贝类派琴虫实时荧光定量PCR检测方法的建立和应用

Establishment and application of real time PCR method for detection of Perkinsus sp.in bivalves

中文关键词: 派琴虫  贝类  实时荧光PCR  检测

英文关键词: Perkinsus sp.  Bivalve  Real time PCR  Detection

中文摘要:

      选择派琴虫保守的核糖体DNA ITS-2区域设计引物和TaqMan探针，通过对反应体系和反应条件进行优化，建立了实时荧光定量PCR检测派琴虫的方法。所构建方法检测质粒模板DNA的动态范围为2.6×101-2.6×107拷贝，敏感度可检测到26拷贝质粒DNA，而且与包拉米原虫、隐孢子虫等其他寄生性原虫无交叉反应，也不受贝类组织DNA的干扰。利用本研究所建立的方法对来自我国山东、福建等不同沿海海域的30份贝类样品进行检测，检出阳性样品3份。研究表明，本研究所构建的派琴虫实时荧光定量PCR检测方法具有快速、灵敏和特异等优点，可满足国内养殖场及进出口水生动物携带派琴虫的检测需要。

英文摘要:

      Perkinsus sp. are parasites for marine bivalves. They can have severe pathogenic effect on their hosts and cause significant economic loses to the bivalve culture industry. To facilitate rapid and sensitive diagnosis of Perkinsus sp. the real time TaqMan PCR method was developed to detect Perkinsus sp. infection in oysters and other bivalves. The primers and TaqMan probe were designed and chosen to amplify the conserved internal transcript spacer 2 (ITS-2) segment of genus Perkinsus sp. ribosomal DNA. The sensitivity and specificity of the developed method were examined using the plasmid containing the targeted ITS-2 fragment as template. The online BLAST analysis showed that the primers and probe were specific enough to distinguish Perkinsus sp. from Bonamia sp. and other protistan parasites while not affected by bivalve tissue DNA at the same time. The dynamic range of the developed method was between 2.6×101and 2.6×107copies, meaning that the target DNA could be reliably detected and quantified for plasmid template DNA at a concentration as low as 26 copies. Thirty samples, collected from different sea areas of China, were tested for Perkinsus infection and 3 samples, collected from Shandong and Fujian coastal areas respectively, gave positive results. This test result was confirmed by traditional FTM culturing method. In conclusion, the real time PCR method is rapid, sensitive and specific for Perkinsus, and can be used to inspect Perkinsosis in the farm and for quarantine use. 