摘要
本研究采用环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP),建立了一种快速、简便的烟草环斑病毒检测方法。根据TRSV外壳蛋白编码基因上的8个位点,共设计了6条引物,通过RT-LAMP扩增得到特征性的梯度条带。特异性试验表明,引物对TRSV的检测具有良好的特异性;灵敏度试验显示RT-LAMP比普通RT-PCR高10倍。通过反应温度和时间的优化,该方法只需在水浴锅中60℃等温扩增60 min,整个检测周期约1.5 h,结果采用SYBR green I染色显示,易于观察和判定。
The study established a rapid and simple Tobacco ringspot virus (TRSV)detection method using Loop-mediated isothermal amplification (LAMP). According to the eight sites on TRSV coat protein coding genes, six primers were designed and characteristic ladder-like bands were obtained by RT-LAMP. Specificity tests showed that the primers were specific for detection of TRSV and sensitivity test showed that RT-LAMP was 10 times higher than RT-PCR. By the optimizing of temperature and time condition, the method just need the pot with a water bath 60℃ isothermal amplification for 60 min, the entire testing cycle is about 1.5 h, and it is very convenient to observe the results by using SYBR green I.
出处
《植物病理学报》
CAS
CSCD
北大核心
2014年第6期629-633,共5页
Acta Phytopathologica Sinica
基金
宁波市科研项目(2011C50080)
国家质检总局项目(2013IK279)
关键词
烟草环斑病毒
环介导等温扩增
检测
Tobacco ringspot virus (TRSV)
Loop-mediated isothermal amplification (LAMP)
detection