牛副流感病毒3型RT-LAMP检测方法的建立及应用

›› 2012, Vol. 39 ›› Issue (11): 31-34.

牛副流感病毒3型RT-LAMP检测方法的建立及应用

师新川, 温永俊, 王凤雪, 胡嘉欣, 杨博超, 王炜, 宋妮, 程世鹏, 武华

中国农业科学院特产研究所,吉林长春 130122

收稿日期:2012-03-27 出版日期:2012-11-20 发布日期:2012-11-22
通讯作者: 武华,研究员,博士生导师。E-mail:wuhua@bvbio.com E-mail:wuhua@bvbio.com
作者简介:师新川(1982-),男,河南人,硕士,研究方向:动物疫苗与分子免疫学。
基金资助:
"863"计划——"新型重大动物疫苗与诊断试剂创制与工艺创新"(2011AA10A213)。

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

SHI Xin-chuan, WEN Yong-jun, WANG Feng-xue, HU Jia-xin, YANG Bo-chao, WANG Wei, SONG Ni, CHENG Shi-peng, WU Hua

Institute of Special Economic Animal and Plant Science,Chinese Academy of Agricultural Sciences,Changchun 130122,China

Received:2012-03-27 Online:2012-11-20 Published:2012-11-22

摘要/Abstract

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1 h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。

关键词: 牛副流感病毒3型; N基因; 反转录-环介导等温扩增

Abstract: 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.

Key words: bovine parainfluenza virus type 3; nucleoprotein gene; reverse transcription loop-mediated isothermal amplification

中图分类号:

S854.4

师新川, 温永俊, 王凤雪, 胡嘉欣, 杨博超, 王炜, 宋妮, 程世鹏, 武华. 牛副流感病毒3型RT-LAMP检测方法的建立及应用[J]. , 2012, 39(11): 31-34.

SHI Xin-chuan, WEN Yong-jun, WANG Feng-xue, HU Jia-xin, YANG Bo-chao, WANG Wei, SONG Ni, CHENG Shi-peng, WU Hua. Establishment and Application of Loop-mediated Isothermal Amplification for Bovine Parainfluenza Virus 3[J]. , 2012, 39(11): 31-34.

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