苜蓿矮化病毒实时荧光定量PCR检测体系的建立  

Establishment of Real-time Fluorescence Quantitative PCR Detection System for Alfalfa Dwarf Virus

作　　者：党开祥 万超越 王思越 李克梅[1] 托伦巴特·毕亚洪 DANG Kai-xiang;WAN Chao-yue;WANG Si-yue;LI Ke-mei;Tuolunbate Biyahong(Key Laboratory of Prevention and Control of Invasive Alien Species in Agriculture&Forestry of the North-western Desert Oasis(Co-constructed by Ministry and Province),Ministry of Agriculture and Rural Affairs/Key Laboratory of the Pest Monitoring and Safety Control of Crops and Forests of the Universities of Xinjiang Uygur Autonomous Region/College of Agriculture,Xinjiang Agricultural University,Urumqi 83005 China;Agricultural and Animal Husbandry Development Center,Ergong Township,Xinjiang,Tacheng 834300,China)

机构地区：[1]新疆农业大学农学院/农业农村部西北荒漠绿洲农林外来入侵生物防控重点实验室(部省共建)/农林有害生物监测与安全防控重点实验室,乌鲁木齐830052 [2]新疆塔城地区塔城市二工镇农业(畜牧业)发展中心,塔城834300

出　　处：《新疆农业大学学报》2023年第3期234-239,共6页Journal of Xinjiang Agricultural University

基　　金：国家重点研发计划课题(2022YFD1401101);国家自然科学基金项目(31760708)。

摘　　要：为灵敏、快速检测紫花苜蓿(Medicago sativa L.)中的苜蓿矮化病毒(ADV),根据该病毒L蛋白保守序列设计特异性引物,建立基于SYBR GreenⅠ染料的实时荧光定量PCR检测体系,并应用该方法对田间样品和种子进行快速检测。结果表明,建立的RT-qPCR检测技术可对苜蓿中的苜蓿矮化病毒含量进行精确检测,最低检测浓度为9.8×10^(2)copies/μL,灵敏度约是常规PCR的100倍。通过对101份苜蓿样品的检测验证,该方法阳性检出率为61%,较常规PCR高6%。挑选的10份苜蓿种子中有3份被检测出携带苜蓿矮化病毒,病毒含量范围为0~3.02×10^(6)copies/μL。表明建立的RT-qPCR检测体系特异性强、灵敏度高,可应用于苜蓿矮化病毒的定量检测。In order to detect alfalfa dwarf virus(ADV)in alfalfa sensitively and rapidly,specific primers were designed based on the conserved L protein sequence of alfalfa dwarf virus,and a real-time fluorescent quantitative PCR(RT-qPCR)detection system based on SYBR GreenⅠfluorescent dye was established,and the method was applied to rapid detection of field samples and seeds with toxicity.The results showed that the established RT-qPCR assay can accurately detect the alfalfa dwarf virus content in alfalfa with a minimum detection concentration of 9.8×10^(2) copies/μL,which is 100 times more sensitive than conventional PCR.The method was validated by testing 101 alfalfa samples,with a positive detection rate of 61%,6%higher than conventional PCR.Three of the ten selected alfalfa seeds were detected as carrying alfalfa dwarf virus,with virus levels ranging from 0 to 3.02×10^(6) copies/μL.Showing that the established RT-qPCR assay system is specific and sensitive and can be applied to the quantitative detection of alfalfa dwarf virus.

关 键 词：紫花苜蓿 苜蓿矮化病毒 实时荧光定量PCR 检测体系 

分 类 号：S435.4[农业科学—农业昆虫与害虫防治]