贝类包拉米虫和派琴虫二重荧光定量PCR方法的建立

Development of Duplex Real-time PCR Assay for Detection of Bonamia and Perkinsus in Shellfish

根据GenBank中包拉米虫和派琴虫保守基因序列,用Primer Express3.0软件设计合成了两对引物和两条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包拉米虫和派琴虫的二重荧光定量PCR方法。该方法对包拉米虫和派琴虫的检测敏感性达到200个模板拷贝数;此外抗干扰能力强,对包拉米虫和派琴虫不同模板浓度进行组合,仍可有效同时检测到这两个原虫。该方法对单孢子虫、马尔太虫、副溶血弧菌、溶藻弧菌和河弧菌等病原体的检测,结果全为阴性。研究建立的包拉米虫和派琴虫荧光定量PCR具有特异、敏感、快速、定量、重复性好等优点,可用于贝类包拉米虫和派琴虫感染的检测。

Two pairs of specific oligonucleotide primers for Bonamia and Perkinsus, and two TaqMan probes specific for each protozoan parasite were designed with Primer Express 3.0 software. The reaction parameters such as the concentration of two pairs of primers, two TaqMan probes and the reaction buffer were optimized to develop a duplex real-time PCR assay for the rapid detection of Bonamia and Perkinsus. The sensitivity of the duplex real-time PCR assay was 200 template copies for Bonamia and Perkinsus .The duplex real-time PCR assay was found to be specific and to be able to detect and differentiate Bonamia and Perkinsus, and no positive results were observed when nucleic acids from Haplosporidium, Marteilia refringens, Vibrio parahaemolyticu, V. Alginolyticu, and V.Fluvialisand were used as duplex real-time PCR templates. This duplex real-time PCR assay was a rapid, sensitive, and specific test fo detection of Bonamia and Perkinsus and could be useful for the control of these protozoan parasites in shellfish.