Detection of Citrus yellow vein clearing virus by Quantitative Real-time RT-PCR

To develop a rapid and reliable detection method for Citrus yellow vein clearing virus (CYVCV), a quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) system based on SYBR Green I was established by using a pair of specific primers designed from its conserved coat protein gene. 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 qRT-PCR was 100-fold higher than that of conventional RT-PCR. An excellent linear correlation (R2 = 0.999) was obtained from two standard curves of cRNA, and the amplification efficiency was 102%. The data from field citrus samples detection showed that the qRT-PCR system could be used in determining the concentration of CYVCV in different citrus species.