Establishment and application of the nested RT-PCR for the detection of a novel largemouth bass birnavirus (LBBV)

Early detection of pathogen is particularly important for the early warning, prevention and control of diseases. In recent years, a new viral disease occurred in farmed Micropterus salmoides in Guangdong Province and its pathogen is largemouth bass birnavirus (LBBV); In order to prevent the virus effectively, specific primers were designed for the conserved gene VP1 of LBBV to construct the recombinant plasmid pMD-LBBV-VP1 in this study. And the sensitivity and specificity of the method were tested after the PCR reaction conditions were optimized. The nested RT-PCR method for detecting the LBBV was established and the 304 clinical samples of dead fish collected from 2017 to 2020 were tested using this method; The results show that the optimal concentration of primers F1/R1 and F2/R2 were 4×10⁻¹² mol. And the optimal annealing temperatures were 64.1 °C and 61.5 °C, respectively. When PCR amplification was performed for 35 cycles, a minimum plasmid concentration of 4.15 copies/µL and a minimum simulated sample concentration of 10² PFU/mL could be detected. Compared with the first round PCR, the sensitivity was increased by 10 000 times. 9 different viruses were detected at the same time and only LBBV showed bright specific bands. Among the 304 diseased fish samples, 14 positive samples were detected in the first round PCR with a detection rate of 4.60% and 28 positive samples were detected in the nested RT-PCR with a detection rate of 9.21%. The nested RT-PCR assay established in this study has high sensitivity and good specificity, which can be used for the early detection and prevention of LBBV.