马铃薯A病毒RT-LAMP检测方法的建立  

Development of A RT-LAMP Assay for Rapid Detection of Potato Virus A

作　　者：刘洪义 辛言言[2] 刘忠梅 杨长志 杨立群 张永江[2] 

机构地区：[1]黑龙江出入境检验检疫局检验检疫技术中心/国家马铃薯病毒检测重点实验室,哈尔滨150001 [2]中国检验检疫科学研究院,北京100123

出　　处：《中国农学通报》2015年第11期143-147,共5页Chinese Agricultural Science Bulletin

基　　金：国家质检总局科技计划项目"环介导等温扩增技术(LAMP)检测马铃薯病毒方法的研究"(2013IK274)

摘　　要：马铃薯A病毒(potato virus A,PVA)是一种侵染马铃薯的病毒,严重影响马铃薯的经济价值和产量,为有效防止该有害生物的传入和扩散,建立马铃薯A病毒RT-LAMP检测方法,根据PVA的外壳蛋白基因序列设计RT-LAMP反应的特异引物,通过实验成功建立了PVA的RT-LAMP特异性检测方法。该方法检测快速,36 min即可检测到扩增曲线;特异性强,可有效区分同样侵染马铃薯的马铃薯A病毒(PVA)、马铃薯V病毒(PVV)、马铃薯Y病毒(PVY)以及同属的百合斑驳病毒(LMo V);灵敏度高,比普通RT-PCR灵敏度高10倍;并可通过肉眼观察浊度或颜色反应直接进行结果判断。该方法适用于现场PVA的快速检测。Potato virus A（PVA） is a kind of quarantine pest which can infect potato and cause the losses ofyield and value of potato. In order to prevent the pest from entry and spreading, the one step loop-mediatedisothermal amplification（RT-LAMP） method for the detection of potato virus A（PVA） was developed. Usingprimer explorer software, RT- LAMP primers were designed originally using a conserved region in the coatprotein coding region of PVA. The amplification curve result on specificity using PVA, PVV, PVY, LMo V,negative control and water as template showed that the assay could amplify PVA specifically, which was thesame as the fluorescent detection reagent（FDR） reaction result. The sensitivity comparison showed that theRT-LAMP was 10 times more sensitive than conventional reverse transcriptase polymerase chain reaction（RT-PCR） using serial dilutions of total RNA extracts. The method can be used to detect PVA rapidly on site.

关 键 词：马铃薯A病毒 反转录-环介导等温扩增 检测 

分 类 号：S412[农业科学—植物保护]