葡萄A病毒RT-LAMP检测方法的建立

Development of a RT-LAMP Assay for Detection of Grapevine virus A

【目的】葡萄A病毒（Grapevine virus A,GVA）是葡萄皱木复合病的重要病原之一,以带毒种苗的嫁接为田间主要传播途径。使用无毒葡萄苗木进行生产是控制该病毒病害的根本措施,而简便灵敏的检测技术是筛选无毒种苗的有效保证。为此,开展针对GVA的反转录环介导等温扩增技术（reverse transcription loop-mediated isothermal amplification,RT-LAMP）研究,旨在建立其RT-LAMP特异性检测方法。【方法】通过比对分析GVA的CP（coat protein）基因序列并经引物设计软件Primer Explore 4.0设计,获得6条检测引物GVA-FIP（5′-CTTACAGCCACGCTCAGAGTCC-CGTGGGAAGTTGGTTGTGT-3′）、GVA-BIP（5′-GCCCGTCAAAGGGGCTACAC-TCATA GGCGTTCTGTGCGA-3′）、GVA-F3（5′-AGAAGATGGGGATAGACCCG-3′）、GVA-B3（5′-CCGCCATTAACACGAGGAA-3′）、GVA-LF（5′-AT CCTTCCCACCAGCTCGG-3′）和GVA-LB（5′-TCAGGCAGATGTGTGAACCT-3′）。将RT-LAMP的反应温度分别设为59、61、63和65℃,根据1 h扩增曲线出现的先后次序确定最佳反应温度。以同样侵染葡萄的沙地葡萄茎痘相关病毒（Grapevine rupestris stem pitting-associated virus,GRSPa V）、葡萄卷叶病毒（Grapevine leafrollassociated virus,GLRa V）、葡萄斑点病毒（Grapevine fleck virus,GFKV）的阳性材料及阴性对照（健康葡萄叶片,用NC表示）的总RNA为模板,对RT-LAMP方法的特异性进行测定。将GVA的总RNA进行10倍梯度稀释,得到10^1、10^2、10^3、10^4、10^5、10^6倍的不同稀释液后用作模板分别进行RT-LAMP和普通RT-PCR检测,比较两者的灵敏度。RT-LAMP产物可进行实时扩增曲线检测,阳性样品在浊度仪上出现扩增曲线,阴性样品无扩增曲线;还可进行染料颜色反应检测,在反应产物中加入SYBR Green I染料,阳性样品产生肉眼可见的绿色,阴性反应为橙色。【结果】建立了GVA的RT-LAMP快速特异性检测方法,最佳反应温度为65℃。该方法约27 min即可检测到扩增曲线,而普通RT-PCR获得检测结果约需1.5 h。特异性试验表明只有GVA的总RNA能够出现扩增曲线,反应液颜色变为绿色,显示为阳性,而其他3种参试病毒和健康对照没有出现扩增曲线,反应液的颜色为橙色,显示为阴性;RT-LAMP检测结果可通过肉眼观察直接判断,比普通RT-PCR结果判断容易。灵敏度试验显示RT-LAMP可检测到10^1、10^2、10^3、10^4和10^5倍的GVA总RNA稀释液模板,而普通RT-PCR只能检测到10^1、10^2、10^3和10^4倍的GVA总RNA稀释液模板,表明前者比后者的灵敏度高10倍。【结论】研究建立的RT-LAMP方法能够快速特异地检测GVA,可为筛选无GVA葡萄苗木提供技术支持;适用于检疫、科研及生产机构在进境检疫、苗木繁育和大田监测工作中对GVA的检测鉴定。

【Objective】Grapevine virus A（GVA） is one of the most important pathogens causing grapevine rugose woodcomplex, which is mainly transmitted by grafting using seedlings with GVA in the field. The production using free-GVA grape seedlings is the radical measure for the control of GVA disease, while the simple and sensitive detection method is the effective guarantee for the free-GVA seedlings screening. The objective of this study is to carry out the study of reverse transcription loopmediated isothermal amplification technology（RT-LAMP）, and to establish the RT-LAMP specific method for the detection of GVA.【Method】 Six specific primers for GVA detection including GVA-FIP（5′-CTTACAGCCACGCTCAGAGTCC-CGTGGGAAG TTGGTTGTGT-3′）, GVA-BIP（5′-GCCCGTCAAAGGGGCTACAC-TCATAGGCGTTCTGTGCGA-3′）, GVA-F3（5′-AGAAGATG GGGATAGACCCG-3′）, GVA-B3（5′-CCGCCATTAACACGAGGAA-3′）, GVA-LF（5′-ATCCTTCCCACCAGCTCGG-3′） and GVA-LB（5′-TCAGGCAGATGTGTGAACCT-3′） were designed using GVA coat protein（CP） gene sequences after a comparison analysis and design using primer design software Primer Explore 4.0. Different RT-LAMP reaction temperatures including 59, 61, 63 and 65℃ were experimented in order to determine the optimal reaction temperature according to the appearing order of the amplification curve within 1 h. The total RNAs of three other grape-infecting viruses including Grapevine rupestris stem pitting-associated virus（GRSPa V）, Grapevine leafroll-associated virus（GLRa V）, Grapevine fleck virus（GFKV） and the negative control（healthy grape leaf named NC） were used in this study to determine the specificity of the RT-LAMP method. The total RNA of GVA was ten-fold serially diluted including 10^1、10^2、10^3、10^4、10^5 and 10^6 dilutions and used as the templates for the sensitivity comparison between RT-LAMP and conventional RT-PCR. The RT-LAMP result could be judged by a real-time amplification curve, the curve of the positive sample appeared at the turbidity meter while no curve was observed in the negative samples. The RT-LAMP result could be also judged by a dye color reaction, the color of the positive sample was green and the color of the negative sample was orange after adding SYBR Green I in the reaction liquid.【Result】The faster and specific RT-LAMP method for the detection of GVA was developed, and the optimal reaction temperature was 65℃. The method could obtain an amplification curve only in 27 min, while the detection results could be obtained in 1.5 h using conventional RT-PCR. Specificity experiments indicated that the amplification curve could only be obtained from the total RNA of GVA and the color of the reaction liquid changed to green, which showed positive; while the other three viruses and the healthy control did not appear on the amplification curve and the color of the reaction liquids were orange, which showed negative. RT-LAMP detection results could be directly observed by the naked eye, which was easier than the results judgment of the conventional RT-PCR. Sensitivity experiments indicated that RT-LAMP could detect 10^1, 10^2, 10^3, 10^4 and 10^5 diluent total RNA templates of GVA, while the conventional RT-PCR could only detect 10^1, 10^2, 10^3 and 10^4 diluent total RNA templates of GVA, which showed the sensitivity of the former was 10 times higher than the sensitivity of the latter.【Conclusion】The RT-LAMP method developed in this study could be used to detect GVA rapidly and specifically, which provided technical support for the screening of GVA-free grape seedlings and was suitable for the detection and identification of GVA in the entry quarantine, seedling breeding and field monitoring works by quarantine, research and production organizations.