摘要
为了建立甘薯羽状斑驳病毒(Sweet potato feathery mottle virus,SPFMV)和甘薯矮化褪绿病毒(Sweet potato chlorotic stunt virus,SPCSV)的逆转录环介导等温扩增(RT-LAMP)检测方法,提高检测灵敏度,实现结果的可视化。根据SPFMV外壳蛋白基因(CP)的核苷酸序列和SPCSV的热休克蛋白基因(Hsp 70)的核苷酸序列设计4条RT-LAMP特异性引物,采取单因素优化试验,对RT-LAMP反应体系中的多个因素包括时间、温度、BST聚合酶、Mg^(2+)、RNase抑制剂、dNTPs和Betaine浓度优化,恒温扩增60 min。经琼脂糖凝胶电泳分析,SYBR Green I可视化显色,结果表明:SPFMV的优化反应体系为:FIP/BIP 2μL、F3/B30.5μL、BST聚合酶1.0μL、dNTPs 0.6μL、MgSO 41.5μL、RNase抑制剂1.0μL、Betaine 7μL,62℃60 min。SPCSV的优化反应体系为:FIP/BIP 2μL、F3/B30.5μL、BST聚合酶1.0μL、dNTPs 0.6μL、MgSO 41.5μL、RNase抑制剂1.2μL、Betaine 7μL,64℃60 min。进一步利用SPFMV全基因组的4个片段(SPFMV-1、SPFMV-2、SPFMV-3、SPFMV-4)、SPCSV-Hsp 70和RGNNV进行特异性检验,分别建立了SPFMV和SPCSV的特异性RT-LAMP检测方法,扩增出了具有RT-LAMP的典型瀑布状条带,与凝胶电泳和SYBR Green I显色结果一致,SPFMV和SPCSV的灵敏度检测下限分别为:1×10^(-6)、1×10^(-3)ng·μL^(-1),该方法检测灵敏度高,实现了结果的可视化。对田间甘薯苗和离体组织培养甘薯苗进行检测验证,SPFMV-RT-LAMP检测方法成功率为100%,SPCSV-RT-LAMP检测方法的成功率为95%,表明研发的SPFMV和SPCSV的RT-LAMP检测方法适用于SPFMV和SPCSV的快速检测。
In order to establish a reverse transcription loop-mediated isothermal amplification(RT-LAMP)method to detect the sweet potato feathery mottle virus(SPFMV)and sweet potato chlorotic stunt virus(SPCSV),improve the detection sensitivity and realize the visualization of the results,four RT-LAMP specific primers were designed according to the nucleotide sequences of SPFMV coat protein gene(CP)and SPCSV heat shock protein gene(Hsp 70)in this study,and then the single factor optimization experiment was used to optimize the multiple factors in the RT-LAMP reaction system including time,temperature,BST polymerase,Mg^(2+),RNase inhibitor,dNTPs and Betaine concentration,with the constant temperature amplification for 60 min.Through the agarose gel electrophoresis analysis,and the SYBR Green I visual color development,the results showed that:the optimal reaction system of SPFMV was as follows:FIP/BIP 2μL,F3/B30.5μL,BST polymerase 1.0μL,dNTPs 0.6μL,MgSO 41.5μL,RNase inhibitor 1.0μL,Betaine 7μL,at 62℃for 60 min.The optimal reaction system of SPCSV was as follows:FIP/BIP 2μL,F3/B30.5μL,BST polymerase 1.0μL,dNTPs 0.6μL,MgSO 41.5μL,RNase inhibitor 1.2μL,Betaine 7μL,at 64℃for 60 min.The four fragments of the whole genome of SPFMV(SPFMV-1,SPFMV-2,SPFMV-3,and SPFMV-4),SPCSV-Hsp 70 and RGNNV were used to test the specificity.Then,the specific RT-LAMP detection methods of SPFMV and SPCSV were established respectively,and the typical waterfall-like bands with RT-LAMP were amplified,which were consistent with the results of gel electrophoresis and SYBR Green I coloration.The lower limits of sensitivity detection of SPFMV and SPCSV were as follows:1×10^(-6)and 1×10^(-3)ng·μL^(-1).The detection sensitivity of this method was high,and the results could be visualized.Field sweet potato seedlings and in vitro tissue culture sweet potato seedlings were tested,and the success rate of SPFMV-RT-LAMP detection method was 100%.The success rate of SPCSV-RT-LAMP detection method was 95%,indicating that the developed RT-LAMP detection method of SPFMV and SPCSV was suitable for the rapid detection of SPFMV and SPCSV.
作者
陈涛
薛婷
杨志坚
陈选阳
陈由强
陈建楠
CHEN Tao;XUE Ting;YANG Zhi-jian;CHEN Xuan-yang;CHEN You-qiang;CHEN Jian-nan(College of Life Science,Fujian Normal University,Fuzhou,Fujian 350117,China;Public Service Platform for Industrialization Development Technology of Marine Biological Medicine and Products of the State Oceanic Administration Fuzhou,Fujian 350117,China;Fujian Key Laboratory of Special Marine Bioresource Sustainable Utilization,Fuzhou,Fujian 350117,China;Fujian Key Laboratory of Crop Biotechnology,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China)
出处
《福建农业科技》
CAS
2023年第6期11-21,共11页
Fujian Agricultural Science and Technology
基金
福建省科技计划项目(2020N5013)
福建省科技计划项目(2020N0003)。
