基于一步法RT-PCR的瓜类单粒种子携带CGMMV的快速检测技术  

Rapid Detection of CGMMV from Individual Cucurbitaceous Seed Based on One-step RT-PCR Methods

作　　者：蒋庆琳 周国辉[2] 

机构地区：[1]桂林市植物保护站,广西桂林541213 [2]华南农业大学资源环境学院,广东广州510642

出　　处：《热带作物学报》2014年第9期1860-1865,共6页Chinese Journal of Tropical Crops

基　　金：广东省动植物防疫检疫研究专项(No.粤农保[2008]63)

摘　　要：为探究直接检测瓜类单粒种子携带黄瓜绿斑驳花叶病毒(CGMMV)的快速检测技术,以一步法RT-PCR为基础,构建同时扩增CGMMV基因组3′端和5′端基因的(一步法)双重RT-PCR、双重IC-RT-PCR和巢式双重IC-RT-PCR技术,灵敏度分别为10-3、10-3、10-4,巢式双重IC-RT-PCR灵敏度比双重RT-PCR、双重IC-RTPCR高10倍。对不同种质、不同来源的种子及种子的不同部位进行检出率比较,巢式双重IC-RT-PCR检出率高于其它2个检测技术,试验种子种表带毒检出率高于种子组织研磨物,商用种子种表带毒检出率低于种子组织研磨物。为有效阻断毒源,建议同时对种表和种子组织研磨物进行检测,种表采用双重RT-PCR检测,种子组织研磨物采用双重IC-RT-PCR或巢式双重IC-RT-PCR检测。A comparative study on three one-step RT-PCR-based methods for rapid detection of CGMMV from individual cucurbitaceous seed is presented.Specific primers based on the 3＇-and 5＇-terminal regions of the CGMMV genome were screened and used for duplex RT-PCR,IC-RT-PCR,and nested-IC-RT-PCR detection.The results showed that the sensitivities of the three methods were 10-3,10-3 and 10-4 respectively,with the latter method being the most sensitive.The nested-IC-RT-PCR method had higher detection rates than the other two methods in the detection of the seeds from different germplasms,sources or parts as well.In addition,the resuhs showed that CGMMV detection rates obtained from the surface of experimental seeds were higher than those from ground seed tissue,and that from the surface of commercial seeds were lower than the latter.For effective quarantine,we suggest both the seed surface and ground seed tissue be subjected to RT-PCR detection,the former by duplex RT-PCR and the latter by duplex IC-RT-PCR or nested-IC-RT-PCR.

关 键 词：黄瓜绿斑驳花叶病毒 双重RT-PCR 双重IC-RT-PCR 巢式双重IC-RT-PCR 种子检测 

分 类 号：S41-31[农业科学—植物保护]