Zhao Julie, Vendramin Niccolò, Cuenca Argelia, Polinski Mark, Hawley Laura M, Garver Kyle A
Pacific Biological Station, Department of Fisheries and Oceans, Nanaimo, BC V9T 6N7, Canada.
Unit for Fish and Shellfish Diseases, National Institute of Aquatic Resources, Technical University of Denmark, 2800 Lyngby-Taarbæk, Denmark.
Pathogens. 2021 Nov 27;10(12):1548. doi: 10.3390/pathogens10121548.
Piscine orthoreovirus (PRV) infects farmed and wild salmon and trout species in North America, South America, Europe, and East Asia. PRV groups into three distinct genotypes (PRV-1, PRV-2, and PRV-3) that can vary in distribution, host specificity, and/or disease potential. Detection of the virus is currently restricted to genotype specific assays such that surveillance programs require the use of three assays to ensure universal detection of PRV. Consequently, herein, we developed, optimized, and validated a real-time reverse transcription quantitative PCR assay (RT-qPCR) that can detect all known PRV genotypes with high sensitivity and specificity. Targeting a conserved region at the 5' terminus of the M2 segment, the pan-PRV assay reliably detected all PRV genotypes with as few as five copies of RNA. The assay exclusively amplifies PRV and does not cross-react with other salmonid viruses or salmonid host genomes and can be performed as either a one- or two-step RT-qPCR. The assay is highly reproducible and robust, showing 100% agreement in test results from an inter-laboratory comparison between two laboratories in two countries. Overall, as the assay provides a single test to achieve highly sensitive pan-specific PRV detection, it is suitable for research, diagnostic, and surveillance purposes.
鱼类正呼肠孤病毒（PRV）感染北美洲、南美洲、欧洲和东亚的养殖及野生鲑鱼和鳟鱼品种。PRV分为三种不同的基因型（PRV-1、PRV-2和PRV-3），其分布、宿主特异性和/或致病潜力可能有所不同。目前病毒检测仅限于基因型特异性检测，因此监测计划需要使用三种检测方法以确保对PRV进行全面检测。因此，我们在此开发、优化并验证了一种实时逆转录定量PCR检测方法（RT-qPCR），该方法能够以高灵敏度和特异性检测所有已知的PRV基因型。针对M2片段5'末端的保守区域，泛PRV检测方法能够可靠地检测所有PRV基因型，RNA拷贝数低至五个时也能检测到。该检测方法仅扩增PRV，不与其他鲑鱼病毒或鲑鱼宿主基因组发生交叉反应，可作为一步法或两步法RT-qPCR进行。该检测方法具有高度的可重复性和稳健性，在两个国家的两个实验室之间进行的实验室间比较中，测试结果显示出100%的一致性。总体而言，由于该检测方法提供了一种单一检测方法来实现高度灵敏的泛特异性PRV检测，因此适用于研究、诊断和监测目的。
