柑橘黄化脉明病毒RT-LAMP检测方法的建立

Establishment of RT-LAMP Assay for Detection of Citrus yellow vein clearing virus

柑橘黄脉病由柑橘黄化脉明病毒(Citrus yellow vein clearing virus,CYVCV)引起,是中国近年来新发生的一种柑橘病害。根据CYVCV的外壳蛋白基因序列设计了一组特异性引物,经过一系列条件优化,建立了该病毒的RT-LAMP检测方法。结果显示该方法能特异扩增CYVCV,与其他6种柑橘病原物(柑橘衰退病毒、温州蜜柑萎缩病毒、鳞皮病毒、柑橘裂皮病类病毒、柑橘碎叶病毒和柑橘黄龙病菌)不发生反应;灵敏度为RT-PCR的10倍;田间样品的检出率为63.33%,与RT-PCR的结果一致,适用于田间样品的检测。在产物中加入荧光染料SYBR GreenⅠ直接用肉眼观察就可判断样品是否感染CYVCV,可省去电泳分析的时间。该方法具有特异性强、灵敏度高、操作简单、快速等特点。

Yellow vein clearing disease caused by Citrus yellow vein clearing virus(CYVCV)was first found in Eureka lemon in Ruili,Yunnan in 2009,and it was observed in several citrus-growing areas of China in recent years. In order to establish the reverse transcription loop-mediated isothermal amplification(RT-LAMP)assay,primers were designed from the conserved region in the coat protein(CP)gene identified by multiple sequence alignment of CP gene sequences of CYVCV available in Gen Bank,and its reaction conditions was optimized. CYVCV was identified by RT-LAMP while Citrus tristeza virus(CTV),Citrus psorosis virus(CPV),Citrus exocortis viroid(CEVd),Satsuma dwarf virus(SDV),Citrus tatter leaf virus(CTLV)and Huanglongbing(HLB)were not amplified by the RT-LAMP assay. Sensitivity of the RT-LAMP assay was 10-fold higher than RT-PCR. The positive rate of 30 samples from Yunnan by using the RT-LAMP was 63.33%,the same as RT-PCR,suggesting the RT-LAMP assay worked as well as RT-PCR. In addition,amplification products were able to detect by visual inspection using SYBR GreenⅠ and there is no need for gel electrophoresis. Hence,this RT-LAMP assay is a specific,sensitive and rapid method for detecting CYVCV.