朱顶红褪绿环斑病毒RT-LAMP快速检测体系的建立  

Development of an RT-LAMP Assay for Rapid Detection of hippeastrum chlorotic ringspot virus(HCRV)

作　　者：赵正婷 盖晓彤 张俊蕾 夏振远[2] 姜宁 刘雅婷[1,4] ZHAO Zhengting;GE Xiaotong;ZHANG Junlei;XIA Zhenyuan;JIANG Ning;LIU Yating(College of Agronomy and biotechnology,Yunnan Agriculture University,Kunming 650201,China;Yunnan Academy of Tobacco Agricultural Sciences,Kunming 650021,China;College of Plant Protection,Yunnan Agriculture University,Kunming 650201,China;College of Tobacco Science,Yunnan Agriculture University,Kunming 650201,China)

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

出　　处：《中国烟草科学》2023年第3期39-46,共8页Chinese Tobacco Science

基　　金：国家自然科学基金项目(32260681);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);云南省教育厅科学研究基金项目(2022Y234)。

摘　　要：为快速检测朱顶红褪绿环斑病毒(hippeastrum chlorotic ringspot virus, HCRV),根据HCRV的核壳体(N)蛋白基因保守核苷酸序列,设计了5组引物进行筛选,并采用单一变量法对RT-LAMP体系反应温度、时间、甜菜碱浓度、dNTPs浓度、Mg2+浓度、内外引物浓度比等进行逐一优化,建立了HCRV逆转录环介导等温扩增(RT-LAMP)检测体系。结果表明,最佳引物组为P1,最适反应温度为64℃,Betaine、dNTPs、Mg2+的最佳终浓度分别为0.8、0.2、6 mmol/L,最佳内外引物浓度比为5∶1,最佳反应时间50 min。检测结果显示优化后的RT-LAMP经SYBR Green I染色可通过肉眼直接判断结果,具有高度特异性,且灵敏度是常规RT-PCR的100倍。本研究为HCRV的检测提供了一种便捷、高效、可靠的方法。In order to quickly detect the hippeastrum chlorotic ringspot virus(HCRV),five sets of primers were designed and screened according to the conserved nucleotide sequence of the nucleocapsid(N)protein gene of HCRV,and the RT-LAMP system reaction temperature,time,betaine concentration,dNTP concentration,Mg2+concentration,and concentration ratio of internal and external primers were optimized by single variable method.A detection system of HCRV reverse transcription loop mediated isothermal amplification(RT-LAMP)was established.The results showed that the optimal primer group was P1,the optimal reaction temperature was 64℃,and the optimal final concentrations of Betaine,dNTPs,and Mg2+were 0.8,0.2,6 mmol/L.The optimal concentration ratio of internal and external primers is 5∶1,and the optimal reaction time is 50 minutes.The test results showed that after staining with SYBR Green I,the optimized RT-LAMP can be directly evaluated by naked eyes,with high specificity and sensitivity,100 times higher than that of conventional RT-PCR.This study provides a convenient,efficient,and reliable method for the detection of HCRV.

关 键 词：朱顶红褪绿环斑病毒 RT-LAMP 特异性 灵敏度 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]