菊花B病毒和番茄不孕病毒RT-RPA双重荧光实时检测体系的构建与性能评价  

Establishment and Performance Evaluation of RT-RPA Real-time Fluorescent Detection System for Chrysanthemum Virus B and Tomato Aspermy Virus

作　　者：姜自红[1] 刘文干 孙钦花[3] JIANG Zihong;LIU Wengan;SUN Qinhua(Food And Environmental Engineering Department,Chuzhou Polytechnic,Chuzhou 239000,China;Anhui Anlong Gene Technology Company,Hefei,230000,China;Environmental Engineering Department,Xuzhou University of Technology,Xuzhou 221000,China)

机构地区：[1]滁州职业技术学院食品与环境工程学院,滁州239000 [2]安徽安龙基因科技有限公司,合肥230000 [3]徐州工程学院环境工程学院,徐州221000

出　　处：《病毒学报》2023年第1期174-184,共11页Chinese Journal of Virology

基　　金：安徽高校自然科学研究重大项目(项目号:KJ2021ZD0161),题目:菊花B病毒和番茄不孕病毒RT-RPA双重荧光检测体系的构建与性能评价。

摘　　要：菊花B病毒(Chrysanthemum virus B, CVB)和番茄不孕病毒(Tomato aspermy virus, TAV)是危害菊花的2种主要优势病毒。为构建一种同步检测CVB和TAV逆转录重组酶聚合酶恒温核酸扩增(Reverse transcriptionrecombinase polymerase amplification, RT-RPA)双重荧光实时检测方法,本研究以CVB和TAV编码外壳蛋白(coat protein, CP)基因保守区域作为靶标设计RIA引物和探针,CVB和TAV探针的5’端分别标记FAM和VIC荧光素,通过对商业化的RT-RPA试剂筛选,引物探针的组合筛选,扩增体系的优化和扩增条件的摸索试验,构建了一种可同时用于CVB和TAV检测的RT-RPA双重荧光实时检测方法。并对该体系进行优化,优化后RT-RPA双重荧光扩增体系中CVB和TAV的最优引物浓度为400 nmol/L,最优探针浓度分别为100 nmol/L和120 nmol/L;最适反应温度为39℃,最适反应时间为15 min。在此扩增体系和条件下,对CVB和TAV的检测敏感性均为1拷贝/μL;对烟草花叶病毒、黄瓜花叶病毒、马铃薯Y病毒、马铃薯X病毒、菊花矮化类病毒和褪绿斑驳类病毒核酸检测结果为阴性,对菊花B病毒、番茄不孕病毒核酸检测结果为阳性;对1.0×10^(4)拷贝/μL和10拷贝/μL的高、低两种浓度的质粒平行检测10次,检测荧光出现阳性扩增拐点所需的时间T变异系数均小于5%,重复性好。采用RTRPA和RT-qPCR方法同步对实验室留存的有症状的100株菊花进行核酸提取并检测,检出6例CVB病毒株和4例TAV病毒株,总符合率100%。因此,本研究建立了一种能同时检测CVB和TAV的快速、灵敏的RT-RPA双重荧光实时检测方法。Chrysanthemum virus B(CVB) and the tomato aspermy virus(TAV) are the two dominant viruses that affect Chrysanthemum species. A reverse transcription-recombinase polymerase amplification(RT-RPA) real-time fluorescent detection system for CVB and the TAV was developed. RIA primers and probes were designed with the conservative region of CVB and TAV coding coat protein gene as target. The 5’ ends of CVB and TAV probes were labeled with FAM and Vic fluorescein, respectively. We carried out screening of commercial RT-RPA reagents, combination screening of primers and probes, optimization of the amplification system, and exploration of amplification conditions. In the optimized amplification system, the concentration of primers(in nmol/L) for CVB and the TAV was 400, the concentration(in nmol/L) of probes was 100 and 120, respectively. The optimal reaction temperature was 39°C and the optimal reaction time was 15 min. Under these conditions, the sensitivity to CVB and the TAV was 1 copy/μL. The results of nucleic-acid test with tobacco mosaic virus, cucumber mosaic virus, potato virus Y, potato virus X, Chrysanthemum dwarf virus and Chrysanthemum chlorotic mottle viroid were all negative, whereas it was positive with CVB and the TAV, 1.0×10^(4) copies/μL and 10 copies/μL were detected 10 times, respectively, and the time T coefficient of variation needed to detect the inflexion of fluorescence amplification was <5%, indicating good repeatability.Using RT-RPA and reverse transcription-quantitative polymerase chain reaction, simultaneously extracted and detected nucleic acids from 100 symptomatic Chrysanthemum plants retained in the laboratory, six cases of CVB and four cases of the TAV were detected, the total coincidence rate was 100%. Therefore, a rapid, sensitive and constant-temperature RT-RPA real-time fluorescent detection system for CVB and the TAV was established. It provideded a reference method for control of Chrysanthemum virus and cultivation of high-quality varieties of Chrysanthemum.

关 键 词：菊花B病毒 番茄不孕病毒 逆转录重组酶聚合酶恒温扩增(RT-RPA) 双重荧光实时检测 

分 类 号：S436.8[农业科学—农业昆虫与害虫防治]