ABSTRACT: Ranaviruses are emerging pathogens that can cause morbidity, mortality and population declines in ectothermic hosts; however, there is no standardized approach to diagnostics. Here, we compared the inter-assay variation and intra-assay precision among 2 commonly used quantitative PCRs (qPCRs), a conventional and a nested PCR assay (used as a gold standard), using laboratory-propagated ranavirus (FV3 and CMTV) and field-collected samples. A qPCR assay (‘Leung’) detected viral DNA in dilutions 2 orders of magnitude lower than other assays regardless of the viral lineage of the cultured isolate (FV3/CMTV). The second qPCR (‘Brunner’) was slightly more sensitive than the conventional PCR (‘Mao’ assay). For field samples, the Leung qPCR detected all known positives, while the Mao assay PCR only detected 2.5% of the positive samples. Amplicon sequences from the 2 conventional PCRs were shown to be useful for inferring viral lineage. Inaccurate results will bias estimates of the distribution and prevalence of ranaviruses, and together these findings emphasize that molecular assays should be chosen carefully in the context of study aims.

中文翻译：

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分子测定的选择确定了鼻病毒检测的可能性以及对流行和发生的推断。

摘要：鼻病毒是新兴的病原体，可导致外热宿主的发病，死亡和人口减少。但是，没有标准化的诊断方法。在这里，我们比较了2种常用定量PCR（qPCR），常规和巢式PCR测定法（用作金标准）之间的测定间变异和测定内精确度，使用实验室传播的鼻病毒（FV3和CMTV）和现场收集的样本。不论培养的分离株（FV3 / CMTV）的病毒谱系为何，qPCR分析法（“ Leung”）均以比其他分析法低2个数量级的稀释度检测病毒DNA。第二个qPCR（“ Brunner”）比常规PCR（“ Mao”测定）敏感性更高。对于田间样品，Leung qPCR检测到所有已知的阳性，而毛测定PCR仅检测到2.5％的阳性样品。显示来自2个常规PCR的扩增子序列可用于推断病毒谱系。不准确的结果将使对鼻病毒的分布和患病率的估计产生偏差，并且这些发现共同强调，应根据研究目的谨慎选择分子测定。