三种无花果病毒的多重RT-PCR检测  

Multiplex RT-PCR Detection of Three Fig Viruses

作　　者：吐逊艾力·艾孜提力 侯婉莹[3] 郭庆元[1,4] 古丽尼沙·卡斯木 代毅 李世访 麦合木提江·米吉提[1,4] Tuxunaili·Aizitili;Hou Wanying;Guo Qingyuan;Gulinisha·Kasimu;Dai Yi;Li Shifang;Maihemutijiang·Mijiti(College of Agriculture,Xinjiang Agricultural University,Urumqi,830052;State Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Science,Beijing,100193;Institute of Tobacco,Chinese Academy of Agricultural Science,Qingdao,266101;Key Laboratory of the Pest Monitoring and Safety Control of Crops and Forests of the Universities of the Xinjiang Uygur Autonomous Region,Urumqi,830052;Xinjiang Academy of Forestry,Urumqi,830052)

机构地区：[1]新疆农业大学农学院,乌鲁木齐830052 [2]中国农业科学院植物保护研究所,植物病虫害生物学国家重点实验室,北京100193 [3]中国农业科学院烟草研究所,青岛266101 [4]新疆自治区高校农作林有害生物监测与安全防控重点实验室,乌鲁木齐830052 [5]新疆林业科学院,乌鲁木齐830052

出　　处：《分子植物育种》2021年第16期5414-5420,共7页Molecular Plant Breeding

基　　金：新疆农业大学作物学重点学科项目(XNCDKY2017003);中央级公益性科研院所基本科研业务费专项(Y2018PT67);自治区天山创新团队计划(2020D14002);新疆维吾尔自治区《百名青年博士引进计划》共同资助。

摘　　要：为了快速检测危害无花果的病毒种类,本研究建立了一种同时检测3种无花果病毒的多重RT-PCR检测体系,包括无花果斑点相关病毒(fig fleck-associated virus, FFkaV)、无花果叶斑相关病毒(fig leaf mottleassociated virus, FLMaV)和无花果花叶病毒(fig mosaic virus, FMV)。根据三种病毒基因保守区域设计特异性引物,分别对cDNA用量、dNTPs用量、EasyPfu DNA聚合酶用量、MgSO_(4)用量、退火温度和引物组合进行优化,并利用RT-PCR进行验证。结果显示,总体积为25μL的反应体系中EasyPfu DNA聚合酶(2.5 U/μL)用量0.7μL、cDNA用量1.0μL、dNTPs (2.5 mmol/L)用量2.5μL、MgSO_(4)(50 mmol/L)用量1.2μL、引物组合为1.5μL,1.0μL,0.3μL、ddH_(2)O为14μL、退火温度56℃时能够扩增出清晰的目的条带,该检测体系的检测极限为6.8 ng/μL的cDNA。田间样品的检测结果表明,该方法能够快速、准确地检测这3种无花果病毒,可用于田间无花果样品的检测。本研究建立的多重RT-PCR检测体系为无花果病毒的检测与防控提供技术支持。To rapidly detect of different fig viruses, we established a multiplex RT-PCR detection system for three major kinds of fig viruses, fig fleck-associated virus(FFkaV), fig leaf mottle-associated virus(FLMaV) and fig mosaic virus(FMV). Specific primers of these three viruses were designed according to their conserved regions. The optimization of the detection system was performed for the amount of cDNA, dNTPs, DNA polymerase and Mg SO4,annealing temperature and the concentration of each primer. The optimal detection system in a volume of 25 μL contains 0.7 μL of EasyPfu DNA polymerase(2.5 U/μL), 1.0 μL of cDNA, 2.5 μL of dNTPs(2.5 mmol/L), 1.2 μL of MgSO_(4)(50 mmol/L), 1.5, 1.0, 0.3 μL of primers of FFkaV, FLMa V, and FMV, and 14 μL of ddH_(2)O. This detection system can efficiently amplify the target bands using annealing temperature of 56 ℃. In addition, the detection limitation of this system was determined by detecting a series of cDNA dilution, resulting in 6.8 ng/μL of c DNA.The detection results of field samples show that this method can rapidly and accurately detect these three fig viruses, and can be used for the detection of field fig samples. The multiple RT-PCR detection system established in this study provides technical support for the detection, prevention and control of fig virus.

关 键 词：无花果 病毒 多重RT-PCR 

分 类 号：S436.639[农业科学—农业昆虫与害虫防治]