Development and Preliminary Application of LAMP Assay for Rapid Detection of Tobacco leaf curl Yunnan virus (TbLCYnV)
ZHAO Zheng-Ting1,2, GE Xiao-Tong1, ZHANG Jun-Lei3, XIA Zhen-Yuan1, LIU Di4, LIU Ya-Ting2,3,4,*, JIANG Ning1,*
1 Yunnan Academy of Tobacco Agricultural Sciences, Kunming 650021, China; 2 College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming 650201, China; 3 College of Plant Protection, Yunnan Agricultural University, Kunming 650201, China; 4 College of Tobacco Science, Yunnan Agricultural University, Kunming 650201, China
Abstract：Tobacco leaf curl Yunnan virus (TbLCYnV) is a new virus of the Begomovirus discovered on tobacco (Nicotiana tabacum) in Baoshan, Yunnan, China. It is one of the important pathogens of tobacco leaf curl disease in Yunnan. In order to quickly detect the TbLCYnV, five sets of primers were designed and screened according to the conserved nucleotide sequence of the coat protein (CP) gene of TbLCYnV, and the loop mediated isothermal amplification (LAMP) system reaction temperature, time, betaine concentration, dNTP concentration, Mg2+ concentration, and concentration ratio of internal and external primers were optimized by single variable method, At the same time, parallel comparisons with traditional PCR were conducted to verify the specificity and sensitivity of LAMP. The practicality of the LAMP system was verified using field collected samples, and the optimal LAMP detection system for TbLCYnV was established. The results showed that the optimal parameters were as follows: Primer group was TbL-5, the reaction temperature was 60 ℃, and the final concentrations of Betaine, dNTPs, and Mg2+ were 1.0, 0.4, 4.0 mmol/L, respectively; The optimal concentration ratio of internal and external primers was 4∶1, and the optimal reaction time was 60 min. The test results show that the optimized LAMP can be directly judged by naked eyes after staining with SYBR GreenⅠ, with high specificity and 10 000 times higher sensitivity than that of conventional PCR. This study provides a convenient, efficient, and reliable method for the detection of TbLCYnV, which is of great significance for the early and rapid diagnosis and prevention and control of TbLCYnV.
[1] 关雪, 胡琼波. 2020. 烟粉虱传播的植物病毒病及其化学防治研究进展[J]. 广东农业科学, 47(06): 63-69. (Guan X, Hu Q B.2020. Research progress of plant virus disease transmitted by bemisia tabaci and chemical control[J]. Guangdong Agricultural Sciences, 47(06): 63-69.) [2] 胡浅浅. 2016. 宏基因组测序研究介体烟粉虱中的双生病毒多样性[D]. 硕士学位论文, 浙江大学, 导师: 谢艳, pp. 1-4. (Hu Q Q.2016. Exploring the diversity of begomoviruses in whiteflies by metagenomic sequencing[D]. Thesis for M.S., Zhejiang University, Suppervisor: Xie Y, pp. 1-4.) [3] 刘勇. 2006. 云南烟草病毒检测及防治研究[D]. 博士学位论文, 浙江大学, 导师: 陶小荣, pp. 51-56. (Liu Y.2006. Identification of viruses infecting tobacco in Yunnan and viral disease control[D]. Thesis for Ph.D., Zhejiang University, Suppervisor: Tao X R, pp. 51-56.) [4] 刘勇, 谢艳, 廖白璐, 等. 2007. 云南省烟草上双生病毒的发生与分布(英文)[J]. 植物病理学报, (06): 566-571. (Liu Y, Xie Y, Liao B L, et al. 2007. Occurrence and distribution of Geminiviruses in tobacco in Yunnan province of China[J]. Acta Phytopathologica Sinica, (06): 566-571.) [5] 卢永灿. 2016. 三种梨病毒实时荧光定量RT-PCR和RT-LAMP检测技术的研究[D]. 硕士学位论文, 华中农业大学, 导师: 洪霓, pp. 38-49. (Lu Y C.2016. The establishment of real-time RT-PCR and RT-LAMP technioues for the detection of three viruses infecting pear plants[D]. Thesis for M.S., Huazhong Agricultural University, Suppervisor: Hong N, pp. 38-49.) [6] 王玉. 2011. 云南粉虱传双生病毒优势种变异及其与卫星分子的共进化[D]. 硕士学位论文, 昆明理工大学, 导师: 张仲凯, pp. 1-8. (Wang Y.2011. Variation of dominant species of WTGs and coevolution with satellite DNA in Yunnan[D]. Thesis for M.S., Kunming University of Science and Technology, Suppervisor: Zhang Z K, pp. 1-8.) [7] 谢艳, 张仲凯, 李正和, 等. 2002. 粉虱传双生病毒的TAS-ELISA及PCR快速检测[J]. 植物病理学报, (02): 182-186. (Xie Y, Zhang Z K, Li Z H, et al. 2002. Rapid detection of whitefly-transmitted Geminiviruses by TAS-ELISA and PCR[J]. Acta Phytopathologica Sinica, (02): 182-186.) [8] 张静雅, 何衍彪. 2019. 植物病毒病检测及防治技术研究进展[J]. 安徽农学通报, 25(12): 79-81. (Zhang J Y, He Y B.2019. Research progress of plant virus disease detection and management technology[J]. Anhui Agricultural Science Bulletin, 25(12): 79-81.) [9] 张仲凯. 2010. 云南双生病毒种群地理分布特征及其对病害发生危害的影响[D]. 硕士学位论文, 中国农业科学院, 导师: 黄兴奇, pp. 1-4. (Zhang Z K.2010. Geographic distribution of Geminiviruses in Yunnan and its effect on the disease occurrence and damage[D]. Thesis for M.S., Chinese Academy of Agricultural Sciences, Suppervisor: Huang X Q, pp. 1-4.) [10] 赵兴能. 2015. 云南烟草上双生病毒的优势种群及危害, 云南省烟草学会2014年学术年会[C]. 中国云南昆明. (Zhao X N.2015 Dominant population and harm of Begomoviruses on tobacco in Yunnan province, 2014 Academic Annual Meeting of Yunnan Tobacco Society[C].Kunming, Yunnan, China, 2014 Academic Annual Meeting of Yunnan Tobacco Society.) [11] 周雪平. 2015. 双生病毒种类鉴定、分子变异及致病机理研究, 中国植物保护学会2015年学术年会[C]. 中国吉林长春. (Zhou X P.2015. Identification of Begomoviruses species, molecular variation, and pathogenic mechanism research, 2015 Academic Annual Meeting of the Chinese Plant Protection Society[C]. Changchun, Jilin, China.) [12] 邹驰. 2021. 双生病毒β卫星分子在菜豆金黄花叶病毒属病毒-植物-烟粉虱三者互作中的机制探究博士学位论文, 浙江大学, 导师: 王晓伟, pp. 1-8. (Zou C.2021. The role of Geminivirus betasatellite in Begomovirus-plant-whitefly tripartite interactions[D]. Thesis for Ph.D., Zhejiang University, Suppervisor: Wang X W , pp. 1-8.) [13] Bhat A I, Aman R, Mahfouz M.2022. Onsite detection of plant viruses using isothermal amplification assays[J]. Plant Biotechnology Journal, 20(10): 1859-1873. [14] Boonham N, Kreuze J, Winter S, et al.2014. Methods in virus diagnostics: From ELISA to next generation sequencing[J]. Virus Research, 186: 20-31. [15] Brown J K, Idris A M, Torres-Jerez I, et al.2001. The core region of the coat protein gene is highly useful for establishing the provisional identification and classification of Begomoviruses[J]. Archives of Virology, 146(8): 1581-1598. [16] Caruso A G, Ragona A, Bertacca S, et al.2023. Development of an in-field real-time LAMP assay for rapid detection of Tomato leaf curl New Delhi virus[J]. Plants-Basel, 12(7): 1487. [17] Gawande S P, Raghavendra K P, Monga D, et al.2019. Rapid detection of Tobacco streak virus (TSV) in cotton (Gossypium hirsutum) based on reverse transcription loop mediated isothermal amplification (RT-LAMP)[J]. Journal of Virological Methods, 270: 21-25. [18] Gomez-Gutierrez S V, Goodwin S B.2022. Loop-mediated isothermal amplification for detection of plant pathogens in wheat (Triticum aestivum)[J]. Frontiers in Plant Science, 13: 857673. [19] Harrison B, Robinson D.1999. Natural genomic and antigenic variation in whitefly-transmitted Geminiviruses (Begomoviruses)[J]. Annual Review of Phytopathology, 37: 369-398. [20] Heithoff D M, Barnes L T, Mahan S P, et al.2022. Assessment of a smartphone-based loop-mediated isothermal amplification assay for detection of SARS-CoV-2 and Influenza viruses[J]. Jama Network Open, 5(1): e2145669. [21] Hogenhout S A, Ammar E, Whitfield A E, et al.2008. Insect vector interactions with persistently transmitted viruses[J]. Annual Review of Phytopathology, 46: 327-359. [22] Khan M, Wang R, Li B, et al.2018. Comparative evaluation of the LAMP assay and PCR-based assays for the rapid detection of Altemaria solani[J]. Frontiers in Microbiology, 9: 2089. [23] Le D T, Vu N T.2017. Progress of loop-mediated isothermal amplification technique in molecular diagnosis of plant diseases[J]. Applied Biological Chemistry, 60(2): 12. [24] Madhu K M, Nair S, Loius V.2023. One-step reverse transcription-loop mediated isothermal amplification (RT-LAMP) for closed-tube colorimetric detection of Banana bract mosaic virus in banana (Musa spp.)[J]. 3 Biotech, 13(5): 131. [25] Notomi T, Okayama H, Masubuchi H, et al.2000. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Research, 28(12): E63. [26] Shchit I Y, Kudryavtseva T Y, Mokrievich A N, et al.2022. Detection of tularemia agent DNA by loop mediated isothermal amplification[J]. Molecular Genetics Microbiology and Virology, 37(4): 202-208. [27] Soroka M, Wasowicz B, Rymaszewska A.2021. Loop-mediated isothermal amplification (LAMP): The better sibling of PCR?[J]. Cells, 10(8): 1931. [28] Supakitthanakorn S, Vichittragoontavorn K, Sunpapao A, et al.2022. Tobacco mosaic virus infection of chrysanthemums in thailand: Development of colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) technique for sensitive and rapid detection[J]. Plants (Basel), 11(14). [29] Tian Z, Yang L, Qi X, et al.2022. Visual LAMP method for the detection of Vibrio vulnificus in aquatic products and environmental water[J]. BMC Microbioloy, 22(1): 256. [30] Wang M, Tang Z, Liao M, et al.2023. Loop-mediated isothermal amplification for detecting the Ile-2041-Asn mutation in fenoxaprop-P-ethyl-resistant Alopecurus aequalis[J]. Pest Management Science, 79(2): 711-718. [31] Wanjala B W, Ateka E M, Miano D W, et al.2021. Loop-mediated isothermal amplification assays for on-site detection of the main sweetpotato infecting viruses[J]. Journal of Virological Methods, 298: 114301. [32] Watts M R, James G, Sultana Y, et al.2014. A loop-mediated isothermal amplification (LAMP) assay for strongyloides stercoralis in stool that uses a visual detection method with SYTO-82 fluorescent dye[J]. The American Journal of Tropical Medicine and Hygiene, 90(2): 306. [33] Zhou X P, Xie Y, Zhang Z K.2001. Molecular characterization of a distinct Begomovirus infecting tobacco in Yunnan, China[J]. Archives of Virology, 146(8): 1599-1606.
