番茄斑萎病毒4种检测方法比较  

Comparison of four methods for detecting Tomato spotted wilt virus

作　　者：赵巍巍[1] 杨家强[2] 周小毛[1] 吴青君[2] 

机构地区：[1]湖南农业大学农药研究所,长沙410128 [2]中国农业科学院蔬菜花卉研究所,北京100081

出　　处：《植物保护》2015年第2期108-113,共6页Plant Protection

基　　金：国家科技支撑计划(2012BAD19B06);北京市自然科学基金(6122028);现代农业产业技术体系北京市叶类蔬菜创新团队建设专项资金(blvt-15);北京市科委课题(Z121100001212006);蔬菜有害生物控制与优质栽培北京市重点实验室

摘　　要：本研究根据番茄斑萎病毒(TSWV)S RNA上的核衣壳蛋白(N)基因保守序列设计特异性引物,比较了4种检测方法的灵敏度。结果表明,特异性引物可扩增出397bp的片段,序列和已发表的TSWV核苷酸序列同源性高达99%,可用于常规PCR和荧光定量RT-PCR(qRT-PCR)检测。qRT-PCR的灵敏度比快速检测试纸条、双抗体夹心酶联免疫吸附法(DAS-ELISA)和常规PCR分别高出15 625倍、3 125倍和125倍,且能够准确定量;常规PCR灵敏度较高,但不能准确定量;DAS-ELISA方法适用于批量定性测定,但检测时间较长;试纸条法检测速度最快,但灵敏度最低,使用时可根据症状程度和试验条件选择适宜的检测方法。Specific primers were designed according to the conserved sequence of the nucleocapsid protein（N）gene of Tomato spotted wilt virus（TSWV）S RNA,and the sensitivities of four methods for detecting TSWV were compared.The results showed that a 397 bp fragment was amplified with the specific primers,and the sequence showed a homology of 99% with the published TSWV nucleotide sequence.The designed primers could be used for conventional PCR and quantitative reverse transcription-PCR（qRT-PCR）methods.The sensitivity of qRT-PCR for detecting TSWV was 15 625 fold,3 125 fold and125 fold higher than that of the rapid detection test strip（RDTT）,double sandwich enzyme-linked immunosorbent assay（DAS-ELISA）and conventional PCR,respectively.Moreover,qRT-PCR method can be accurately quantified.Conventional PCR showed a higher sensitivity,but it cannot be accurately quantified.DAS-ELISA is suitable for batch qualitative detection but it needs a longer detection time.RDTT is the fastest method,but the sensitivity is the lowest.Suitable detection methods can be selected according to the symptom degree and the experimental conditions.

关 键 词：番茄斑萎病毒 DAS-ELISA 常规PCR 快速检测试纸条 QRT-PCR 灵敏度 

分 类 号：S432.41[农业科学—植物病理学]