番茄斑萎病毒NASBA检测方法的建立

Development of a nucleic acid sequence-based amplification assay for detection of Tomato spotted wilt virus

为建立一种快速检测番茄斑萎病毒(Tomato spotted wilt virus,TSWV)的方法,以TSWV-CP1/TSWV-CP2为引物对TSWV的N基因进行PCR扩增及序列测定,在TSWV N基因的高度保守区设计特异性扩增引物NA-P1/NA-P2进行核酸序列依赖性扩增(NASBA)反应,并对NASBA方法的特异性和灵敏度进行验证。结果表明,建立的NASBA方法最佳反应时间为1.5 h。该方法特异性较好,只有TSWV阳性样品中出现了预期大小为235 bp的扩增产物,与烟草环斑病毒(Tobacco ring spot virus,TRSV)、番茄黑环病毒(Tomato black ring virus,TBRV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)、番茄花叶病毒(Tomato mosaic virus,ToMV)、番茄黄化曲叶病毒(Tomato yellow curl virus,ToYCLV)无交叉反应。灵敏度验证中,实时荧光RT-PCR的灵敏度最高,为1.56×10^(-5)ng/μL感病植物RNA模板,NASBA次之,为1.56×10^(-4)ng/μL,普通RT-PCR最低,为1.56×10^(-3)ng/μL。在对9份实际样品检测中,NASBA的阳性检出率与实时荧光RT-PCR、普通RT-PCR相同,均为33%,高于ELISA检测的22%。表明NASBA方法适用于实际样品检测,可对TSWV进行快速检测。

To develop a novel method for detecting the Tomato spotted wilt virus（TSWV） at low concentration,universal primers（TSWV-CP1/TSWV-CP2） were used for gene amplification and sequencing. Specific PCR primers（NA-P1/NA-P2）,based on the highly conserved region of N gene sequence of TSWV,were designed for nucleic acid sequence-based amplification（NASBA）. The suitable reaction time of NASBA for detecting TSWV was 1. 5h. The performance of NASBA was compared with real-time RT-PCR and conventional RT-PCR assays for detection of TSWV. The detection limits of NASBA,real-time RT-PCR and conventional RT-PCR assays were 1.56×10^（-4）,1.56×10^（-5）,and 1. 56 × 10^（-3）ng/μL for TSWV RNA,respectively. Real-time RT-PCR assay provided the most sensitive detection for TSWV. To further evaluate the specificity by using NASBA assay for the detection of TSWV,the Tobacco ring spot virus（TRSV）,Tomato black ring virus（TBRV）,Cucumber mosaic virus（CMV） and Tomato yellow curl virus（ ToYCLV） were also detected by NASBA. NASBA,real time RTPCR,conventional RT-PCR and ELISA assay were applied to detect nine clinical samples in order to assess the practical application effect. The results showed that NASBA was a very specific method for the detection of TSWV and had the same positive rate of 33% as real time RT-PCR and conventional RT-PCRassay. In comparison of the three methods,ELISA showed a low positive rate of 22%. Therefore,the NASBA method is useful,and may provide a potential alternative tool for rapid detection of TSWV.