H9亚型禽流感病毒环介导等温扩增检测方法的建立与荧光指示剂的应用  

Detection of H9 avian influenza virus by Loop-mediated Isothermal Amplification and Application of Fluorescent Reagent

作　　者：蒋菲[1] 黄书林[1] 徐威[1] 张跃伟[1] 刘金华[1] 吴文学[1] 

机构地区：[1]中国农业大学动物医学院,北京100193

出　　处：《农业生物技术学报》2011年第1期191-196,共6页Journal of Agricultural Biotechnology

基　　金：农业部重点项目(No.2008-G57);2009年国家大学生创新性实验计划;中国农业大学URP计划资助

摘　　要：利用反转录环介导等温核酸扩增技术(RT-LAMP),建立了一种特异、灵敏、便捷的H9亚型禽流感病毒(H9subtype of avian influenza virus,H9-AIV)的检测方法。该方法使用了对应于H9亚型禽流感病毒血凝素(hemagglutinin HA)基因的8个不同区域的6条特异引物,在63℃的等温条件下反应,最低可检测到103拷贝的目的基因重组质粒片段,较RT-PCR方法敏感10倍。通过对15种H亚型禽流感病毒、新城疫病毒(Newcastle disease virus,NDV)、传染性支气管炎病毒(Infectious bronchitis virus,IBV)的检测表明,该方法具有良好的特异性。在反应体系中使用钙黄绿素与Mn2+的混合溶液作为荧光指示剂可以用于RT-LAMP结果判定,并且其判断结果与浊度判断结果一致。对109份禽咽喉拭子及泄殖腔拭子临床样品进行检测,RT-LAMP与RT-PCR检出阳性样本数分别为61份和46份,表明RT-LAMP方法阳性检出率高于RT-PCR。Reverse transcription loop-mediated isothermal amplification （RT-LAMP） was used to develop a specific,sensitive and convenient method for detection of H9 subtype of Avian influenza virus（H9-AIV）. Six primers directing to eight recognition sites on the hemagglutinin（HA） gene of H9-AIV were used,and at least as 103 copies of the target gene fragment could be detected after reaction for 60 minutes at 63℃,with a sensitivity of 10-fold higher than that of RT-PCR. High species-specificity of the RT-LAMP method was confirmed by the assay of H1~H15 hemagglutinin（HA） subtypes Avian influenza viruses,Newcastle disease virus（NDV） and Infectious bronchitis virus（IBV）. A mixture of calcein and Mn2＋ was used as fluorescent reagent to make the result of RT-LAMP visible,and similar results could be obtained as the turbidity test. Furthermore,109 clinical samples were assayed by RT-LAMP and RT-PCR,of which 61 and 46 samples were confirmed as positive by the RT-LAMP and RT-PCR,respectively. So we could draw a conclusion that the sensitivity of RT-LAMP was preferable to the RT-PCR assay.

关 键 词：反转录环介导等温扩增 H9亚型禽流感病毒 RT-PCR 荧光指示剂 

分 类 号：S852.659.5[农业科学—基础兽医学]