摘要
为快速检测烟草番茄斑萎病毒(tomato spotted wilt virus,TSWV),基于TSWV核外壳蛋白(nucleocapsid protein,NP)保守序列设计5组引物进行筛选,采用单一变量法对反应温度、时间及反应体系中dNTPs、Mg^(2+)、甜菜碱的含量和内外引物比等参数进行优化,建立烟草TSWV反转录环介导等温扩增(loop-mediated isothermal amplification,RT-LAMP)检测体系,并采用RT-PCR(revers transcription-polymerase chain reaction)检测方法进行平行比对试验,验证优化后RT-LAMP的特异性、灵敏性及实用性。结果表明,烟草TSWV RT-LAMP检测体系最佳引物组为TS-N-4,在25μL的反应体系中各组分最佳加入量为缓冲液2.5μL、100 mmol·L^(-1) MgSO40.5μL、10 mmol·L^(-1) dNTPs 0.5μL、10 mmol·L^(-1) FIP/BIP 1.5μL、10 mmol·L^(-1) F3/B30.5μL、10 mmol·L^(-1) LF/LB 1.5μL、5 mmol·L^(-1) Betaine 6μL、Bst 2.0 WarmStar DNA Polymerase(8000 U·mL-1)0.5μL、M-MLV酶(10000 U·mL^(-1))0.125μL、RNA(≥64.7 fg)1μL,DEPC H2O补至25μL;最佳反应温度和时间分别为58℃、60 min。优化后的RT-LAMP灵敏度是RT-PCR的1000倍,且田间样品检测结果与RT-PCR相符。建立的RT-LAMP方法特异性强、灵敏度高、操作简单,对于烟草TSWV的检测及监控有重要意义。
To rapidly detect tobacco tomato spotted wilt virus(TSWV),5 sets of primers were designed based on the conserved sequence of TSWV nucleocapsid protein(NP)for screening,and parameters such as reaction temperature and time,volumetric molar concentrations of dNTPs,Mg^(2+),betaine,and internal and external primer ratios in the reaction system were optimized using the univariate method to establish the tobacco TSWV reverse transcription loopmediated isothermal amplification(RT-LAMP),the specificity,sensitivity,and practicality of the optimized RTLAMP were verified by parallel comparison tests using the revers transcription-polymerase chain reaction(RT-PCR)assay.The results showed that the best primer set for the tobacco TSWV RT-LAMP assay was TS-N-4,and the optimal addition of each component in the 25μL reaction system were 2.5μL of buffer,0.5μL of MgSO4(100 mmol·L^(-1)),0.5μL of dNTPs(10 mmol·L^(-1)),1.5μL of FIP/BIP(10 mmol·L^(-1)),0.5μL of F3/B3(10 mmol·L^(-1)),1.5μL LF/LB(10 mmol·L^(-1)),6μL of Betaine(5 mmol·L^(-1)),0.5μL of Bst 2.0 WarmStar DNA Polymerase(8000 U·mL^(-1)),0.125μL of M-MLV enzyme(10000 U·mL^(-1)),0.125μL of RNA(≥64.7 fg),and added DEPC H2O to 25μL.The optimum reaction temperature and time were 58℃ and 60 min,respectively.The optimized RT-LAMP was 1000 times more sensitive than RT-PCR,and the results of field samples were consistent with RT-PCR.The RT-LAMP method established in this study was specific,sensitive,and simple to operate,which was important for the detection and monitoring of TSWV in tobacco.
作者
张俊蕾
盖晓彤
赵正婷
刘弟
王金凤
姜宁
刘雅婷
ZHANG Junlei;GE Xiaotong;ZHAO Zhengting;LIU Di;WANG Jinfeng;JIANG Ning;LIU Yating(School of Plant Protection,Yunnan Agricultural University,Kunming 650201,China;Yunnan Academy of Tobacco Agricultural Sciences,Kunming 650021,China;School of Agriculture and Biotechnology,Yunnan Agricultural University,Kunming 650201,China;School of Tobacco Science,Yunnan Agricultural University,Kunming 650201,China)
出处
《中国农业科技导报》
CAS
CSCD
北大核心
2024年第8期140-150,共11页
Journal of Agricultural Science and Technology
基金
云南省烟草公司科技计划项目(2021530000242031)
云南省科技厅基础研究专项(202101AU070129)
国家重点研发计划项目(2021YFD1400200)
国家自然科学基金项目(32260681)。
关键词
番茄斑萎病毒
NP基因
反转录-环介导等温扩增
快速检测
tomato spotted wilt virus
NP gene
reverse transcription-loop-mediated isothermal amplification
rapid detection
