度分别为5.5、12.8、17.2拷贝/μl,特异性强。结论建立了多重荧光定量PCR检测伯氏疏螺旋体、弓形虫和恶性疟原虫方法,缩短了检测时间,在疾病防控等方面有很好的应用前景。

Objective To develop a multiplex fluorescenct quantitative PCR assay for rapid and simultaneous detection of Borrelia burgdorferi, Toxoplasma gondii and Plasmodium falciparm carried by rodents. Methods Specific primers and probes were designed to amplify the 23S rRNA gene of B. burgdorferi, the B1 gene of T. gondii and the SSU gene of P. falciparm. The sensitivity of the assay was detected by the fold dilution method. The other eight strains of rodent-borne bacteria were used to examine the specificity of the assay. The method was evaluated to detect B. burgdo（feri, T. gondii and P. falciparm simultaneously in mice blood. Results A highly sensitive and specific multiplex fluorescent quantitative PCR assay was established for detection of B. burgdorferi, T. gondii and P.falciparm. The sensitivity was 5.5 eopies/μl for B. burgdorferi, 12.8 eopies/μl for T. gondii and 17.2 copies/μl for P. falciparm. Conclusion A multiplex fluorescent quantitative PCR assay was developed for detection of B. burgdorferi, T. gondii and P. falciparm, significantly reducing the time needed for simultaneous detection of the three rodent-borne pathogens.