基于烟草属特异性基因<i>Ntsp</i>151的环介导等温扩增检测

张轲; 杨金初; 赵旭; 童治军; 李萌; 徐永明; 张展; 孙九喆

doi:10.12187/2023.02.014

April 2023

Article Contents

ZHANG Ke, YANG Jinchu, ZHAO Xu, et al. Detection of loop-mediated isothermal amplification based on Nicotiana-specific gene Ntsp151[J]. Journal of Light Industry, 2023, 38(2): 110-117. doi: 10.12187/2023.02.014

Citation: ZHANG Ke, YANG Jinchu, ZHAO Xu, et al. Detection of loop-mediated isothermal amplification based on Nicotiana-specific gene Ntsp151[J]. Journal of Light Industry, 2023, 38(2): 110-117. doi: 10.12187/2023.02.014 shu

Detection of loop-mediated isothermal amplification based on Nicotiana-specific gene Ntsp151

ZHANG Ke ¹ ,
YANG Jinchu ² ,
ZHAO Xu ² ,
TONG Zhijun ³ ,
LI Meng ⁴ ,
XU Yongming ² ,
ZHANG Zhan ² ,
SUN Jiuzhe ²

1. Yunnan Tobacco Quality Inspection & Supervision Station, Kunming 650106, China;
2. Technology Center, China Tobacco Henan Industrial Co., Ltd., Zhengzhou 450000, China;
3. Yunnan Academy of Tobacco Agriculture Science/Key Laboratory of Tobacco Biotechnological Breeding/National Tobacco Genetic Engineering Research Center, Kunming 650021, China;
4. College of Food and Bioengineering, Zhengzhou University of Light Industry, Zhengzhou 450001, China

Received Date: 2022-12-17

Abstract

The loop-mediated isothermal amplification (LAMP) primers were designed for the conserved region of the selected Nicotiana-specific gene Ntsp151 sequence, and a visual LAMP detection method for tobacco materials based on the SYBR Green I was established. The genomic DNA extraction method and reaction conditions were optimized in the detection process. Furthermore, the specificity and sensitivity of the LAMP reaction system were evaluated. The results showed that the genomic DNA extracted by Chelex-100 could be directly amplified and fluorescently colored by LAMP; the optimal reaction conditions of the LAMP primers were amplification temperature 63 ℃, reaction time 60 min, Mg²⁺ concentration 6 mmol/L; LAMP detection method had good specificity and the results of chromogenic amplification and fluorescence detection of 17 non-tobacco materials and 1 tobacco material were consistent and had good specificity. The sensitivity test showed that the minimum detection limit of the LAMP reaction system was 10² copies/μL. The LAMP detection based on Nicotiana-specific gene Ntsp151 was result-visualized, highly sensitive, and specific, which was suitable for field inspection.
- Nicotiana-specific gene,
- Ntsp151,
- loop-mediated isothermal amplification,
- visualization,
- genome
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沈阳化工大学材料科学与工程学院沈阳 110142

Detection of loop-mediated isothermal amplification based on Nicotiana-specific gene Ntsp151

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