摘要
根据逆转录环介导等温扩增(RT-LAMP)原理,针对水疱性口炎病毒(VSV)糖蛋白G基因序列中的6个区域设计内外各1对特异引物,建立扩增VSV糖蛋白(G)基因的RT-LAMP方法。扩增产物电泳呈特异的阶梯状条带分布,扩增产物加SYBR GREEN I染色呈特征性的黄绿色,肉眼可直接观察判定。同时,还建立了可以进行定量检测的实时RT-LAMP方法。特异性试验显示,本方法可快速检验鉴别VSV与口蹄疫病毒(FMDV)和猪水泡病病毒(SVDV)。敏感性试验显示,建立的实时RT-LAMP方法检测VSVRNA的最低检测量为0.01 PFU,比实时荧光RT-PCR显著提高。建立的LAMP方法可检测p-VSVNJ质粒DNA的最低量为6.36×10-3pg/μL(1.4×103copies/μL),比PCR也显著提高。综合表明,本研究建立RT-LAMP检测VSV的方法具有特异、敏感、快速、简便的特点,具有开发应用前景。
A rapid, one-step, reverse transcription loop-mediated isothermal amplification （RT-LAMP） assay was developed in this study to enable vesicular stomatitis virus （VSV） to be detected in an hour in a single tuber without thermal cycle. The amplified product appeared a ladder-like pattern on the gel and was green after the addition of SYBR Green I dye. The real-time RT-LAMP assay was also developed for quantitation of VSV. The specificity analysis of RT-LAMP showed that the target product could be not amplified from other virus that causes vesicular diseases, such as foot-and-mouth disease virus （FMDV） and swine vesicular disease virus （SVDV）. In sensitivity, real-time RT-LAMP assay could detect 10-2 PFU of VSV-NJ, which was approximately 10-fold higher than real-time RT-PCR. Also, LAMP could detect p-VSVG plasmid at a dilution of 6. 36×10^-3 pg/μL or 1.4×10^3 copies/μL, which was about 10-fold higher than ordinary PCR. In conclusion, the RT-LAMP developed in this study is more convenient, sensitive and rapid than PCR for the detection of VSV.
出处
《畜牧与兽医》
北大核心
2009年第7期24-28,共5页
Animal Husbandry & Veterinary Medicine
基金
广东出入境检验检疫局科技项目(2003GDK-09)
