同时检测海洋贝类包纳米虫和派琴虫的双重TaqMan MGB探针实时荧光PCR方法

Use of the duplex TaqMan MGB probe for simultaneous detection of Perkinsus and Bonamia in marine shellfish

根据包纳米虫(Bonamia sp.)SSU rDNA和派琴虫(Perkinsus sp.)ITS rDNA的保守区序列,设计特异性引物和Taqman MGB探针,建立了同时检测上述两种贝类原虫的双重实时荧光PCR方法.该方法可检测到包纳米虫基因的446拷贝质粒,以及派琴虫基因的171拷贝质粒,具有较高的灵敏度.以10倍系列稀释的包纳米虫阳性标准品质粒和派琴虫阳性标准品质粒为模板,测得该方法的定量标准曲线相关系数分别为0.999和1.000,显示出很好的扩增效率.同时该方法与尼氏单孢子虫(Haplosporidium nelsoni)、沿岸单孢子虫(H.costale)等其他贝类原虫,以及副溶血性弧菌(Vibrio parahaemolyticus)、迟缓爱德华氏菌(Edwardsiella tarda)和鮰爱德华氏菌(E.ictaluri)等海水养殖常见致病菌之间无交叉反应,表现出较高的特异性.福建莆田的湄州湾、平海湾、兴化湾等地采集的296份贝类临床样本的检测证明,该方法是一种有效的快速检测鉴定上述2种贝类原虫的方法,具有良好的灵敏度和特异性,可广泛应用于海洋贝类原虫感染情况调查,以及贝类疫病检疫监控等领域.

A duplex TaqMan MGB real-time PCR method was optimized to simultaneously detect Perkinsus sp. and Bonamia sp.. The primers and TaqMan MGB probes were designed and chosen to amplify the conserved SSU segment of genus Bonamia sp. ribosomal DNA and ITS segment of genus Perkinsus sp. ribosomal DNA. The duplex realtime PCR identified and differentiated the two protozoan parasite groups. The sensitivity of the duplex real-time PCR assay was 446 and 171 template copies and it had higher sensitivity. Tenfold serial dilutions of the plasmid DNAs of Bonamia sp. and Perkinsus sp. were quantified the actual copy numbers using the duplex real-time PCR. The correlation coefficient of calibration curves were 0. 999 and 1. 000,respectively. Meanwhile,this method had no cross reaction with other species of protozoa in mollusks and the common pathogenic bacteria in mariculture. The method showed advantages of rapid and high efficiency when applied to detect 296 clinical specimens from Meizhou bay, Pinghai bay and Xinghua bay of Fujian. This assay is proved to be sensitive and specific and can be widely used for the protozoan infection survey,disease surveillance and the quarantine of shell fish.