文章摘要
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
投稿时间:2020-11-05  修订日期:2021-01-05
DOI:
中文关键词: 双RNA病毒  大口黑鲈  巢式RT-PCR
英文关键词: Birnavirus  Largemouth Bass  Nested RT-PCR
基金项目:国家自然科学基金(31872589)
作者单位邮编
罗明菊 上海海洋大学水产与生命学院 510380
李宁求 中国水产科学研究院珠江水产研究所 
林强 中国水产科学研究院珠江水产研究所 
牛银杰 中国水产科学研究院珠江水产研究所 
刘礼辉 中国水产科学研究院珠江水产研究所 
梁红茹 中国水产科学研究院珠江水产研究所 
罗霞 中国水产科学研究院珠江水产研究所 
付小哲 中国水产科学研究院珠江水产研究所 510380
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中文摘要:
      【目的】建立一种新型双RNA病毒的巢式RT-PCR检测方法;【方法】本研究针对大口黑鲈双RNA病毒(largemouth bass birnavirus, ,LBBV)保守基因VP1,设计特异性引物, 构建pMD-LBBV-VP1重组质粒,经过优化PCR反应条件,对方法的灵敏度、特异性进行检测,并采用该方法对实验室2017~2020年收集的304株病样进行检测;【结果】巢氏引物F1/R1、F2/R2最佳工作浓度均为4 pmol,最佳退火温度分别为64.1 ℃、61.5 ℃。当巢氏PCR扩增35个循环时,可以检测最低质粒浓度为4.15 个拷贝/μL,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10 000倍。在304个病鱼样品中,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%。【结论】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。
英文摘要:
      [Objective] The aim was to establish a nested RT-PCR assay of a novel double RNA virus. [Method] In this study, specific primers were designed for conserved gene VP1 of Largemouth Bass Birnavirus (LBBV) and pMD-LBBV-VP1 recombinant plasmid was constructed. The sensitivity and specificity of the method were detected after the PCR reaction conditions was optimized. And the 304 clinical samples of dead fish collected from 2017 to 2020 were tested using this method. [Result] The optimal concentration of primers F1/R1 and F2/R2 were 4 pmol. 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 RT-PCR, the sensitivity was increased by 10 000 times. Among the 304 diseased fish samples, 14 positive samples were detected in the first round RT-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%. [Conclusion] 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.
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