H5亚型禽流感病毒环介导等温扩增检测方法的建立

发布时间：2018-06-01 12:10

  本文选题：反转录环介导等温扩增(RT-LAMP) + H亚型禽流感病毒(H-AIV)　； 参考：《农业生物技术学报》2016年09期

【摘要】：本研究利用反转录环介导等温扩增(reverse transcription loop-mediated isothermal amplification,RT-LAMP)技术建立了H5亚型禽流感病毒(Avian influenza virus,AIV)检测方法。首先,针对H5亚型禽流感病毒血凝素(hemagglutinin,HA)基因8个保守区域设计了3对扩增引物。然后,添加到RT-LAMP反应体系中,并加入优选的冻干保护剂进行冷冻干燥。取等温扩增冻干试剂,用双蒸水溶解并恢复至原体积后,在63℃反应1 h,可检出102拷贝的目的基因,比RT-PCR方法敏感约10倍,并且证明冷冻干燥对等温扩增反应敏感性没有影响。另外,利用等温扩增冻干试剂对禽流感病毒、新城疫病毒(Newcastle disease virus,NDV)和传染性支气管炎病毒(Infectious bronchitis virus,IBV)进行检测,结果显示特异性良好。为了便于临床应用,在反应结束后加入由SYBR Green I与羟基萘酚蓝(hydroxynaphthol blue,HNB)组成的显色液,可通过肉眼观察颜色变化判定RT-LAMP结果,与琼脂糖凝胶电泳方法判定结果一致。为考察等温扩增冻干试剂的临床应用效果,对326份临床样品进行了检测。结果表明,RT-LAMP与RT-PCR检出阳性样本数分别为38份和31份,表明RT-LAMP方法阳性检出率高于RT-PCR方法。因此,本研究建立的H5亚型流感病毒RT-LAMP反应体系,可以冻干处理并经显色观察结果,便于研制成试剂盒产品,其敏感性高、特异性好、操作简捷,利于在基层兽医实验室和养殖场推广应用。
[Abstract]:In this study, a reverse transcription loop-mediated isothermal amplification assay (RT-LAMPP) technique was used to detect avian influenza virus (Avian influenza virus) of H5 subtype. Firstly, three pairs of primers were designed for 8 conserved regions of hemagglutinin (H5) gene of H5 subtype avian influenza virus. Then, it was added to RT-LAMP reaction system and freeze-dried protective agent was added. Isothermal amplification freeze-dried reagent was extracted, dissolved and restored to its original volume with double distilled water. After 1 hour reaction at 63 鈩，

本文编号：1964133