牛副流感病毒3型RT-LAMP检测方法的建立及应用  

Establishment and Application of Loop-mediated Isothermal Amplification for Bovine Parainfluenza Virus 3

作　　者：师新川[1] 温永俊[1] 王凤雪[1] 胡嘉欣[1] 杨博超[1] 王炜[1] 宋妮[1] 程世鹏[1] 武华[1] 

机构地区：[1]中国农业科学院特产研究所,吉林长春130122

出　　处：《中国畜牧兽医》2012年第11期31-34,共4页China Animal Husbandry & Veterinary Medicine

基　　金："863"计划--"新型重大动物疫苗与诊断试剂创制与工艺创新"(2011AA10A213)

摘　　要：本试验根据GenBank中登录的牛副流感病毒3型(BPIV-3)基因序列,利用在线软件Primer Explorer V4Software和Primer Premier 5.0,针对BPIV-3 NP基因序列的保守区设计并筛选了一套环介导逆转录等温核酸扩增(RT-LAMP)引物,建立BPIV-3特异性检测的RT-LAMP方法。在Bst DNA聚合酶作用下,63℃恒温反应1h即可完成扩增过程,扩增产物通过浑浊度比较、凝胶电泳和肉眼可视化进行判定。结果表明,该方法比RT-PCR敏感度更高,最低检出量可达0.069fg/μL。该方法可用于牛副流感病毒3型的实验室检测和临床初步诊断。A set of primers used for reverse transcription loop-mediated isothermal amplification（RT-LAMP） detection was designed based on the conserved nucleoprotein gene of bovine parainfluenza virus 3（BPIV-3） complete genome sequence submitted in GenBank.The usefulness of RT-LAMP for rapid preclinical detection of BPIV-3 infection was evaluated.The reaction could be finished in 1 h under isothermal condition at 63 ℃.This RT-LAMP assay had a detection limit of 0.069 fg/μL per reaction,was higher sensitivities than that of RT-PCR.The specificity of this assay could be easily confirmed by agarose gel electrophoresis,color reaction or turbidity comparison.As a result,the RT-LAMP assay was an ideal method for detecting BPIV-3 in laboratory and primary diagnosis of clinical infection.

关 键 词：牛副流感病毒3型 N基因 反转录-环介导等温扩增 

分 类 号：S854.4[农业科学—临床兽医学]