Establishment and application of real-time quantitative PCR system to differentiate RB and VT genotypes of citrus tristeza virus
In order to quantitatively detect the RB and VT genotypes of citrus tristeza virus(CTV),a geno-type-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 u-sing 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=-3.325 5 x+37.845 3 and y=-3.273 7x+36.283 9,respectively,with R2 values of 0.999 1 and 0.995 4,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 specifici-ty and sensitivity,and suitable for the detection of samples from the field.
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