改良逆转录环介导等温扩增检测技术快速实时检测H9亚型禽流感病毒  

Rapid and Real-time Detection of H9 Subtype Avian Influenza Virus by Improved Reverse Transcriptase Loop-mediated Isothermal Amplification Assay

作　　者：但晓雅[1,2] 董英[1] 邹明强[2] 薛强[2] 杜景娇[2] 

机构地区：[1]江苏大学,镇江212013 [2]中国检验检疫科学研究院,北京100123

出　　处：《生物技术通报》2012年第8期125-129,共5页Biotechnology Bulletin

基　　金：国际科技合作项目(2009DFA32720);"十一五"国家科技支撑计划(2009BAK61B04)

摘　　要：利用改良逆转录环介导等温扩增(RT-LAMP)技术建立一种快速、灵敏的检测方法用于H9亚型禽流感病毒检测。根据H9亚型禽流感病毒血凝素(HA)基因保守区序列中的8个区段设计6条特异性引物,在恒温条件下进行核酸扩增反应,并以琼脂糖凝胶电泳和目视检查绿色荧光两种方法对扩增结果进行判定。结果表明,RT-LAMP的最小检测限为100 fg,灵敏度比PT-PCR高100倍,且与H5亚型、H7亚型禽流感病毒,新城疫病毒(NDV)无交叉反应。目视检查绿色荧光与常规琼脂糖凝胶电泳的判定结果一致。整个扩增检测过程在35 min内即可完成。利用临床样本对RT-LAMP法进行验证,结果与RT-PCR一致。由实时浊度分析得到的标准曲线,计算出临床样本中的病毒质粒拷贝数均在2×107-2×104之间。因此,本研究建立的RT-LAMP方法快速、灵敏、特异性强,是H9亚型禽流感病毒的一种高效检测方法。It was to develop a rapid and high sensitive detection method for H9 subtype avian influenza virus by the improved reverse transcriptase loop-mediated isothermal amplification （ RT-LAMP ） . According to the sequences of H9 subtype AIV hemagglutinin gene, six primers specific to the eight site of HA gene were designed, and the reactions were under isothermal conditions. Detection of gene amplification could be accomplished by agarose gel electrophoresis as well as visualized by adding fluorescent reagent. The results showed that the detection limit of RT-LAMP assay was 100 fg, which was lO0-fold higher than that of RT-PCR. The assay had no cross reaction with HS, H7 subtype AIV and Newcastle disease virus （ NDV ） . Detection results were identical to using gel electrophoresis and fluorescent reagent. The amplification method can be completed within 35 rain. The comparative evaluation of the RT-LAMP assay for clinical diagnosis revealed 100% concordance with RT-PCR. The copies of virus in clinical samples was 2 ×10^7-2× 10^4, as determined from standard curve based on the time of positivity. These results suggested that the newly developed RT-LAMP assay is a sensitive and specific method for rapid detection of H9 subtype AIV infield conditions.

关 键 词：H9亚型禽流感病毒 逆转录环介导等温扩增 快速检测 实时 

分 类 号：S854.43[农业科学—临床兽医学]