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园艺学报 ›› 2015, Vol. 42 ›› Issue (1): 183-190.doi: 10.16420/j.issn.0513-353x.2014-0740

• 新方法 • 上一篇    下一篇

T3基因型柑橘衰退病毒实时荧光定量RT-PCR检测体系的建立及应用

陶珍珍1,2,李中安2,贾 敏1,唐 萌2,唐科志2,周常勇2,*,周 彦2,*   

  1. 1西南大学植物保护学院,重庆 400715;2中国农业科学院柑桔研究所,重庆 400712
  • 出版日期:2015-01-25 发布日期:2015-01-25
  • 基金资助:

    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(CSTC2012jjA80029);重庆市应用开发计划项目(CSTC2014yykfA8005);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)

Development and Application of a Quantitative RT-PCR Approach for Quantification of T3 Genotype of Citrus tristeza virus

TAO Zhen-zhen1,2,LI Zhong-an2,JIA Min1,TANG Meng2,TANG Ke-zhi2,ZHOU Chang-yong2,*,and ZHOU Yan2,*   

  1. 1College of Plant Protection,Southwest University,Chongqing 400715,China;2Citrus Research Institute,Chinese Academy of Agricultural Sciences,Chongqing 400712,China
  • Online:2015-01-25 Published:2015-01-25

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。

关键词: 柑橘衰退病毒, T3基因型, 实时荧光定量RT-PCR

Abstract: 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 ORF1a. 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 cRNA. 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 studythe variation of T3 genotype.

Key words: Ctrus tristeza virus(CTV), T3 genotype, quantitative RT-PCR

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