贝类折光马尔太虫PCR检测方法的建立

根据基因库中折光马尔太虫的基因保守序列,设计了1对特异性引物,通过对PCR扩增条件的优化,研究建立了检测贝类折光马尔太虫的PCR方法。该方法对折光马尔太虫模板进行扩增,得到与试验设计相符的478 bp的特异性扩增带,而对派琴虫、单孢子虫、嗜水气单胞菌、荧光假单胞菌、副溶血弧菌、溶藻弧菌和河弧菌等病原体的扩增,结果全为阴性。敏感性试验结果表明,该技术最低能检测到1 pg的折光马尔太虫DNA。用该PCR对广西沿海的119份牡蛎病料进行检测,折光马尔太虫的阳性率为1.68%,结果提示了中国南方沿海的养殖贝类中存在折光马尔太虫的感染,建立的PCR方法可以用于贝类折光马尔太虫的临床快速检测。

Development of a Polymerase Chain Reaction Assay for Detection of Marteilia re fringens in Shellfish

According to the gene sequences in GenBank of Marteilia refringens,one pair of specific primers were designed for amplifying the specific fragments of Marteilia refringens.The reaction parameters were optimized to develop the polymerase chain reaction method for detection of Marteilia refringens.The 478 bp long specific DNA fragment of Marteilia refringens was amplified,but not from other pathogenic such as Perkinsus sp.,Haplosporidium sp.,Aeromonas hydrophila,Pseudomonas fluorescens,Vibrio parahaemolyticu,Vibrio alginolyticu,and Vibrio fluvialis by this PCR.The sensitivity results showed that as little as 1 pg DNA of Marteilia refringens was detected by this PCR.119 Oyster samples of Guangxi coastal were detected by this PCR.As a result,the positive rate was 1.68%.It suggested that Marteilia refringens existed in the cultivated shellfish in south China and the PCR method could be used as a sensitive tool to detect Marteilia refringens in clinical samples.