摘要
根据不同基因型柑橘衰退病毒(Citrus tristeza virus,CTV)ORF1a的序列差异,设计T3基因型CTV的特异性引物T3-4F/R,通过优化得到最佳反应条件建立T3基因型CTV分离株的SYBR GreenⅠ实时荧光定量RT-PCR,并进行灵敏性、重复性试验。应用该方法测定柑橘植株中T3基因型CTV分离株含量。建立了一种特异性检测T3基因型CTV分离株的SYBR GreenⅠ实时荧光定量RT-PCR检测方法,其灵敏性比普通RT-PCR方法高100倍。标准曲线循环阈值与模板浓度呈良好的线性关系,扩增效率为97.1%,相关性系数为0.992。组内和组间变异系数均小于2.94%,表明该方法重复性好。田间样品中T3基因型CTV含量差异较大,最高含量是最低含量的1 250倍。本研究中建立的实时荧光定量RT-PCR检测方法能够准确检测柑橘植株内的T3基因型CTV分离株,可用于研究T3基因型的CTV的变化规律。
This study established a quantitative RT-PCR method with primers T3-4F/R based on the conserved nucleotide sequence of Citrus tristeza virus(CTV)gene ORF1 a. The results showed that the method was at least hundred times higher than that with conventional PCR. A good linear correlation(R2 = 0.992)obtained from two standard curve of c RNA. The amplification efficiency was 97.1%. Three-time repeats revealed that the coefficients of variation between the intra- and inter-assay were both within 2.94%,indicating a reliating reproducibility detection method to CTV. There was noticeable differences among the content of T3 genotype in field samples,the highest content can reach to 1 250 times the lowest. The method was used for accurate determination of T3 genotype in the plant,and could be used to study the variation of T3 genotype.
出处
《园艺学报》
CAS
CSCD
北大核心
2015年第1期183-190,共8页
Acta Horticulturae Sinica
基金
国家公益性行业(农业)科研专项(201203076-01)
重庆市自然科学基金项目(CSTC2012jj A80029)
重庆市应用开发计划项目(CSTC2014yykf A8005)
柑橘学重庆市市级重点实验室开放基金项目(CKLC201101)
中央高校基本科研业务费资助项目(XDJK2014C027
XDJK2014A001)
