Lyons M Maille, Smolowitz Roxanna, Dungan Christopher F, Roberts Steven B
Department of Marine Sciences, University of Connecticut, 1080 Shennecossett Road Groton, Connecticut 06340, USA.
Dis Aquat Organ. 2006 Sep 14;72(1):45-52. doi: 10.3354/dao072045.
Quahog Parasite Unknown (QPX) is a thraustochytrid pathogen responsible for catastrophic mortalities of the northern quahog (hard clam) Mercenaria mercenaria. A real-time quantitative polymerase chain reaction (qPCR) assay was developed to assist research efforts on QPX ecology and pathology. Sensitivity of the assay was evaluated with serial dilutions of QPX-cultured cells to determine the lowest concentration of DNA that remained detectable in both the presence and absence of extraneous environmental substances. QPX cells were quantified before DNA extraction to calibrate standard curves to cell counts. Based on our results, the qPCR assay is able to quantify QPX within the range of 1 to several thousand organisms per reaction. Specificity of the assay was assessed by testing 29 thraustochytrid-like protists isolated from suspension-feeding bivalves from China, Oregon, Maryland, and Virginia. Application of the assay was demonstrated with positive qPCR results from naturally contaminated environmental samples including marine aggregates (i.e. marine snow), clam pseudofeces, and inflammatory nodules from infected clams. This quantitative assay for QPX will provide a valuable tool for characterizing QPX parasite abundances in coastal environments and for improving clam disease diagnostics.
圆蛤寄生虫未知种（QPX）是一种破囊壶菌病原体，可导致北方圆蛤（硬壳蛤） Mercenaria mercenaria 大量死亡。开发了一种实时定量聚合酶链反应（qPCR）检测方法，以辅助QPX生态学和病理学研究。通过对QPX培养细胞进行系列稀释来评估该检测方法的灵敏度，以确定在有无外来环境物质的情况下仍可检测到的最低DNA浓度。在DNA提取前对QPX细胞进行定量，以将标准曲线校准为细胞计数。根据我们的结果，qPCR检测方法能够在每个反应1至数千个生物体的范围内对QPX进行定量。通过检测从中国、俄勒冈州、马里兰州和弗吉尼亚州的悬浮摄食双壳类动物中分离出的29种类似破囊壶菌的原生生物，评估了该检测方法的特异性。通过对包括海洋聚集体（即海雪）、蛤类假粪便和受感染蛤类的炎性结节在内的自然污染环境样本进行qPCR阳性结果，证明了该检测方法的应用。这种针对QPX的定量检测方法将为表征沿海环境中QPX寄生虫丰度和改善蛤类疾病诊断提供有价值的工具。
