山东农业科学2024，Vol.56Issue(5)：154-162,9.DOI:10.14083/j.issn.1001-4942.2024.05.020
基于CP基因的云南烟草花叶病毒RT-LAMP快速检测体系的构建
Development of Reverse Transcription Loop-Mediated Isothermal Amplification(RT-LAMP)System for Rapid Detection of Yunnan Tobacco Mosaic Virus Based on CP Gene
摘要
Abstract
In order to rapidly detect tobacco mosaic virus(TMV)in Yunnan tobacco,five sets of prim-ers were designed for screening based on the conserved nucleotide sequence of the coat protein(CP)gene from Yunnan tobacco TMV isolate,the reaction temperature,time,betaine concentration,dNTPs concentra-tion,Mg2+concentration,and the ratio of internal to external primer concentrations were optimized by the sin-gle variable method,and then a reverse transcription loop mediated isothermal amplification(RT-LAMP)de-tection system for Yunnan tobacco TMV was established.The results showed that the optimal primer group was the fourth group,the optimal reaction temperature was 60℃,and the optimal final concentrations of Betaine,dNTPs and Mg2+were 0.6,0.4 and 2.0 mmol/L,respectively.The optimal concentration ratio of internal prim-er to external primer was 4∶1,and the optimal reaction time was 40 minutes.The results of optimized RT-LAMP could be visualized after stained by SYBR Green Ⅰ,which showed high specificity to TMV and the sensitivity was 10 times that of conventional RT-PCR.This study provided a convenient,efficient and reliable method for the detection of TMV in Yunnan tobacco.关键词
烟草花叶病毒/CP基因/RT-LAMP/特异性/灵敏度Key words
Tobacco mosaic virus(TMV)/CP Gene/RT-LAMP/Specificity/Sensitivity分类
农业科技引用本文复制引用
赵正婷,盖晓彤,张俊蕾,卢灿华,姜宁,刘雅婷..基于CP基因的云南烟草花叶病毒RT-LAMP快速检测体系的构建[J].山东农业科学,2024,56(5):154-162,9.基金项目
国家自然科学基金项目(32260681) （32260681）
云南省烟草公司科技计划项目(2021530000242031) （2021530000242031）
云南省科技厅基础研究专项(202101AU070129) （202101AU070129）
