Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Guangzhou, 510380, China.
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Guangzhou, 510380, China; Guangzhou Key Laboratory of Aquatic Animal Diseases and Waterfowl Breeding, College of Animal Sciences and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou, Guangdong, 510225, China.
Microb Pathog. 2019 Apr;129:146-151. doi: 10.1016/j.micpath.2019.02.001. Epub 2019 Feb 4.
To distinguish between three types of Siniperca chuatsi rhabdovirus (SCRV) viral RNA (vRNA, cRNA, and mRNA) and investigate SCRV transcription and replication dynamics in Chinese perch brain CPB cells, a novel, strand-specific, reverse transcriptase quantitative real-time PCR (RT-qPCR) assay was established. The method is based on strand-specific reverse transcription, using tagged primers to add a 'tag' sequence at the 5' end. We used the 'tag' sequence as the forward primer and a strand-specific reverse primer to quantify the three types of RNA. Three types of synthetic viral RNA were used as reference standards for validation and quantification. These assays were optimized to produce a standard curve from 10 to 10 copies/μL, with an efficiency of 91-101% and an R value of 0.9949-0.9999. The coefficients of variation for repeatability and reproducibility were less than 2.85% and 5.52%, respectively. Using this method, specific target RNA was detected at a 3500-70,000 fold higher level than other types of RNA. This method was also used to evaluate the dynamics of vRNA, cRNA and mRNA synthesis in CPB cells infected with SCRV. The results indicate that the intracellular dynamics of vRNA, cRNA and mRNA are different. In the earliest phase of SCRV infection, all three types of viral RNA increased very slowly. The copy number of vRNA and mRNA increased exponentially from 4 h post infection, while cRNA increased from 6 h post infection. The amount of cRNA was lower than vRNA and mRNA throughout the infection. The novel, strand-specific RT-qPCR method developed in this study provides critical data to aid the understanding of transcription and replication during SCRV infection.
为了区分三种中华鳋鱼呼肠孤病毒（SCRV）病毒 RNA（vRNA、cRNA 和 mRNA），并研究 SCRV 在中华鳜脑 CPB 细胞中的转录和复制动态，建立了一种新型的、链特异性、逆转录定量实时 PCR（RT-qPCR）检测方法。该方法基于链特异性逆转录，使用标记引物在 5'端添加'标记'序列。我们使用'标记'序列作为正向引物和链特异性反向引物来定量三种 RNA。三种类型的合成病毒 RNA 被用作验证和定量的参考标准。这些检测方法经过优化，可在 10 至 10 拷贝/μL 的范围内产生标准曲线，效率为 91-101%，R 值为 0.9949-0.9999。重复性和再现性的变异系数分别小于 2.85%和 5.52%。使用该方法，特异性靶 RNA 的检测灵敏度比其他类型的 RNA 高 3500-70000 倍。该方法还用于评估 SCRV 感染 CPB 细胞中 vRNA、cRNA 和 mRNA 合成的动力学。结果表明，vRNA、cRNA 和 mRNA 的细胞内动力学不同。在 SCRV 感染的最早阶段，所有三种类型的病毒 RNA 增加非常缓慢。vRNA 和 mRNA 的拷贝数从感染后 4 小时开始呈指数增长，而 cRNA 则从感染后 6 小时开始增加。在整个感染过程中，cRNA 的数量始终低于 vRNA 和 mRNA。本研究中建立的新型链特异性 RT-qPCR 方法提供了关键数据，有助于了解 SCRV 感染过程中的转录和复制。
