蜜蜂幼虫芽孢杆菌TaqMan荧光定量PCR检测方法的建立

Development of Taq Man real-time PCR assay for detection of Paenibacillus larvae of honey bees

为建立蜜蜂幼虫芽孢杆菌(P.larvae)荧光定量PCR检测方法,本研究根据Gen Bank中幼虫芽孢杆菌16S r RNA基因保守序列设计特异性引物和探针,经反应体系及条件优化,建立了检测P.larvae Taq Man荧光定量PCR方法。结果表明:该方法与其它病原无交叉反应;最低可检出1.3×10拷贝/μL的阳性质粒,比普通PCR灵敏度高100倍;重复试验显示该方法的批内和批间变异系数均小于3%。应用该方法对50份实验室模拟样品和100份临床样品进行了检测,与预期结果一致。本研究建立的方法可用于美洲幼虫腐臭病(AFB)的早期快速检测及P.larvae定量分析。

To establish a sensitive detection protocol of american foulbrood, a TaqMan real-time PCR assay was developed for the bacteria with a pair of specific primers and a TaqMan probe targeting the conserved region of the 16S rRNA gene of P.larvae in this study. With the optimized reaction conditions, the established assy was highly specific for detecting P.larvae, but no amplification from other honey bees bacteria. In addition the detected limit of this method was 1.3 ×10 copies/μL pXT-16S recombinant plasmid, which was 100-fold more sensitive than that of conventional PCR. Moreover, the method was highly reproducible and coefficient of variation was less than 3% for both intra- and inter-assays. Fifty simulation samples and one hundred clinical samples were tested by the TaqMan real-time PCR, the result was consistent with the prediction in the theory. These data suggested that the developed TaqMan real-time PCR assay could be applied in early rapid detection and quantitative analysis of P.larvae infections.