Abstract: 【Objective】Grapevine virus A (GVA) is one of the most important pathogens causing grapevine rugose wood complex, 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 loop- mediated 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 (GRSPaV), Grapevine leafroll-associated virus (GLRaV), 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 10¹, 10², 10³, 10⁴, 10⁵ and 10⁶ 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 10¹, 10², 10³, 10⁴ and 10⁵ diluent total RNA templates of GVA, while the conventional RT-PCR could only detect 10¹, 10², 10³ and 10⁴ 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.

Key words: Grapevine virus A, RT-LAMP, detection