H5亚型禽流感病毒的逆转录环介导等温扩增(RT-LAMP)检测技术  

Rapid diagnosis of H5 subtype avian influenza virus by RT-loop-mediated isothermal amplification

作　　者：谢青梅[1] 曹永长[2] 张祥斌[1] 李少璃[1] 冀君[1] 马静云[1] 毕英佐[1] 

机构地区：[1]华南农业大学动物科学学院,广东广州510642 [2]中山大学生命科学学院,广东广州510275

出　　处：《中国兽医学报》2010年第1期41-46,共6页Chinese Journal of Veterinary Science

基　　金：广东省重大科技专项资助项目(2005A20901004;2006A20901003;2007A020400006)

摘　　要：根据GenBank中H5N1亚型流感病毒HA基因保守区的序列，设计1组对应HA序列6个区域的4条特异性引物进行核酸扩增，建立RT～LAMP反应体系，优化LAMP反应条件，最佳等温扩增反应条件为65℃保温60min。扩增后产物加入核酸染色剂SYBRI和4mmol／L Mg^2＋，结果可视。扩增产物可用特异性内切酶KpnI进行酶切鉴定。用已建立的RT—LAMP技术检测临床样品，检测结果与RT—PCR方法一致。RT—LAMP技术整个检测过程只需1～2h，不需要PCR仪和成像系统，仅需要一恒温水浴锅即可完成试验。通过特异性、敏感性试验，验证了建立的RT-LAMP技术可以作为一种操作简单，灵敏性、特异性高的检测H5亚型禽流感病毒的方法。In this study,a RT-LAMP （reverse transcription loop-mediated isothermal amplification） assay was developed to rapidly detect and diagnose H5 subtype avian influenza virus（AIV）. A set of four specific primers were successfully designed to recognize six distinct genomic sequences according to the conserved region of the H5 subtype AIV HA hemagglutinin gene,including one forward inner primer,one back inner primer and two outer primers. The optimum reaction temperature and time were verified to be 65℃ and 60 min, respectively. Compared to PCR,amplification efficiency of the LAMP method is extremely high,does not i＇equire complicated thermal cycling steps and denaturation,and all amplification steps are completed within about an hour. In addition,we added 4 mmol/L Mg^2＋ to precipitate RT-LAMP products, and enzymetic digestion and cross-specificity test indicated the RT-LAMP method is a simple,rapid,accurate, sensitive and specific method for detecting H5 subtype AIV. RT-LAMP assay, which is a good alternative method for on-farm diagnosis of the disease.

关 键 词：H5亚型禽流感病毒 HA RT—LAMP 

分 类 号：S852.65[农业科学—基础兽医学]