牛副流感病毒3型RT-LAMP检测方法的建立及应用

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 V4 Software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0.069 fg/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 preciinical 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 elec-trophoresis, 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.