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为了快速、灵敏地检测柑橘脉突病毒(Citrus vein enation virus,CVEV),通过设计特异性引物(EVq F4/EVq R4),优化反应条件,建立了CVEV的实时荧光定量RT-PCR检测体系。该方法特异性良好;检测灵敏度比常规RT-PCR高100倍;标准曲线循环阈值与模板浓度呈良好的线性关系,相关系数为0.992,扩增效率101.8%;检测组内和组间变异系数均小于2.85%,重复性较好。利用所建立的实时荧光定量方法对柑橘植株进行检测发现,‘代代酸橙’和‘象山红’植株中CVEV分布不均匀,其中根部病毒滴度最高,分别为162.52和45.32拷贝·ng~(-1) RNA,皮及叶部病毒滴度相对较低。
In order to detect Citrus vein enation virus (CVEV) rapidly and sensitively, a real-time fluorescence quantitative RT-PCR detection system of CVEV was established by designing specific primers (EVq F4 / EVq R4) and optimizing the reaction conditions. The sensitivity of the method was 100 times higher than that of the conventional RT-PCR. The standard curve circulating threshold had a good linear relationship with the template concentration, the correlation coefficient was 0.992, and the amplification efficiency was 101.8%. The coefficient of variation Less than 2.85%, better reproducibility. Using the established real-time fluorescence quantitative method to detect citrus plants, the results showed that the distribution of CVEV was not uniform among the generations of ’Lonicean’ and ’Xiangshanhong’ plants, with the highest root virus titer of 162.52 and 45.32 copies · ng ~ -1) RNA, skin and leaf virus titer is relatively low.