贝类包纳米虫病焦磷酸测序检测方法的建立与应用

摘要

One pair of specific primers were designed and used to pyrosequencing the conserved region of Bonamia DNA with PyroMark software basing on pyrosequencing platform and Bonamia gene characteristics.PCR pyrosequencing method was established with the recombinant plasmid of Bonamia spp.constructed with OIE PCR method as the template.The obtained sequence can be identified as the sequence of Bonamia spp.through Blast analysis,and enzyme digestion site analysis identified the specie as Bonamia spp..The imported oysters were detected with the newly developed method and PCR-RFLP as well as was recommended by OIE.The result showed that the developed PCR pyrosequencing method can be used to correctly identifying Bonamia spp.infection and species.The detection results were con sistent with that yielded with OIE method.Therefore,this method can be applied to the detection and identification of Bonamia spp.infection in port.%以焦磷酸测序技术为检测平台,在研究包纳米虫基因特性的基础上,选择包纳米虫(Bonamia spp.)DNA序列的保守区域,利用焦磷酸测序软件设计专用引物.以OIE推荐的PCR方法构建的阳性质粒作为模板,建立了包纳米虫的PCR焦磷酸测序检测方法,确定测得序列为包纳米虫序列,经酶切位点分析可鉴别感染种类.以建立的包纳米虫PCR-焦磷酸测序方法对进口牡蛎(Ostrea gigas thunberg)样品进行检测,同时以OIE推荐的PCR-RFLP方法进行对照检测,检测结果表明,本研究建立的PCR-焦磷酸测序检测方法可以在基因序列水平上准确鉴定样品是否为包纳米虫感染以及感染种类,检测结果与OIE推荐方法的检测结果一致.研究结果表明,本研究建立的检测方法可应用于口岸包纳米虫感染情况的检测鉴定.