CAJ | 학술논문

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#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．
근거포납미충（Bonamia sp．）SSU rDNA화파금충（Perkinsus sp．）ITS rDNA적보수구서렬，설계특이성인물화Taqman MGB탐침，건립료동시검측상술량충패류원충적쌍중실시형광PCR방법．해방법가검측도포납미충기인적446고패질립，이급파금충기인적171고패질립，구유교고적령민도．이10배계렬희석적포납미충양성표준품질립화파금충양성표준품질립위모판，측득해방법적정량표준곡선상관계수분별위0.999화1.000，현시출흔호적확증효솔．동시해방법여니씨단포자충（Haplosporidium nelsoni）、연안단포자충（H．costale）등기타패류원충，이급부용혈성호균（Vibrio parahaemolyticus）、지완애덕화씨균（Edwardsiella tarda）화#애덕화씨균（E．ictaluri）등해수양식상견치병균지간무교차반응，표현출교고적특이성．복건보전적미주만、평해만、흥화만등지채집적296빈패류림상양본적검측증명，해방법시일충유효적쾌속검측감정상술2충패류원충적방법，구유량호적령민도화특이성，가엄범응용우해양패류원충감염정황조사，이급패류역병검역감공등영역．
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 seg-ment of genus Bonamia sp.ribosomal DNA and ITS segment of genus Perkinsus sp.ribosomal DNA.The duplex real-time 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 corre-lation 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.