马铃薯卷叶病毒实时荧光定量PCR检测技术研究  

Study on Real Time Fluorescence Quantitative PCR Detection Technology of Potato Leaf Roll Virus

作　　者：陈兆贵 叶新友 邢澍祺 侯红因 CHEN Zhao-gui;YE Xin-you;XING shu-qi;HOU Hong-yin(School of Life Sciences,Huizhou University,Huizhou 516007,PRC)

机构地区：[1]惠州学院生命科学学院,广东惠州516007

出　　处：《湖南农业科学》2018年第9期9-12,共4页Hunan Agricultural Sciences

基　　金：广东省教育厅科技创新项目(KJCX0176);广东省科技计划项目(2013B020301009);广东省省级大学生创新训练项目(201710577040)

摘　　要：研究利用Primer Premier 5.0软件对GenBank数据库中已登录的马铃薯卷叶病毒全基因组序列进行比对,以管家基因Actin作为内参基因,以马铃薯卷叶病毒基因作为目的基因,设计特异性强的引物,采用相对定量法,建立马铃薯卷叶病毒的实时荧光定量检测体系,并利用建立的检测方法对不同马铃薯植株中的马铃薯卷叶病毒(PLRV)进行检测。结果表明:建立的马铃薯卷叶病毒实时荧光定量PCR检测体系获得的Real-time PCR扩增基线平整,指数扩增明显,斜率大,重复性好,变异系数小;马铃薯卷叶病毒cDNA被稀释10~4倍后依然能被检测出来,具有较好的灵敏度;此检测方法成功检测出患病植株中含有马铃薯卷叶病毒,并对其表达情况进行了相对定量。该方法具有高效、特异性强以及能够定量检测等优点,为从分子生物学水平检测马铃薯卷叶病毒提供了一种新手段。Primer Premier 5.0 software was used to align the whole genome sequence of potato leaf roll virus in GenBank database. The housekeeper gene Actin was used as the internal reference gene and the potato leaf roll virus gene was used as the target gene. Designed primers with strong specifcity, the real-time fuorescence quantitative detection system of potato leaf roll virus was established, and the PLRV in different potato plants was detected by the established detection method. The results showed that the real-time PCR amplifcation baseline obtained by the established fuorescence quantitative PCR detection system for potato leaf roll virus was fat, the exponential amplifcation was obvious, the slope was large, the repeatability was good, and the coeffcient of variation was small. Potato leaf roll virus cDNA can be detected after being diluted for 104 times and has good sensitivity. Potato leaf roll virus was successfully detected in diseased plants by this method, and its expression was quantifed. This method has the advantages of high effciency, strong specifcity and quantitative detection, and provides a new method for the detection of potato leaf roll virus from the molecular biological level.

关 键 词：实时荧光定量PCR 马铃薯卷叶病毒 检测 

分 类 号：Q78[生物学—分子生物学] S435.32[农业科学—农业昆虫与害虫防治]