玉米矮花叶病毒微滴数字RT-PCR检测方法的建立  

Establishment of droplet digital RT-PCR method for detecting maize dwarf mosaic virus

作　　者：范奇璇 赵振兴 冯黎霞 李献锋 周涛 张永江[2] Fan Qixuan;Zhao Zhenxing;Feng Lixia;Li Xianfeng;Zhou Tao;Zhang Yongjiang(Key Laboratory of Surveillance and Management for Plant Quarantine Pests of Ministry of Agriculture and Rural Affairs,College of Plant Protection,China Agricultural University,Beijing 100193,China;Chinese Academy of Inspection and Quarantine,Beijing 100176,China;Guangzhou Customs Technology Center,Guangzhou 510623,Guangdong Province,China)

机构地区：[1]中国农业大学植物保护学院,农业农村部植物检疫性有害生物监测防控重点实验室,北京100193 [2]中国检验检疫科学研究院,北京100176 [3]广州海关技术中心,广州510623

出　　处：《植物保护学报》2022年第5期1450-1456,共7页Journal of Plant Protection

基　　金：国家重点研发计划(2021YFD1400100,2021YFD1400103);海关总署项目(2021HK165);博士后基金(2021M693037)。

摘　　要：为准确检测进境玉米可能携带的玉米矮花叶病毒(maize dwarf mosaic virus,MDMV),基于MDMV外壳蛋白(coat protein,CP)保守基因序列,筛选3对引物和2组探针,并优化退火温度,构建基于微滴数字RT-PCR(droplet digital reverse transcription-polymerase chain reaction,ddRT-PCR)的MDMV精准检测方法。该方法可特异性检测MDMV,对MDMV CP阳性质粒样品的检测灵敏度可以达到1×10^(3)拷贝/μL,对69份进口玉米种子检测,检出5批次玉米种子携带MDMV,而荧光定量RT-qPCR仅检出4批次玉米种子,表明建立的ddRT-PCR相对于RT-qPCR有更高的灵敏度和可靠性,可用于玉米上MDMV的检测鉴定。In order to detect maize dwarf mosaic virus(MDMV), an important quarantine virus that threatens maize production, in imported maize accurately, a highly sensitive detection method based on droplet digital reverse transcription-polymerase chain reaction(ddRT-PCR) was established. Three pairs of primers and two probes were screened according to the conserved gene sequence of MDMV coat protein(CP), the annealing temperature was optimized. The results showed the established method detects MDMV specifically, and the detect limit could reach to 1×10^(3) copies/μL for MDMV CP plasmids. Five lots out of 69 imported maize seed lots were tested MDMV positive using ddRT-PCR assays while only four lots were positive using RT-qPCR, showing the ddRT-PCR method could be used for the detection and identification of MDMV in maize and is more sensitive and reliable than RT-qPCR.

关 键 词：玉米矮花叶病毒 微滴数字RT-PCR 检测 

分 类 号：S435.131[农业科学—农业昆虫与害虫防治]