黄瓜绿斑驳花叶病毒核酸试纸条检测技术的建立  

Development of Nucleic Acid Test Strip for Detection of Cucumber Green Mottle Mosaic Virus

作　　者：霍亚云 张永江[2] 李为民[1] 朱罗罗 胡林 尤其敏 

机构地区：[1]中国农业科学院生物技术研究所,北京100081 [2]中国检验检疫科学研究院,北京100176 [3]杭州优思达生物技术有限公司,浙江杭州310012

出　　处：《北方园艺》2017年第10期100-103,共4页Northern Horticulture

基　　金：国家"十二五"科技支撑计划资助项目(2015BAD08B01);国家"十三五"重点研发计划资助项目(2016YFF0203203)

摘　　要：以黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)阳性样品为研究对象,根据其外壳蛋白基因设计3条引物和2条探针,探针5′端分别用生物素和荧光进行标记;利用这套引物和探针在59℃进行RT-PCR恒温扩增90min,扩增产物用核酸试纸条进行检测。用其它5种同样侵染葫芦科作物的植物病毒作为对照进行试验,以期确定试纸条检测方法的特异性。将梯度稀释后的总RNA作为模板进行检测,以确定该方法的灵敏度。结果表明:该试验建立的核酸试纸条检测方法对CGMMV具有特异性,能够有效区分CGMMV及同样侵染葫芦科作物的黄瓜花叶病毒(CMV)、辣椒轻斑驳病毒(PMMoV)、烟草花叶病毒(TMV)、番茄花叶病毒(ToMV)和小西葫芦黄花叶病毒(ZYMV);同时该方法也具有较高的灵敏度,可检测低至24.300ng·μL^(-1)的总RNA。因此该试验建立的核酸试纸条法可用于CGMMV的快速检测。Positive sample of CGMMV was used as research object.Three primers and two probes were designed according to the coat protein of CGMMV and probes were labeled with Biotin and FAM at 5′ terminus separately.The nucleic acid strip was used to show the detection result after isothermal RT-PCR at the temperature of 59 ℃ for 90 minutes.The other five viruses that also infect cucurbitaceae crops were used to assay the specificity of this method.Diluted nucleic acid solution with certain concentration was used as template to confirm the sensitivity of this method.The results showed that nucleic acid strip was a special method to distinguish CGMMV from the other five plant virus which also infect cucurbitaceae crops，CMV，PMMoV，TMV，ToMV and ZYMV.And the sensibility of this method was 24.300 ng？·L^-1 of total RNA.Thus，nucleic acid strip was a rapid，special and sensitive detection method for CGMMV and can be used in routine testing.

关 键 词：黄瓜绿斑驳花叶病毒 核酸试纸条 检测 

分 类 号：S431.9[农业科学—农业昆虫与害虫防治]