基于重组酶聚合酶扩增技术的玉米矮花叶病毒快速检测方法的建立  

Establishment of a fast detection method for maize dwarf mosaic virus by recombinase polymerase amplification

作　　者：赵振兴 范奇璇 冯黎霞 李献锋 张永江[1] ZHAO Zhenxing;FAN Qixuan;FENG Lixia;LI Xianfeng;ZHANG Yongjiang(Chinese Academy of Inspection and Quarantine,Beijing 100176,China;College of Plant Protection,China Agricultural University,Beijing 100193,China;Guangzhou Customs Technology Center,Guangzhou 510623,China)

机构地区：[1]中国检验检疫科学研究院,北京100176 [2]中国农业大学植物保护学院,北京100193 [3]广州海关技术中心,广州510623

出　　处：《植物保护》2022年第6期83-89,共7页Plant Protection

基　　金：国家重点研发计划(2021YFC2600400,2021YFC2600402);海关总署项目(2021HK165);中国博士后科学基金(2021M693037)。

摘　　要：玉米Zea mays L.是重要的粮饲兼用作物。重要检疫性病毒玉米矮花叶病毒maize dwarf mosaic virus(MDMV)一直威胁玉米生产,为预防外来有害生物入侵,本研究以玉米可能携带的MDMV为目标,根据MDMV外壳蛋白(coat protein,CP)保守基因序列,基于重组酶聚合酶扩增技术(recombinase polymerase amplification,RPA),设计了3对RPA引物,筛选并优化了反应体系,建立了MDMV的快速检测方法,最终的RPA反应体系中引物终浓度为0.4μmol/L,反应温度为42℃,反应时间为20 min。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10^(5)拷贝/μL(≈369 fg/μL),高于RT-PCR的检测灵敏度。该方法具有快速准确等优点,可用于玉米种子或植株中携带MDMV的检测。Zea mays L.is an important food and forage crop.Maize dwarf mosaic virus(MDMV)as an important quarantine virus threatens maize production.In this study,to establish a rapid detection method based on recombinase polymerase amplification(RPA),three pairs of RPA primers were designed according to the conserved gene sequence of MDMV CP,the RPA primers were screened,and the reaction system was optimized.The final concentration of primers was 0.4μmol/L,reaction temperature was 42℃and reaction time was 20 min.The results showed the established method detects MDMV specifically,and the detect limit could reach to 10^(5) copies/μL(≈369 fg/μL)for MDMV CP plasmids,which is more sensitive than RT-PCR method.In conclusion,this study established a rapid and accurate RPA detection method for MDMV in maize samples,which could be used for the detection and identification of MDMV in maize.

关 键 词：玉米矮花叶病毒 重组酶聚合酶扩增 检测 

分 类 号：S435.131.4[农业科学—农业昆虫与害虫防治]