桑花叶萎缩类病毒滚环扩增检测技术体系的建立

Construction of Rolling Circle Amplification Detection System to Mulberry Mosaic Dwarf Viroid

为建立一种快速、灵敏、简单的检测类病毒的方法,以桑花叶萎缩类病毒为研究对象,根据其基因组设计了1条特异性连接环化的寡核苷酸链探针和2条PCR扩增检测引物,对收集到的6份桑花叶型萎缩类病毒样品进行了滚环扩增。结果所有样品的RCA扩增产物的核酸电泳,条带呈阶梯状分布,但大部分聚集于上样孔附近,与理论分析相符,表明该方法适用于桑花叶萎缩类病毒的快速检测。进一步利用人工合成的模拟DNA分子测试该检测方法的灵敏度,研究显示,利用RCA技术可以成功检测到10~3拷贝的模式DNA分子。

In order to establish a rapid, sensitive and simple method for detection of viroid, one specific oligonucleotide probe for linking cyclization and two primers for mulberry mosaic dwarf viroid testing, were designed according to the genome sequence of MMDVd. The results of the rolling circle amplification of the six collected MMDVd samples showed that the bands of the RCA products in nucleic acid electrophoresis presented ladder distribution, but most gathered near the sample hole, which was consistent with the theoretical results and it indicated that the method was appropriate for the rapid detection of MMDVd. The sensitivity of the method was tested by a synthetic DNA molecule. The results showed that the sensitivity of this method could reach the level of 10-3 copies of the DNA molecules.