贝类派琴虫PCR检测方法的建立及初步应用

Development and application of polymerase chain reaction assay for detection of Perkinsus sp.in shellfish

根据派琴虫的基因保守序列设计了特异性引物,对派琴虫的DNA进行PCR扩增并将产物克隆到pMD18-T载体后测序。结果表明,扩增大小为596bp,与预期扩增序列同源性为99.8%。该PCR方法检测灵敏度高,最低可检测1pg的派琴虫DNA;特异性强,对荧光假单胞菌、嗜水气单胞菌、大肠杆菌、葡萄球菌、副溶血弧菌、沙门氏菌、诺瓦克样病毒等贝类病原体的扩增均为阴性。用该PCR技术对广西沿海的104份牡蛎、49份贻贝和20份文蛤病料进行检测,阳性率分别为14.6%、10.6%和15.0%。结果显示,派琴虫广泛存在于中国南方沿海的养殖贝类中,建立的PCR方法可用于贝类派琴虫的临床快速检测。

According to the gene sequence of Perkinsus sp.in GenBank,one pair of specific primers were designed to amplify the specific fragments of Perkinsus sp.in shellfish.The obtained 596bp PCR product shared 99.8 % identity with the published sequence.This PCR assay was specific and no PCR product was detected from other pathogenic bacteria such as Pseudomonas fluorescens,Aeromonas hydrophila,Escherichia coli,Staphylococcus,Vibrio parahaemolyticus,Salmonella,and Norwalk virus.The sensitivity determination showed that the lowest amount of Perkinsus sp.detected by this PCR was 1pg DNA.In our study,104 Oyster,49 Mussel,and 20 Clam samples collected from Guangxi coasts were tested by this PCR method.The percentage of positive results were 14.6%,10.6%,and 15.0% respectively,suggesting that Perkinsus sp.exists widely in the cultivated shellfish in southern China and this PCR assay is a sensitive tool to detect Perkinsus sp.in clinical samples.