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选用柑橘黄化脉明病毒(Citrus yellow vein clearing virus,CYVCV)和柑橘衰退病毒(Citrus tristeza virus,CTV)两种病毒外壳蛋白保守序列及内参基因Ubiquitin的特异性引物,优化影响二重RT-PCR反应的Mg~(2+)浓度、dNTPs浓度、引物浓度和退火温度,建立了针对两种病毒的一步法二重RT-PCR检测体系。二重RT-PCR获得CYVCV、CTV及Ubiquitin的特异性片段大小分别为614、373和194 bp,克隆测序和序列对比结果显示它们与已报道的病毒序列具有较高的同源性。该体系最低能从40 ng·μL~(-1)总核酸中检测出CYVCV,从4 ng·μL~(-1)总核酸中检测出CTV,其灵敏度与单一RT-PCR检测灵敏度一致。利用该体系对33份田间样品进行检测,CYVCV和CTV感染率分别为54.5%和66.7%,复合侵染率高达36.4%。该一步法二重RT-PCR技术体系可用于大量田间样品中CYVCV和CTV的快速同步检测。
The conserved sequence of coat protein of Citrus yellow vein clearing virus (CYVCV) and Citrus tristeza virus (CTV) and the specific primers of Ubiquitin were selected to optimize the double-stranded RT-PCR Reaction of Mg 2+ concentration, dNTPs concentration, primer concentration and annealing temperature, established a one-step two-dimensional RT-PCR detection system for the two viruses. The specific fragment sizes of CYVCV, CTV and Ubiquitin obtained by double RT-PCR were 614, 373 and 194 bp, respectively. The results of sequence comparison and sequencing showed that they had high homology with the reported sequences. The system could detect CYVCV from 40 ng · μL -1 total nucleic acid, and CTV was detected from 4 ng · μL -1 total nucleic acid. The sensitivity of this system was consistent with that of single RT-PCR. 33 field samples were tested by this system. The infection rates of CYVCV and CTV were 54.5% and 66.7% respectively, and the rate of composite infection was as high as 36.4%. The one-step multiplex RT-PCR system can be used for rapid and simultaneous detection of CYVCV and CTV in a large number of field samples.