应用实时荧光RT-PCR检测柑橘黄化脉明病毒

为更快速准确检测柑橘黄化脉明病毒（Citrus yellow vein clearing virus，CYVCV），根据病毒外壳蛋白基因的保守序列设计特异性引物，通过体系优化得到最佳反应条件，建立基于SYBR GreenⅠ的CYVCV实时荧光RT-PCR检测方法。该方法可特异性检测CYVCV，而测试的柑橘衰退病毒、碎叶病毒等5种柑橘病毒均不能检测出。灵敏度较普通RT-PCR提高了100倍，标准曲线循环阈值与模板浓度呈良好的线性关系，扩增效率和相关性系数分别为102%和0.999。对田间样品的检测结果表明，建立的实时荧光RT-PCR适用于检测不同柑橘品种中CYVCV的含量。

Detection of Citrus yellow vein clearing virus Based on a Real-time RT-PCR Approach

To develop a rapid and reliable detection method，a real-time RT-PCR system based on SYBR GreenⅠwas established by using a pair of specific primers designed from conserved coat protein gene of Citrus yellow vein clearing virus（CYVCV）. The sensitivity，specificity and applicability of the system were evaluated accordingly. The results showed that amplicons were produced from CYVCV isolates，whereas no amplicons from non-CYVCV citrus virus samples including Citrus tristeza virus （CTV）and Citrus tatter leaf virus（CTLV）were obtained. The sensitivity of the real-time RT-PCR was 100-fold higher than that of conventional RT-PCR. An excellent linear correlation（R2 = 0.999）obtained from two standard curves of cRNA and the amplification efficiency was 102%. The data from field citrus samples detection showed that the real-time RT-PCR system could be used to determine the concentration of CYVCV in the different citrus species.