[目的]建立一种快速、灵敏、特异的检测甘蔗条点病毒(sugarcane striate virus,SStrV)的SYBR Green I 荧光定量PCR方法.[方法]从SStrV基因组保守区域序列设计特异性扩增引物,构建含有SStrV基因组序列的重组质粒pMD19-T-SStrV-qN作为阳性质粒标准品,以其为模板建立SStrV荧光定量PCR检测方法,并对该方法的灵敏性、特异性、稳定性进行了测试,随后用该方法对甘蔗不同组织部位中SStrV载量进行检测.[结果]将含有SStrV基因组序列的重组质粒按 10 倍比稀释成标准品,将其作为模板进行荧光定量PCR,获得标准曲线y=-3.337 x+38.197,相关系数r2=0.999,说明Cq值与标准品浓度拷贝数的对数呈线性关系;建立的荧光定量PCR最低可以检测到 13 拷贝重组质粒/μL,是普通PCR灵敏度的 100 倍.该方法能特异的检测SStrV,特异性高,组内和组间的变异系数在 0.13%-0.94%之间,表明该方法重复性良好.SStrV的载量在甘蔗的不同组织部位中差异较大,+4 叶中SStrV的载量最高,与其他组织部位达到了显著差异.[结论]建立了能灵敏特异检测SStrV的SYBR Green I 荧光定量PCR方法,明确了+4 叶是甘蔗中SStrV检测的最佳采样部位.
Establishment and Application of Real-time PCR for Sugarcane Striate Virus
[Objective]It is to establish a rapid,sensitive and specific SYBR Green I quantitative PCR method for the detection of sugarcane striate virus(SStrV).[Method]A specific amplification primer was designed from the conserved sequence of the SStrV genome sequence,the recombinant plasmid pMD19-T-SStrV-qN containing SStrV gene was constructed as a positive plasmid standard.Using it as template,the SStrV fluorescence quantitative PCR assay was established.And the sensitivity,specificity,stability,and subsequently of this method were tested,then the SStrV loads in different tissue sites of sugarcane were detected.[Result]The recombinant plasmid containing SStrV genome sequence was diluted into a standard at a 10-fold ratio,and they were used as a template for real-time PCR,the standard curve y=-3.337 x+38.197 was obtained,and the correlation coefficient r2=0.999 was obtained,indicating that the Cq value was linearly related to the logarithm of the copy number of the standard concentration.With this established real-time PCR,the least detection limit was 13 copies of the recombinant plasmid/μL,which was 100 times more sensitive than ordinary PCR.The method specifically detected SStrV with high specificity,and the coefficient of variation within and between groups was n 0.13%-0.94%,indicating that the method was of good repeatability.The load of SStrV was significantly different among different tissue sites of the sugarcane,and the load of SStrV was the highest in+4 leaves,which was significantly different from other tissue sites.[Conclusion]The SYBR Green I quantitative PCR method is established to provide an efficient quantitative detection method for the diagnosis of SStrV,and it is determined that+4 leaves are the best sampling site for the detection of SStrV in sugarcane.
国家科技期刊平台
NSTL
万方数据
