家蚕核型多角体病毒LAMP可视化检测技术的建立  

Establishment of loop-mediated isothermal amplification(LAMP)assay for visual detection of Bombyx mori nucleopolyhedrovirus

作　　者：梁湘[1,2] 李俊[1] 陈慧珠[1] 卓秋红 龙羽燕 屈达才[1] 

机构地区：[1]广西大学农学院,南宁530004 [2]亚热带农业生物资源保护与利用国家重点实验室,南宁530004

出　　处：《南方农业学报》2017年第1期163-168,共6页Journal of Southern Agriculture

基　　金：广西科学研究与技术开发计划项目(桂科重14121002-4-1)

摘　　要：【目的】建立针对家蚕核型多角体病毒(Bm NPV)的环介导等温扩增(LAMP)可视化检测技术,为生产现场进行家蚕血液型脓病早期诊断提供技术支持。【方法】以Bm NPV多角体蛋白基因polh为扩增靶标,分别设计5组内/外引物和5条环引物,根据扩增效率筛选最佳引物组合;优化LAMP反应条件,并进行LAMP检测的特异性、敏感性及临床样本检测试验;使用羟基萘酚蓝(HNB)染色对结果进行比色观察,对简化样品处理的条件进行摸索。【结果】使用环引物后LAMP对Bm NPV基因组DNA的最低检测浓度为7 fg/μL,检测灵敏度得到有效提高,且反应时间缩短。建立的LAMP检测方法对靶标DNA扩增具有特异性,对样本的检出率高于常规PCR,其最低检测限是常规PCR的100倍。在反应前加入HNB,结果易判断且能减少交叉污染。感染家蚕血淋巴进行100℃煮沸处理后即可直接用于LAMP反应,既简化了操作步骤,又降低了检测成本。【结论】针对Bm NPV建立的LAMP可视化检测技术具有灵敏、快捷、可靠的特点,适合用于生产现场的Bm NPV感染早期诊断。【Objective 】The present study established loop-mediated isothermal amplification（LAMP） for visual detection of Bombyx mori nucleopolyhedrovirus（Bm NPV）, in order to provide support for early diagnosis of nuclear polyhedrosis at work field. 【Method】Taking Bm NPV polyhedrin gene polh as amplification target, five groups of inner primer/outer primer and five pieces of loop primer were designed in order to screen the best primer combination based on amplification efficiency. LAMP reaction conditions were optimized and assays on specificity, sensibility as well as clinical sample detection were conducted. The amplification result was observed by colorimetric determination with hydroxynaphthol blue（HNB） staining. The conditions for simplifying treatment were analyzed. 【Result】When using LAMP of loop primer, the lowest detection line of Bm NPV genomic DNA could reach 7 fg/μl, the sensitivity of detection was improved effectively and reaction time was reduced. The established LAMP detection specifically amplified the target DNA, and its sample detection rate was superior to conventional PCR. The lowest detection line was as 100 times as that of conventional PCR. The result could be easily judged and the cross-contamination could be reduced by adding HNB before reaction. The hemolymph of infected silkworm boiled at 100 ℃ could be directly applied to LAMP, which could simplify operation steps and reduce detecting cost. 【Conclusion】The visual LAMP detection targeting Bm NPV established in this study is sensitive,quick and reliable, which is appropriate to use for early diagnosis of Bm NPV infection at work field.

关 键 词：家蚕核型多角体病毒(Bm NPV) 家蚕血液型脓病 LAMP 早期诊断 可视化 

分 类 号：S884.51[农业科学—特种经济动物饲养]