十足目虹彩病毒(DIV1)环介导等温扩增(LAMP)检测方法的建立及应用

Establishment and Application of the LAMP Detection Method for Decapod Iridescent Virus 1 (DIV1)

中文关键词: 十足目虹彩病毒(DIV1)  虾血细胞虹彩病毒(SHIV)  环介导等温扩增(LAMP)  检测方法

英文关键词: Decapod iridescent virus 1 (DIV1)  Shrimp hemocyte iridescent virus (SHIV)  Loop-mediated isothermal amplification (LAMP)  Detection method

中文摘要:

      本研究以十足目虹彩病毒(Decapod iridescent virus 1, DIV1)主要衣壳蛋白基因为靶序列设计引物，建立了DIV1的环介导等温扩增(Loop-mediated isothermal amplification, LAMP)检测方法，并以pMD18-DIV1质粒标准品为模板对该方法的检测灵敏度、检测特异性等进行了评估。结果显示，此方法最适反应温度为64.4℃，优化后的25 μl反应体系中包含2.5 μl 10×Isothermal amplification buffer、4.0 mmol/L Mg2+、1.2 mmol/L dNTPs、6.4 U Bst 2.0 WarmStart® DNA聚合酶、0.8 μmol/L EvaGreen®和4.4 μl ddH2O。该方法检测灵敏度下限为3.54×102拷贝/反应；与虾肝肠胞虫(EHP)、致急性肝胰腺坏死病副溶血弧菌(VpAHPND)、对虾偷死野田村病毒(CMNV)、传染性皮下及造血组织坏死病病毒(IHHNV)、白斑综合征病毒(WSSV)、桃拉综合征病毒(TSV)和黄头病毒(YHV)等主要虾类病原没有交叉反应；具有较好的重复性和稳定性。以GeneFinder®替换EvaGreen®并将其预置于反应管内，结合上述扩增方法可实现对DIV1的现场快速高灵敏检测。本研究建立的DIV1-LAMP实时荧光定量和现场检测方法具有灵敏、特异和快速等特点，为近几年新发虾类病原DIV1的定性、定量以及现场快速检测提供了新的技术选择，有利于对虾养殖业中开展DIV1的监测、预警和防控。

英文摘要:

      The loop-mediated isothermal amplification (LAMP) detection method of Decapod iridescent virus 1 (DIV1) was established and optimized based on primers designed from the DIV1 capsid protein gene in present study. Analytic sensitivity of the newly established method was assessed using the pMD18-DIV1 plasmid standard as a template, and the detection specificity was also determined. The results showed that the optimal reaction temperature for the DIV1-LAMP method was 64.4℃, and the optimized 25 μl reaction system contained 2.5 μl 10×isothermal amplification buffer, 4.0 mmol/L Mg2+, 1.2 mmol/L dNTPs; 6.4 U Bst 2.0 WarmStart® DNA polymerase and 0.8 μmol/L EvaGreen®. The detection limit of the new method was 3.54×102 copies/reaction; it did not cause a cross-reaction with major shrimp pathogens such as Enterocytozoon hepatopenaei (EHP), Vibrio causing acute hepatopancreatic necrosis disease (VpAHPND), covert mortality nodavirus (CMNV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), white spot syndrome virus (WSSV), Taura syndrome virus (TSV), or yellow head virus (YHV). The newly developed method showed good repeatability and stability. The on-site, rapid, highly sensitive detection method of DIV1 was established by replacing EvaGreen® with GeneFinder® which was preset in the cap of the reaction tube. The DIV1-LAMP real-time fluorescence and on-site LAMP detection method established in this study is sensitive, specific, and rapid. This novel method will provide new technical options for the qualitative, quantitative, and rapid on-site detection of the emerging DIV1, which will benefit the shrimp farming industry by facilitating the monitoring, early warning, and prevention of DIV1.