文章摘要
虾血细胞虹彩病毒(Shrimp hemocyte iridescent virus, SHIV)LAMP检测方法的建立及应用
Establishment and application of LAMP detection method for shrimp hemocyte iridescent virus (SHIV)
投稿时间:2020-05-24  修订日期:2020-05-31
DOI:
中文关键词: 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: Shrimp hemocyte iridescent virus (SHIV)  loop-mediated isothermal amplification (LAMP)  detection method
基金项目:
作者单位E-mail
邹莹 中国水产科学研究院黄海水产研究所 农业农村部海水养殖病害防治重点实验室 青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室 青岛市海水养殖流行病学与生物安保重点实验室 青岛 15216481627@163.com 
郭晓萌 中国水产科学研究院黄海水产研究所 农业农村部海水养殖病害防治重点实验室 青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室 青岛市海水养殖流行病学与生物安保重点实验室 青岛  
万晓媛 中国水产科学研究院黄海水产研究所 农业农村部海水养殖病害防治重点实验室 青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室 青岛市海水养殖流行病学与生物安保重点实验室 青岛  
邱亮 中国水产科学研究院黄海水产研究所 农业农村部海水养殖病害防治重点实验室 青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室 青岛市海水养殖流行病学与生物安保重点实验室 青岛  
张庆利 中国水产科学研究院黄海水产研究所 农业农村部海水养殖病害防治重点实验室 青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室 青岛市海水养殖流行病学与生物安保重点实验室 青岛 zhangql@ysfri.ac.cn 
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中文摘要:
      本研究以虾血细胞虹彩病毒(Shrimp hemocyte iridescent virus, SHIV)主要衣壳蛋白基因为靶基因设计引物,建立、优化了SHIV的LAMP检测方法,并以pMD18-SHIV质粒标准品为模板分析了所建立方法的灵敏度,测定了其检测特异性。结果显示,此方法最适反应温度为64.4℃,优化后的25 μL反应体系中含2.5 μL 10×Isothermal Amplification Buffer,4.0 mM Mg^2+,1.2 mM dNTPs;6.4 U Bst 2.0 WarmStart? DNA Polymerase和0.8 μM Eva Green和4.4 μL ddH2O。该方法检测灵敏度下限为3.54×10^2拷贝/反应;与EHP、VpAHPND、CMNV、IHHNV、WSSV、TSV和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Eva Green并将其预置于反应管内,可以实现对SHIV的现场快速高灵敏检测。本研究建立的SHIV实时荧光和现场LAMP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SHIV的定性、定量以及现场快速检测提供了新的技术选择,有利于对虾养殖业开展SHIV的监测、预警和防控。
英文摘要:
      LAMP detection method of shrimp hemocyte iridescent virus (SHIV) was established and optimized based on the primers designed from the capsid protein gene of SHIV in this study. Analytic sensitivity of the newly established method were assessed by using the pMD18-SHIV plasmid standard as a template, and the detection specificity was also determined. The results showed that the optimal reaction temperature for SHIV-LAMP method was 64.4°C, and the optimized 25 mL reaction system contained 2.5 mL 1 × Isothermal Amplification Buffer, 4.0 mM Mg2+, 1.2 mM dNTPs; 0.8 mL Bst 2.0 WarmStart? DNA Polymerase and 0.8 mL Eva Green. The detection limit of the new method was 3.54 × 102 copies/reaction; it did not causing cross-react with major shrimp pathogens such as EHP, VpAHPAD, CMNV, IHHNV, WSSV, TSV and YHV; the new developed method showed good repeatability and stability. The on-site using, rapid, highly sensitive detection method of SHIV was established by replacing Eva Green with equal volume Genefinder and preseting it in the reaction tube cap. The SHIV real-time fluorescence and on-site LAMP detection method established in this study possessed the characteristics of sensitivity, specificity and rapidity, which provided new technical options for the qualitative, quantitative and rapid on-site detection of new shrimp pathogen SHIV in recent years, which was beneficial to the shrimp farming industry to carry out SHIV monitoring, early warning and prevention.
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