贝类单孢子虫和折光马尔太虫二重荧光定量PCR方法的建立

Development of duplex real-time PCR assay for detection of Haplosporidium sp. and Marteilia refringens in shellfish

根据GenBank中单孢子虫和折光马尔太虫基因序列,用Primer Express2.0软件设计了两对引物和两条TaqMan探针。对反应条件和试剂浓度进行优化,建立了能够同时检测单孢子虫和折光马尔太虫的二重荧光定量PCR方法。该方法对单孢子虫和折光马尔太虫的检测敏感性达到40个模板拷贝数;此外抗干扰能力强,对单孢子虫和折光马尔太虫不同模板浓度进行组合,仍可有效地同时检测到这两个原虫。该方法对派琴虫、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。研究建立的单孢子虫和折光马尔太虫荧光定量PCR具有特异、敏感、快速、定量、重复性好等优点,可用于贝类单孢子虫和折光马尔太虫感染的检测。

Two sets of specific oligonucleotide primers for Haplosporidium sp.and Marteilia refringens,along with two TaqMan probes specific for each protozoan parasite were designed with Primer Express 2.0 software.The reaction parameters such as the concentration of two pair of primers,two TaqMan probes and the reaction buffer were optimized to develop duplex real-time PCR assay for the rapid detection of Haplosporidium sp.and Marteilia refringens.The sensitivity of duplex real-time PCR assay was 40 template copies for Haplosporidium sp.and Marteilia refringens.The duplex real-time PCR assay was found to be specific and to be able to detect and differentiate Haplosporidium sp.and Marteilia refringens,and no positive results were observed when nucleic acid from Perkinsus sp.,Pseudomonas fluorescens,Vibrio parahaemolyticu,V.Alginolyticu,V.Fluvialis and V.Mimicus were used as duplex real-time PCR templates.This duplex real-time PCR assay is a rapid,sensitive,and specific test for detection of Haplosporidium sp and Marteilia refringens and will be useful for the control of these protozoan parasites in shellfish.