3种牡蛎原虫的流行情况调查及多重PCR检测方法

应用聚合酶链式反应(PCR)方法对福建、广东和海南等地沿海的养殖牡蛎进行包拉米虫、派琴虫和单孢子虫检测.结果表明,这些地区的牡蛎均不同程度地感染这些原虫,经鉴定病原为牡蛎包拉米虫、奥尔森派琴虫和尼氏单孢子虫.根据基因库中奥尔森派琴虫和尼氏单孢子虫的基因序列设计多对特异性引物,检测包拉米虫的引物采用世界动物卫生组织推荐引物,通过对多重PCR反应条件的优化,建立可同时检测这3种原虫的多重PCR方法.运用该方法对样品中的牡蛎包拉米虫、奥尔森派琴虫和尼氏单孢子虫进行扩增,结果得到与试验设计相符的303、480和749 bp 3条特异性扩增条带,对其他贝类病原核酸的扩增均为阴性.多重PCR方法最低能检测到10 pg牡蛎包拉米虫、奥尔森派琴虫和尼氏单孢子虫DNA,表明该方法适用于这3种原虫的快速检测和鉴别诊断.

Prevalence survey and multiplex PCR detection method of three protozoa in oyster

Three protozoa Bonamia sp.,Perkinsus sp.and Haplosporidium sp.in cultured oysters which had been collected from the seaside of Fujian,Guangdong and Hainan province were detected using polymerase chain reaction method.It was revealed that all of the oysters had been infected by these protozoa with various degrees.The pathogens were identified as Bonamia ostreae,Perkinsus olseni and Haplosporidium nelsoni in these areas.Specific primers were designed according to the conserved regions from the gene of P.olseni and H.nelsoni in GeneBank.The primers used in detecting Bonamia sp.were recommend by World Organization for Animal Health.A multiplex polymerase chain reaction(multiplex PCR) was developed to detect these parasites simultaneously.The specific amplified band of Bonamia sp.,P.olseni and H.nelsoni were 303,480 and 749 bp respectively,and no specific band was amplified from other shellfish pathogens in this multiplex PCR system.As little as 10 pg DNA of the parasites above could be detected using this method.It was showed that the method is applicable to all of the three parasites in rapid detection and differential diagnosis.