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Development of strand-specific real-time RT-PCR for the analysis of SCRV transcription and replication dynamics

Lin, Qiang, Zhao, Yongliang, Fu, Xiaozhe, Liu, Lihui, Liang, Hongru, Niu, Yinjie, Chen, Xi, Huang, Zhibin, Lin, Li, Li, Ningqiu
Microbial pathogenesis 2019 v.129 pp. 146-151
Siniperca chuatsi, brain, messenger RNA, quantitative polymerase chain reaction, reference standards, reverse transcriptase polymerase chain reaction, reverse transcription
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 102 to 107 copies/μL, with an efficiency of 91–101% and an R2 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.