柑橘脉突病毒实时荧光定量RT-PCR检测体系的建立与应用

Development and Application of a Quantitative RT-PCR Approach for Quantification of Citrus vein enation virus

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 fast by detect Citrus vein enation virus（CVEV） with sensitivity and specificity,a Reverse Transcription Quantitative Polymerase Chain Reaction（RT-qPCR） assay was developed with selective primer pairs（EVqF4/EVqR4） and optimized conditions.Using the method,only sample infected with CVEV showed positive,while those with other five citrus pathogens were negative.The sensitivity of the method was 100 times higher than the conventional RT-PCR.There was a good linear（R^2 = 0.992）relationship between the threshold cycle and CVEV template concentration,while the amplification efficiency of the RT-qPCR was 101.8%.The coefficients of variation of the intra- and inter-assay were both within 2.85%,indicating a good reproducibility of the method.The results also showed that CVEV was uneven distributed in sour orange plants and hybrid citrus plants,and the amount of the virus in roots was the highest（162.52 and 45.32 copies ？ ng^（-1） RNA,respectively）,followed by that in barks and leaves.