贝类派琴虫LUX荧光PCR检测方法的建立

Development of LUX PCR detection assay for Perkinsus infection of mollusks

为满足贝类派琴虫病的快速检测需要,本研究选择派琴虫保守的核糖体DNAITS-2区域设计引物,通过对反应体系和反应条件的优化,首次建立了LUX荧光PCR检测派琴虫的方法。试验表明,所建立方法检测质粒模板DNA的动态范围为1.0×101-1.0×108,敏感性为10拷贝质粒DNA。采用模拟阳性样品试验,可检测到100拷贝质粒DNA。而且与贝类的包拉米原虫、隐孢子虫等其它寄生性原虫无交叉反应,也不受组织DNA的干扰。50份采集样品的检测结果与RFTM培养方法一致。本研究所构建的派琴虫LUX荧光PCR检测方法具有快速、灵敏、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。

To meet the demand for rapid diagnosis of perkinsosis, a real time LUX PCR for detection of Perkinsus was developed. The primers were designed and chosen to amplify the conserved internal tmnscripted spacer 2 （ITS-2） region of genus Perkinsus ribosomal DNA. Results indicated that the dynamic range of the developed method was from 1.0× 10^1 copies to 1.0× 10^8 copies plasmid DNA, and the detection limit was 10 copies of plasmid DNA, and 100 copies of plasmid DNA from artificially contaminated clam tissue. The Test results of 50 samples collected from China were in line with RFTM culture. The developed method was specific enough to distinguish Perkinsus from Bonamia spp and other parasitic protozoa while not interfered by mollusk tissue DNA. In conclusion, the real-time LUX PCR assay is rapid, sensitive, specific and can be used to rapidly detect Perkinsus in the farm and the entry-exit products.