一株新型大口黑鲈双RNA病毒巢式RT-PCR检测方法的建立及应用  

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

作　　者：罗明菊 李宁求[2] 林强[2] 牛银杰 刘礼辉[2] 梁红茹[2] 罗霞[2] 付小哲[2] LUO Mingju;LI Ningqiu;LIN Qiang;NIU Yinjie;LIU Lihui;LIANG Hongru;LUO Xia;FU Xiaozhe(College of Fisheries and Life Science,ShangHai Ocean University,ShangHai 201306,China;Key Laboratory of Fishery Drug Development,Ministry of Agriculture and Rural Affairs,Key Laboratory of Aquatic Animal Immune Technology of Guangdong Province,Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou 510380,China)

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]中国水产科学研究院珠江水产研究所,农业农村部渔用药物创制重点实验室,广东省水产动物免疫技术重点实验室,广东广州510380

出　　处：《水产学报》2021年第9期1584-1591,共8页Journal of Fisheries of China

基　　金：国家重点研发计划(2019YDF0900105,2018YFD0900501);国家自然科学基金(31872589);广东省农业产业技术体系创新团队(2019KJ140,2019KJ141)。

摘　　要：为有效预防大口黑鲈双RNA病毒(largemouth bass birnavirus,LBBV),实验针对LBBV保守基因VP1设计特异性引物,构建重组质粒pMD-LBBV-VP1,通过优化PCR反应条件,提高检测方法的灵敏度和特异性,建立了大口黑鲈双RNA病毒的巢式RTPCR检测方法,并采用该方法对实验室2017—2020年收集的304株样本进行检测。结果显示,巢氏引物F1/R1、F2/R2最佳工作浓度均为4×10-12 mol,最佳退火温度分别为64.1℃和61.5℃,当巢氏RT-PCR扩增35个循环时,可以检测质粒最低浓度为4.15个/μL拷贝数,最低模拟样品浓度为102 PFU/mL,与第1轮PCR相比,灵敏度均提高了10000倍,同时检测9种不同病毒,仅LBBV出现明亮特异性条带,在304个样品中,第1轮PCR检出阳性样品14株,检出率为4.60%,巢式RT-PCR检出阳性样品28株,检出率为9.21%;本实验建立的巢式RT-PCR检测方法灵敏度高且特异性好,可用于LBBV的早期检测及防控。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 ℃ and 61.5 ℃,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.

关 键 词：大口黑鲈 双RNA病毒 巢式RT-PCR 检测方法 

分 类 号：S941.4[农业科学—水产养殖]