贝类派琴虫和单孢子虫双重PCR检测方法的建立  

Development of a duplex PCR assay for detection of Perkinsus and Haplosporidium in Shellfish

作　　者：谢丽基[1] 谢芝勋[1] 庞耀珊[1] 刘加波[1] 邓显文[1] 谢志勤[1] 

机构地区：[1]广西兽医研究所,广西南宁530001

出　　处：《畜牧与兽医》2010年第1期8-11,共4页Animal Husbandry & Veterinary Medicine

基　　金：国家百千万人才工程人选专项资金项目(No.945200603);广西科技攻关项目(桂科攻0630001-3M)

摘　　要：根据基因库中派琴虫和单孢子虫的基因序列,分别设计了2对特异性引物,通过对双重PCR扩增条件的优化,建立了可同时检测鉴别这2种原虫的双重PCR。对同一样品中的派琴虫和单孢子虫模板进行扩增,结果均同时得到2条大小与试验设计相符的596bp(派琴虫)和244bp(单孢子虫)的特异性扩增带,对其他贝类病原核酸的扩增结果为阴性。敏感性试验结果表明,最低能检测到10pg的派琴虫和单孢子虫DNA。用该双重PCR对广西沿海的104份牡蛎、49份贻贝和20份文蛤病料进行检测,派琴虫的阳性率分别为14.6%,10.6%和15%,而未检出单孢子虫,结果提示派琴虫广泛存在于中国南方沿海的养殖贝类中,建立的PCR方法可以用于贝类派琴虫和单孢子虫的临床快速检测。A duplex transcription polymerase chain reaction was optimized to simultaneously detect two pathogens, Perkinsus sp. and Haplosporidium sp. in shellfish. Two pairs of specific primers were designed according to the conserved regions on the sequences of Perkinsus and Haplosporidium in GenBank. The results showed that two specific bands could be amplified from all the samples with Perkinsus and Haplospo- ridium, 596 bp for the former and 244 bp for the latter by this duplex PCR, but no bands with the same sizes were amplified from other shell- fish pathogens. As little as 10 pg DNA of Perkinsus and Haplosporidium could be detected. One hundred and four oyster, 49 mussel and 20 clam samples from Guangxi coastal were collected and detected by this PCR. As a result, the positive rates for Perkinsus were 14. 6%, 10. 6% and 15%, respectively, but no infections for Haplosporidium were found in the above samples. It suggested that Perkinsus was common in the cultivated shellfish in south China and this PCR method could be used to detect Perkinsus and Haplosporidium in clinical samples.

关 键 词：派琴虫 单孢子虫 双重PCR 

分 类 号：S852.7[农业科学—基础兽医学]