关键词: 柑橘, 柑橘衰退病毒, 柑橘碎叶病毒, 柑橘黄化脉明病毒, 柑橘叶斑驳病毒, 多重PCR

Abstract:

Citrus tristeza virus（CTV），Citrus tatter leaf virus（CVLT），Citrus yellow vein clearing virus（CYVCV），and Citrus leaf blotch virus（CLBV）are important graft-transmissible pathogens of citrus. Rapid and accurate detection methods are of great significance for the prevention and control of viral diseases. Four compatible sets of primers specific for each virus were designed based on conserved sequences of coat protein gene for multiplex PCR assay. The crucial factors of multiple PCR including primer concentration and annealing temperature were optimized for the highest sensitivity and specificity. Four specific fragments were simultaneously amplified in one PCR reaction. Their molecular weights were determined to be 889（CTLV），612（CYVCV），462（CTV）and 294 bp（CLBV）. The sensibility assay showed that the sensibility of this one-step multiplex PCR is about 10 times lower than that of regular single RT-PCR. Finally，this detection system was used to detect these viruses from 59 field samples. The results revealed that the detection rate of these viruses was between 11.9% and 54.2%. In conclusion，this one-step multiplex PCR system is suitable for rapid detection of the 4 citrus viruses from field samples with accuracy，rapidity and sensitivity.

Key words: citrus, Citrus tristeza virus, Citrus tatter leaf virus, Citrus yellow vein clearing virus, Citrus leaf blotch virus, one-step multiplex PCR