Stilwell Natalie K, Whittington Richard J, Hick Paul M, Becker Joy A, Ariel Ellen, van Beurden Steven, Vendramin Niccolò, Olesen Niels J, Waltzek Thomas B
Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610, USA.
Dis Aquat Organ. 2018 May 7;128(2):105-116. doi: 10.3354/dao03214.
Ranaviruses are globally emerging pathogens negatively impacting wild and cultured fish, amphibians, and reptiles. Although conventional and diagnostic real-time PCR (qPCR) assays have been developed to detect ranaviruses, these assays often have not been tested against the known diversity of ranaviruses. Here we report the development and partial validation of a TaqMan real-time qPCR assay. The primers and TaqMan probe targeted a conserved region of the major capsid protein (MCP) gene. A series of experiments using a 10-fold dilution series of Frog virus 3 (FV3) MCP plasmid DNA revealed linearity over a range of 7 orders of magnitude (107-101), a mean correlation coefficient (R2) of >0.99, and a mean efficiency of 96%. The coefficient of variation of intra- and inter-assay variability ranged from <0.1-3.5% and from 1.1-2.3%, respectively. The analytical sensitivity was determined to be 10 plasmid copies of FV3 DNA. The qPCR assay detected a panel of 33 different ranaviral isolates originating from fish, amphibian, and reptile hosts from all continents excluding Africa and Antarctica, thereby representing the global diversity of ranaviruses. The assay did not amplify highly divergent ranaviruses, members of other iridovirus genera, or members of the alloherpesvirus genus Cyprinivirus. DNA from fish tissue homogenates previously determined to be positive or negative for the ranavirus Epizootic hematopoietic necrosis virus by virus isolation demonstrated a diagnostic sensitivity of 95% and a diagnostic specificity of 100%. The reported qPCR assay provides an improved expedient diagnostic tool and can be used to elucidate important aspects of ranaviral pathogenesis and epidemiology in clinically and sublinically affected fish, amphibians, and reptiles.
蛙病毒是全球范围内新出现的病原体，对野生和养殖的鱼类、两栖动物及爬行动物产生负面影响。尽管已开发出传统和诊断性实时聚合酶链反应（qPCR）检测方法来检测蛙病毒，但这些检测方法通常未针对已知的蛙病毒多样性进行测试。在此，我们报告了一种TaqMan实时qPCR检测方法的开发及部分验证情况。引物和TaqMan探针靶向主要衣壳蛋白（MCP）基因的保守区域。一系列使用蛙病毒3（FV3）MCP质粒DNA 10倍稀释系列的实验显示，在7个数量级（107 - 101）范围内呈线性，平均相关系数（R2）>0.99，平均效率为96%。批内和批间变异系数分别在<0.1 - 3.5%和1.1 - 2.3%范围内。分析灵敏度确定为10个FV3 DNA质粒拷贝。该qPCR检测方法检测了一组33种不同的蛙病毒分离株，这些分离株源自除非洲和南极洲以外各大洲的鱼类、两栖动物和爬行动物宿主，从而代表了蛙病毒的全球多样性。该检测方法未扩增高度分化的蛙病毒、其他虹彩病毒属成员或异疱疹病毒属鲤病毒属成员。通过病毒分离先前确定为蛙病毒流行性造血坏死病毒阳性或阴性的鱼类组织匀浆DNA，诊断灵敏度为95%，诊断特异性为100%。所报道的qPCR检测方法提供了一种改进的便捷诊断工具，可用于阐明临床和亚临床感染的鱼类、两栖动物和爬行动物中蛙病毒发病机制和流行病学的重要方面。
