Abstract:

Citrus yellow vein clearing virus（CYVCV）and Citrus tristeza virus（CTV），which are
transmittedby insects，are often mixed infectionon citrus plants. Specific primers for RT-PCR were used
according to the nucleotide sequences of coat protein genes（for both CYVCV and CTV）and Ubiquitin
gene to conduct One-step duplex RT-PCR. Simultaneously，key reaction factors such as the concentration
（Mg2+，dNTPs and primers）and annealing temperature were optimized.The specific fragments of 614 bp
（CYVCV），373 bp（CTV）and 194 bp（Ubiquitin）were amplified successfully from the sample by the
duplex RT-PCR system. Cloning and sequencing results showed that they shared high nucleotide identity
with sequences deposited in GenBank. In addition，the sensibility assay showed that this One-step duplex
RT-PCR could detect CYVCV and CTV from 40 ng · μL-1 and 4 ng · μL-1 of total nucleic acids，and its sensitivity was consistent with that of the regular One-step RT-PCR. Finally，this detection system was
used to detect the infection of both CYVCV and CTV from 33 field samples. Results also revealed that the
infection rate of CYVCV and CTV were 54.5% and 66.7%，respectively，and the co-infected rate came out
to be 36.4%. In conclusion，this One-step duplex RT-PCR system was suitable for rapid detection of both
CYVCV and CTV from a large number of field samples.

Key words: citrus, Citrus yellow vein clearing virus, Citrus tristeza virus, duplex RT-PCR, detection