流行性乙型脑炎病毒环介导等温扩增快速检测方法的建立

【作者】 杜鹃；

【导师】 宋永； 华荣虹；

【作者基本信息】 黑龙江大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 流行性乙型脑炎(Japanese encephalitis, JE)是由乙型脑炎病毒(Japanese encephalitis virus, JEV)引起的严重的人畜共患病,对人类健康和畜牧业生产危害巨大。目前,乙型脑炎病毒的检测方法有病毒分离鉴定、免疫学检测、分子生物学检测等。这些检测方法虽然为乙型脑炎的诊断提供了强大的技术支持,但存在着耗时长,费用高等缺点。环介导等温扩增法(Loop-mediated isothermal amplification, LAMP)是一种新型的分子生物学检测技术,若能利用该技术检测流行性乙型脑炎病毒可以克服以上缺点。本研究根据JEV基因序列设计了3对特异性引物,采用LAMP方法对流行性乙型脑炎病毒核酸进行等温扩增,建立优化了两步法和一步法两种检测方法。两步法LAMP检测体系中含有0.2μMF3、B3,2μM FIP、BIP,0.8μM FLP、BLP, 0.4mM dNTP,20 mM Tris-HCI (pH8.8),10 mM KCl,10 mM (NH4)2SO4,6 mM Mg2+,0.1%Triton X-100,1.1 mM Betaine,8U BST聚合酶,于65℃水浴锅中放置60min。一步法RT-LAMP检测体系中0.2μM F3、B3,2μM FIP、BIP,0.8μM FLP、BLP,0.8mM dNTPs,20mM Tris-HCl(pH8.8), 10mMKCl,10 mM (NH4)2SO4, 6mMMg2+,0.1%Triton X-100,1.1 mM Betaine,8 U BST聚合酶,1 U AMV反转录酶,于65℃水浴锅中放置70min即可完成反应。两种检测方法均可在扩增产物中加入SYBR Green I染料,直接或在紫外光下观察颜色变化判定结果。试验表明LAMP方法比RT-PCR灵敏度高10倍,并具有较高的特异性,对猪细小病毒、猪小环状病毒、伪狂犬病毒、猪瘟病毒等类似病毒均无交叉反应。且LAMP试剂在-20℃下具有良好的稳定性。更多还原

【Abstract】 Japanese encephalitis (JE), a serious zoonose, is caused by Japanese encephalitis virus (JEV). It has great hazards on health of humanbeing and livestock breeding. Now, there are some methods to detect JEV, such as virus isolation and characterization, immunological tests and molecular biological examination. They have supplied powerful technology to the detection of JEV, but there are also some defects as for long time comsuing and high expenses. Loop-mediated isothermal amplification(LAMP) is a newly developed molecular technology, it could overcome the above defects if it could be used in JEV detection.In this study, three pairs of primers were designed based on the JEV gene sequence. Two LAMP reaction systems, two-step and one-step, were optimized to amplify nucleic acids of JEV. The two-step system amplification coulld be obtained in 60 min at 65℃by employing 0.2μM F3, B3; 2μM FIP, BIP; 0.8μM FLP, BLP; 0.4 mM dNTP; 20 mM Tris-HCl (pH8.8); 10 mM KCl; 10 mM (NH4)2SO4; 6 mM Mg2+; 0.1% Triton X-100; 1.1 mM Betaine; 8 U BST. The one-step system amplification could be obtained in 70 min at 65℃by employing 0.2μM F3, B3; 2μM FIP, BIP; 0.8μM FLP, BLP; 0.8 mM dNTPs; 20mM Tris-HCl (pH8.8); 10mM KCl; 10 mM (NH4)2SO4; 6 mM Mg2+; 0.1% Triton X-100; 1.1 mM Betaine; 8U BST; 1U AMV. Both of the results could be visualized directly or under the UV light with adding SYBR Green I dye. The sensitivity of this method was about 10 times higher than RT-PCR. There was no crossing-reaction with other similar viruses including PPV, PCV, PRV and CSFV which indicated the good specificity. LAMP reagents had excellent stability during stroage at-20℃.更多还原