柑桔衰退病毒RB和VT基因型实时定量PCR检测方法建立和应用  

Establishment and application of real-time quantitative PCR system to differentiate RB and VT genotypes of citrus tristeza virus

作　　者：周俊[1] 韩镕琰 张晓男 方书洁 杨欣悦 易龙[1] ZHOU Jun;HAN Rongyan;ZHANG Xiaonan;FANG Shujie;YANG Xinyue;YI Long(College of Life Sciences,Gannan Normal University/National Navel Orange Engineering Research Center,Ganzhou,Jiangxi,341000,China)

机构地区：[1]赣南师范大学生命科学学院/国家脐橙工程技术研究中心,江西赣州341000

出　　处：《中国南方果树》2024年第4期18-24,共7页South China Fruits

基　　金：江西省教育厅科学技术研究项目(GJJ201431);江西省科技项目(20225BCJ22005);国家自然科学基金地区基金项目(31860488)资助。

摘　　要：为定量检测样品中柑桔衰退病毒(citrus tristeza virus,CTV)RB和VT基因型含量,以RB基因型的p33基因、VT基因型的ORF1a基因为靶标,建立了RB和VT基因型的特异性实时定量PCR方法。该方法检测RB和VT基因型质粒浓度下限均为2×10^(1) copies/μL,灵敏度为普通PCR的1000倍;对RB和VT基因型进行检测,质粒拷贝数对数(x)与Ct值(y)的标准曲线方程分别为y=-3.3255 x+37.8453和y=-3.2737 x+36.2839,R^(2)分别为0.9991和0.9954,扩增效率分别为99.85%和102.05%;方法的重复性良好,组内和组间Ct值变异系数均小于2.07%。对田间样品检测发现,不同样品之间RB基因型含量差异和VT基因型含量差异均较大。该方法特异性强,灵敏度高,适用于田间样品检测。In order to quantitatively detect the RB and VT genotypes of citrus tristeza virus(CTV),a genotype-specific real-time quantitative PCR method was established by targeting the p33 gene of RB genotype and the ORF1a gene of VT genotype.The minimum plasmid concentration for detecting RB and VT genotypes using this method is 2×10 copies/μL,with a sensitivity 1,000 times higher than that of conventional PCR.For detection of the RB and VT genotypes,the standard curve equations for plasmid copy number logarithm(x)and Ct value(y)were y=-3.3255 x+37.8453 and y=-3.2737x+36.2839,respectively,with R^(2) values of 0.9991 and 0.9954,and amplification efficiencies of 99.85%and 102.05%,respectively.This method had good reproducibility,with intra-and inter-group Ct value variation coefficients less than 2.07%.The results of detecting field samples showed that there were significant differences in the proportion of RB genotypes and VT genotypes among different samples.The method established in this study has high specificity and sensitivity,and suitable for the detection of samples from the field.

关 键 词：柑桔衰退病毒 RB基因型 VT基因型 实时定量RT-PCR 

分 类 号：S436.66[农业科学—农业昆虫与害虫防治]