贝类单孢子虫半套式PCR检测方法的建立及初步应用

根据GenBank中单孢子虫的基因保守序列,设计了3条特异性引物,通过对半套式PCR扩增条件的优化,本研究建立了检测贝类单孢子虫的半套式PCR方法。该方法对单孢子虫模板进行扩增,得到与预期相符的333 bp特异性条带,而对折光马尔太虫、派琴虫、副溶血弧菌、溶藻弧菌和河弧菌等病原体的扩增,结果均为阴性。敏感性试验结果表明,该技术最低能检测到0.011 fg单孢子虫质粒DNA。用该半套式PCR对青岛沿海的贝类样品进行检测,结果从牡蛎和菲律宾蛤仔中分别检出单孢子虫6和8份,提示青岛沿海的养殖贝类中存在单孢子虫感染。结果表明,本研究建立的半套式PCR方法可用于贝类单孢子虫的临床快速检测。

Establishmeat and Preliminary Application of A Semi-nested Polymerase Chain Reaction Assay for Detection of Haplosporidium in Shellfish

According to the gene sequences in GenBank of Haplosporidium sp, three specific primers were designed for amplifying the specific fragments of Haplosporidium sp. The reaction parameters were optimized to develop the semi-nested PCR method for detection of Haplosporidium sp. The 333 bp specific DNA fragment of Haplosporidium sp was amplified, but not from other pathogenics such as Marteilia refringens,Perkinsus sp, Vibrio parahaemolyticu, Vibrio alginolyticu and Vibrio fluvialis by this semi-nested PCR. The sensitivity results showed that as little as 0.01 fg DNA plasmid of Haplosporidium sp was detected by this semi-nested PCR. Shellfish samples of Shandong coastal were detected by this semi-nested PCR. As the result, 6 Oyster and 8 Manila clam were Haplosporidium sp positive. It suggested that Haplosporidium sp existed in the cultivated shellfish in Shandong and this semi-nested PCR could be used as a sensitive tool to detect Haplosporidium sp in clinical samples.