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| 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 |
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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.
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Received: 02 August 2023
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Corresponding Authors:
* liuyating@163.com; jiangning1990@163.com
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