蜜蜂蜂房蜜蜂球菌TaqMan荧光定量PCR检测方法的建立

Development of TaqMan real-time PCR assay for detection of European foulbrood pathogen of honey bees

为建立快速检测蜜蜂欧洲幼虫腐臭病(EFIB)病原菌蜂房蜜蜂球菌(M.pluton)的方法,本研究根据M.pluton 16S rRNA基因序列,设计并合成特异性引物和TaqMan探针,经反应参数优化,建立了检测M.pluton TaqMan荧光定量PCR方法。结果表明,该方法与其它蜜蜂常见病病原均无交叉反应;该方法的灵敏度为1.0×10~1拷贝/μL的阳性质粒,比普通PCR灵敏1 000倍;重复性试验表明该方法的批内和批间变异系数均小于1%。应用该方法对100份实验室模拟样品和150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为蜂及蜂产品中M.pluton的检验检疫提供新的、有效的技术手段。

To establish a rapid and sensitive detection method of European foulbrood,a TaqMan real-time PCR was developed with a pair of primers and the probe targeting the conserved region of 16 S rRNA gene of Melissococcus pluton.Under the optimized reaction parameters,the results showed that the method had no cross-amplifications with other honey bees pathogen,and was specific for detecting M.pluton with a detection limit of 1.0×10~1 copies/μL pXT-16 S recombinant plasmid,which was1000 times more sensitive than that of conventional PCR.The repeatability tests indicated that the variation coefficient of intraand inter-assay were less than 1%.The method was applied to detect 100 simulation samples and 150 clinical samples,the result was consistent the prediction in the theory.The development of this detection method could be used to inspection and quarantine of bee and bee products.