贝类单孢子虫荧光定量PCR检测方法的建立

根据基因库中单孢子虫的基因保守序列,设计了1对特异性引物和1条TaqMan探针,对反应条件和试剂浓度进行优化,建立了检测单孢子虫的荧光定量PCR方法。该方法对单孢子虫的检测敏感性达到40拷贝数,比常规PCR敏感性高100倍;对派琴虫、折光马尔太虫、荧光假单胞菌、副溶血弧菌、溶藻弧菌、河弧菌和拟态弧菌等病原体的检测结果全为阴性。表明该研究建立的单孢子虫荧光定量PCR具有特异、敏感、快速、定量和重复性好等优点,可用于临床单孢子虫感染的快速检测。

Application of Real-time PCR for Detection of Haplosporidium sp. in Shellfish

A pair of specific primers and a TaqMan probe were designed and synthesized according to the conserved gene sequences of Haplosporidium sp., and then reaction parameters and reagent concentration were optimized to develop a real-time quantitative PCR assay for detecting Haplosporidium sp.. The result showed that the real-time PCR could detect 40 template copies of plasmid DNA in Haplosporidium sp, and its sensitivity was 100 times higher than that of conventional PCR. The detection results of Perkinsus sp., Marteilia refringens, Pseudomonas fluorescens, Vibrio parahaemolyticu, Vibrio alginolyticu, Vibrio fluvialis and Vibrio mimicus were all negative by real-time PCR. As a result of the sensitivity and specificity of real-time PCR with a relatively rapid and simple procedure, it could be used for clinical rapid detection of Haplosporidium sp. infection.