南美红辣椒脉斑驳病毒RT-LAMP检测体系的建立及优化  

Establishment and optimization of RT-LAMP detection system for chilli veinal mottle virus

作　　者：张俊蕾 盖晓彤 赵正婷 刘弟 陈敏 姜宁 刘雅婷 ZHANG Junlei;GAI Xiaotong;ZHAO Zhengting;LIU Di;CHEN Min;JIANG Ning;LIU Yating(Yunnan Academy of Tobacco Agricultural Sciences,Kunming 650021,China;School of Plant Protection,Yunnan Agricultural University,Kunming 650201,China;School of Agriculture and Biotechnology,Yunnan Agricultural University,Kunming 650201,China;School of Tobacco Science,Yunnan Agricultural University,Kunming 650201,China;Zhaotong Plant Protection and Inspection Station,Yunnan Province,Zhaotong 657000,China)

机构地区：[1]云南省烟草农业科学研究院,昆明650021 [2]云南农业大学植物保护学院,昆明650201 [3]云南农业大学农学与生物技术学院,昆明650201 [4]云南农业大学烟草学院,昆明650201 [5]云南省昭通市植保植检站,昭通657000

出　　处：《植物保护》2023年第6期208-216,共9页Plant Protection

基　　金：云南省烟草公司科技计划(2021530000242031);云南省科技厅基础研究专项(202101AU070129);国家重点研发计划(2021YFD1400200)。

摘　　要：南美红辣椒脉斑驳病毒(chilli veinal mottle virus,ChiVMV)是一种严重危害茄科作物的正单链RNA病毒,也是云南烟草上的主要病毒之一。本研究采用单一变量法,建立并优化检测ChiVMV的反转录环介导等温扩增(reverse transcription-loop-mediated isothermal amplification,RT-LAMP)体系。对RT-LAMP的反应时间、反应温度及体系中Mg 2+、dNTPs、甜菜碱浓度和内外引物浓度比进行优化,同时用RT-PCR对优化后的RT-LAMP检测体系进行灵敏度验证。结果表明,最佳引物组为CH-CP-3,在25μL反应体系中各组分最佳加入量为:缓冲液2.5μL,MgSO 4(100 mmol/L)0.5μL,dNTPs(10 mmol/L)0.5μL,FIP/BIP(10 mmol/L)2μL,F3/B3(10 mmol/L)0.5μL,LF/LB(10 mmol/L)1.5μL,甜菜碱(5 mmol/L)5μL,Bst 2.0 WarmStar DNA Polymerase(8000 U/mL)0.5μL,M-MLV酶(10000 U/mL)0.125μL,RNA(≥50.7 fg)1μL,DEPC H 2O补至25μL;最佳反应温度及时间为63℃50 min,优化后的RT-LAMP检测结果与RT-PCR检测结果一致,且其灵敏度是RT-PCR的100倍。本研究建立的ChiVMV RT-LAMP检测方法具有快速、灵敏和操作简便等优点,为开发ChiVMV RT-LAMP检测试剂盒及其实际应用提供了重要基础。Chilli veinal mottle virus(ChiVMV)is a positive-sense single-stranded RNA virus that causes severe damage to solanaceous crops and is also one of Yunnan’s major tobacco pathogens.A single-variable method was used in this study to establish and optimize the reverse transcription-loop-mediated isothermal amplification(RT-LAMP)system for ChiVMV detection.The RT-LAMP system’s reaction time,temperature,and concentrations of Mg 2+,dNTPs,betaine,and concentration ratio of internal and external primers were optimized,and the sensitivity of the optimized RT-LAMP system was validated using RT-PCR.The results showed that the best primer set was CH-CP-3,and the optimal reaction system(25μL)contained:buffer 2.5μL,MgSO 4(100 mmol/L)0.5μL,dNTPs(10 mmol/L)0.5μL,FIP/BIP(10 mmol/L)2μL,F3/B3(10 mmol/L)0.5μL,LF/LB(10 mmol/L)1.5μL,betaine(5 mmol/L)5μL,Bst 2.0 WarmStar DNA polymerase(8000 U/mL)0.5μL,M-MLV enzyme(10000 U/mL)0.125μL,RNA(≥50.7 fg)1μL and DEPC H 2O to 25μL.The optimal reaction temperature and time were 63℃50 min.The optimized RT-LAMP detection result were consistent with RT-PCR detection,and its sensitivity was 100 times that of RT-PCR.The RT-LAMP method built for ChiVMV detection in this study has the advantages of fast,sensitive,and simple,which lay an important foundation for the development of ChiVMV RT-LAMP detection kit for practical application.

关 键 词：南美红辣椒脉斑驳病毒 反转录环介导等温扩增 灵敏度 检测 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]