贝类折光马尔太虫PCR检测方法的建立

摘要

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 polymer-ase 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 fluviaUs 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.%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.