摘要
菊花容易受到病毒感染而造成品质下降,目前国内对菊花病毒的检测主要根据外观表现或者定性PCR检测,无法准确判定病毒载量。为构建一种可同时用于检测菊花B病毒(Chrysanthemum virus B,CVB)、番茄不孕病毒(Tomato aspermy virus,TAV)和菊花褪绿斑驳类病毒(Chrysanthemum chloritic mottle viroid,CChMVd)的实时荧光定量RT-PCR检测方法,本研究分别以保守区域作为靶标设计相应的引物探针,通过优化扩增体系中CVB、TAV、CChMVd 3种病毒/类病毒探针浓度、引物浓度、Mg^(2+)浓度、dNTPs浓度,摸索扩增程序中反转录时间、退火温度和扩增循环数,构建了一种可同时用于CVB、TAV、CChMVd的3重实时荧光定量RT-PCR检测体系,优化后的扩扩增体系中CVB、TAV和CChMVd的探针浓度分别为100 nmol/L、120 nmol/L和80 nmol/L,引物浓度分别为200 nmol/L、240 nmol/L和160 nmol/L,Mg^(2+)浓度为3.0 mmol/L;dNTPs浓度200μmol/L;最适反转录时间为25 min,退火温度为60℃,循环数为40。敏感性实验结果表明,该反应体系对3种病毒/类病毒的敏感性为1.0×^(10)3拷贝/mL,敏感性好;定量线性范围为1.0×^(10)3拷贝/mL~1.0×^(10)10拷贝/mL,线性范围宽;特异性好,对菊花矮化类病毒、烟草花叶病毒和黄瓜花叶病毒核酸检测结果为阴性;对1.0×^(10)4拷贝/mL的低浓度参考品平行检测10次,定量结果lg值偏差(CV%)为4.81%,重复性好。在南京农业大学"中国菊花种质资源保存中心"基地随机选择菊花20株进行本研究试剂检测,检出6例CVB病毒株和4例TAV病毒株,其病毒载量为2.5×^(10)4拷贝/mL~5.5×^(10)7拷贝/mL,随机选择1株CVB病毒株定量PCR,产物进行TA克隆后经测序与NCBI Blast比对,其与MH678704.1的同源性为100%。因此,本研究建立了一种能同时检测CVB、TAV、CChMVd 3种菊花常见病毒/类病毒的灵敏、快速、可定量的检测方法。
Chrysanthemum is easy to be infected by virus and the quality of the flower is reduced.At present,the detection of chrysanthemum virus is mainly based on the appearance or qualitative PCR detection,and the virus load can not be accurately determined.We wished to design a real-time quantitative reverse transcriptionpolymerase chain reaction(RT-PCR)system for fluorescence-based detection of chrysanthemum virus B(CVB),tomato aspermy virus(TAV)and chrysanthemum chlorotic mosaic virus/viroid(CChMVd).The corresponding primers were designed with the conservative region as the target.We optimized the concentrations of Mg^(2+),dNTPs,primers and probes of three viruses/viroid in the amplification system.Next,we explored the duration of reverse transcription,annealing temperature,and the number of amplification cycles.In this way,a triplex fluorescence quantitative RT-PCR system for simultaneous detection of CVB,TAV and CChMVd was developed.In the optimized amplification system,the concentration of probes for CVB,TAV and CChMVd was 100 nmol/L,120 nmol/L and 80 nmol/L,respectively,the concentration of primers was 200 nmol/L,240 nmol/L and 160 nmol/L,respectively.The final concentration of Mg^(2+)was 3.0mmol/L,and the final concentration of dNTPs was 200μmol/L,The optimal duration of reverse transcription was 25 min,annealing temperature was 60°C and the number of cycles was 40.A sensitivity test showed that the triplex reaction system was sensitive to 1.0×^(10)3 copies/mL.The nucleic-acid test for the chrysanthemum dwarf virus,tobacco mosaic virus,and cucumber mosaic virus was negative,with good specificity.The linear range of quantitative detection was 1.0×^(10)3 copies/mL to 1.0×^(10)10 copies/mL.The reference material for a low concentration(1.0×^(10)4 copies/mL)was detected 10 times,and the deviation in the quantitative 1g value(CV%)was 4.81%with good repeatability.Twenty Chrysanthemum strains from the China Chrysanthemum Germplasm Resources Conservation Center of Nanjing Agricultural University were selected rando
作者
姜自红
殷培峰
JIANG Zihong;YIN Peifeng(Food and Environmental Engineering Department,Chuzhou Polytechnic,Chuzhou 239000,China;School of Biological Science and Food Engineering,Chuzhou University,Chuzhou 239000,China)
出处
《病毒学报》
CAS
CSCD
北大核心
2021年第1期169-180,共12页
Chinese Journal of Virology
基金
安徽省高校优秀拔尖人才培育资助项目(项目号:gxfx2017223),题目:高校优秀青年骨干人才国内外访学研修
滁州职业技术学院院级科技创新团队项目(项目号:YJCXTD-2017-01),题目:琅琊山野生花卉资源开发及利用。
