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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。
Citrus yellow vein disease is caused by Citrus yellow vein clearing virus (CYVCV) and is a new citrus disease in China in recent years. A set of specific primers was designed according to the coat protein gene sequence of CYVCV. After a series of conditions were optimized, the RT-LAMP detection method of the virus was established. The results showed that this method could specifically amplify CYVCV and did not react with the other six citrus pathogens (Citrus recession virus, Satsuma peak wilt virus, lepromatosis virus, Citri distemper virus, Citrus broken leaf virus and Citrus Huanglongbing) ; The sensitivity was 10 times that of RT-PCR; the detection rate of field samples was 63.33%, which was consistent with the results of RT-PCR and was suitable for the field samples. In the product by adding fluorescent dye SYBR Green Ⅰ directly observed with the naked eye can determine whether the sample is infected with CYVCV, eliminating the need for electrophoresis analysis time. The method has the characteristics of strong specificity, high sensitivity, simple operation, fastness and the like.