贝类折光马尔太虫半套式PCR检测方法的建立及初步应用

Development and Primary Application of Asemi-nested Polymerase Chain Reaction assay for detection of Marteilia refringens in Shellfish

根据Genbank中折光马尔太虫的基因保守序列,设计了3条特异性引物,通过对半套式PCR扩增条件的优化,研究建立了检测贝类折光马尔太虫的半套式PCR方法。该方法对折光马尔太虫模板进行扩增,得到与实验设计相符的228bp的特异性扩增带,而对单孢子虫、派琴虫、副溶血弧菌、溶藻弧菌和河弧菌等病原体的扩增,结果全为阴性。敏感性试验结果表明,该技术最低能检测到0.01fg的折光马尔太虫质粒DNA。用该半套式PCR对福建沿海的贝类样品进行检测,结果从溢蛏中检出折光马尔太虫7份,结果提示福建沿海养殖的贝类中存在折光马尔太虫感染,建立的半套式PCR方法可以用于贝类折光马尔太虫的临床快速检测。

According to the gene sequences of Marteilia refringens in GenBank, three specific primers were designed for amplifying the specific fragments of Marteilia refringens. The reaction parameters were optimized to develop the semi-nested PCR method for detection of Marteilia refringens. The specific 228 pb DNA fragment of Marteilia refringens was amplified, but not from other pathogenic such as Haplosporidium sp, Perkinsus sp, Vibrio parahaemolyticu, Vibrio alginolyticu, and Vibrio fluvialis by this semi-nested PCR. The sensitivity results showed that as little as 0.01fg DNA plasmid of Marteilia refringens was detected by this semi-nested PCR. Shellfish samples of Fujian coastal were detected by this semi-nested PCR. As a result, seven razor clam samples were positive for Marteilia refringens, suggesting that Marteilia refringens existed in the cultivated shellfish in Fujian and this semi-nested PCR could be used as a sensitive tool to detect Marteilia refringens in clinical samples.