Robledo J A, Gauthier J D, Coss C A, Wright A C, Vasta G R
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore 21202, USA.
J Parasitol. 1998 Dec;84(6):1237-44.
We examined the species-specificity and sensitivity of a polymerase chain reaction (PCR)-based assay for Perkinsus marinus and compared its overall performance with the fluid thioglycollate medium (FTM) assay on oyster (Crassostrea virginica) hemolymph, mantle, and rectum samples. Our results indicated that the PCR-based methodology is species-specific because Perkinsus olseni, Perkinsus atlanticus, and Perkinsus spp. DNAs were not amplified with the PCR primers developed for P. marinus diagnosis. The sensitivity of the PCR method, as assessed through spike/recovery experiments, was established by the detection of as few as 1 cell of P. marinus in 30 mg of oyster tissue. Tissue samples from naturally infected oysters analyzed both by the FTM and PCR assay suggested that the latter was more sensitive for the diagnosis of P. marinus. Positive results for P. marinus infection ranged from 70% to 83% by FTM and from 92% to 100% by PCR, depending on the tissue examined. Therefore, species-specificity and sensitivity of the NTS-based PCR assay validate its use as a tool for assessment of P. marinus in mollusks.
我们检测了一种基于聚合酶链反应（PCR）的针对海洋派琴虫的检测方法的物种特异性和灵敏度，并将其整体性能与用于牡蛎（弗吉尼亚牡蛎）血淋巴、外套膜和直肠样本的液体硫乙醇酸盐培养基（FTM）检测方法进行了比较。我们的结果表明，基于PCR的方法具有物种特异性，因为为海洋派琴虫诊断开发的PCR引物未扩增出奥尔森派琴虫、大西洋派琴虫和派琴虫属的DNA。通过加标/回收率实验评估的PCR方法的灵敏度，是通过在30毫克牡蛎组织中检测到少至1个海洋派琴虫细胞来确定的。对自然感染牡蛎的组织样本进行FTM和PCR检测分析表明，后者对海洋派琴虫的诊断更敏感。根据所检测的组织不同，FTM检测海洋派琴虫感染的阳性结果为70%至83%，PCR检测为92%至100%。因此，基于非转录间隔区（NTS）的PCR检测方法的物种特异性和灵敏度证实了其作为评估软体动物中海洋派琴虫的工具的用途。
