柑橘黄化脉明病毒巢式RT-PCR检测方法的建立及应用

Development and application of nested RT-PCR assay for detection of Citrus yellow vein clearing virus

为探索柑橘黄化脉明病毒（Citrus yellow vein clearing virus,CYVCV）引起的柑橘新病害—柑橘黄脉病的早期快速检测技术,针对CYVCV核酸结合蛋白基因的保守序列设计2对特异性引物,通过优化退火温度,建立了CYVCV巢式RT-PCR检测方法,并对采自不同柑橘品种的54个CYVCV疑似样品进行了检测。结果表明,CYVCV巢式RT-PCR检测中,以55℃和60℃分别作为第1轮和第2轮扩增的退火温度时检测效果最佳;该方法检测样品中病毒总核酸的最低浓度为2.40μg/L,灵敏度较RT-PCR提高100倍。在CYVCV疑似样品检测中,巢式RT-PCR和RT-PCR的阳性检出率分别为59.26%和57.41%,前者更适用于检测不同来源的CYVCV。当尤力克柠檬、锦橙北碚-447、天草和台湾椪柑嫁接接种CYVCV后,巢式RT-PCR比RT-PCR提前10~30 d检测出病毒。表明所建立的CYVCV巢式RT-PCR检测方法适用于田间病树的早期诊断。

Yellow vein clearing disease caused by Citrus yellow vein clearing virus（CYVCV） is an emerging viral disease in China.To resolve the defect that the existing detection methods were difficult to detect CYVCV at the early stage,two pairs of primers were designed within conserved region of the nucleic acid binding protein gene,and a rapid and specific nested RT-PCR detection method for CYVCV was established.The results showed that 55℃ and 60℃ were the optimized annealing temperature to the first and second PCR reaction,respectively,and nested RT-PCR had a detection limit of 2.40 μg /L of total nucleic acid and the sensitivity was 100-fold higher than RT-PCR.In the detection of 54 CYVCVsuspected samples,the positive rates by using the nested RT-PCR and RT-PCR were 59.26% and57.41%,respectively.The results suggested that the nested RT-PCR could detect CYVCV more effectively in various citrus cultivars.Furthermore,after CYVCV isolates were graft-inoculated on Eureka lemon,Jincheng Beibei-447,Amakusa and Taiwan ponkan,they were detected 10 to 30 days earlier by nested RT-PCR than RT-PCR.The present study demonstrated that the nested RT-PCR is a valuable tool for early detection of CYVCV in different citrus cultivars.