贝类派琴虫二温式PCR检测方法的建立及初步应用

Development and Primary Application of A Two-temperature Polymerase Chain Reaction assay for detection of Perkinsus sp in Shellfish

根据GenBank中派琴虫的基因保守序列,设计了一对特异性引物,通过对二温式PCR扩增条件的优化,研究建立了检测贝类派琴虫的二温式PCR方法。该方法对派琴虫模板进行扩增,得到与试验设计相符的276bp的特异性扩增带,而对马尔太虫、单孢子虫、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1fg的派琴虫质粒DNA。用该二温式PCR对贝类样品进行检测,结果从广西的牡蛎检出派琴虫27份,连云港的菲律宾蛤仔检出派琴虫12份,温州的溢蛏中检出6份,结果提示,建立的半套式PCR方法可以用于贝类派琴虫的临床快速检测。

According to the gene sequences in GenBank of Perkinsus sp, one pair of specific primers were designed for amplifying the specific fragments of Perkinsus sp. The reaction parameters were opti- mized to develop the two-temperature PCR method for detection of Perkinsus sp. The 276 pb-long specific DNA fragment of Perkinsus sp was amplified, but not from other pathogenic such as Marteilia refringens ,Haplosporidium sp , Vibrio parahaemolyticu , Vibrio Alginolyticu , and Vibrio Fluvialis by this two-temperature PCR. The sensitivity results showed that as little as lfg DNA plasmid of Perkinsus sp was detected by this two-temperature PCR. Shellfish samples were detected by this two-temperature PCR. As a result, 27 Oyster from Guangxi, 12 Manila clam from Lianyungang and 6 razor clam from Wenzhou were Perkinsus sp positive. It suggested that this two-temperature PCR could be used as a sensitive tool to detect Perkinsus sp in clinical samples.