梁湘　李俊　陈慧珠　卓秋红　龙羽燕　屈达才

摘要：【目的】建立针对家蚕核型多角体病毒（BmNPV）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BmNPV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BmNPV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BmNPV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BmNPV感染早期诊断。

关键词： 家蚕核型多角体病毒（BmNPV）；家蚕血液型脓病；LAMP；早期诊断；可视化

中图分类号： S884.51 文献标志码：A 文章编号：2095-1191（2017）01-0163-06

Abstract：【Objective】The present study established loop-mediated isothermal amplification（LAMP） for visual detection of Bombyx mori nucleopolyhedrovirus（BmNPV）， in order to provide support for early diagnosis of nuclear polyhedrosis at work field. 【Method】Taking BmNPV 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 BmNPV 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 BmNPV established in this study is sensitive， quick and reliable， which is appropriate to use for early diagnosis of BmNPV infection at work field.

Key words： Bombyx mori nucleopolyhedrovirus（BmNPV）； nuclear polyhedrosis； LAMP； early diagnosis； visua-

lization

0 引言

【研究意義】家蚕血液型脓病是广西养蚕业中最常见及危害最严重的一种病毒性传染病，由于传染性强、病死率高，且目前尚无有效的治疗方法，对蚕茧生产造成巨大经济损失。家蚕血液型脓病病原为家蚕核型多角体病毒（Bombyx mori nucleopolyhedrovirus，BmNPV），其特点是病毒粒子可在多角体结构的保护下长期保存感染活力，病毒多角体在自然环境中广泛富集，极易对桑叶和蚕作环境造成污染。……