基于烟草属特异性基因Ntsp151的环介导等温扩增检测
Detection of loop-mediated isothermal amplification based on Nicotiana-specific gene Ntsp151
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摘要: 针对烟草属特异性基因Ntsp151序列保守区设计环介导等温扩增(LAMP)引物,基于SYBR Green I荧光染料对烟草材料进行可视化LAMP检测,对检测过程中的DNA提取方法和反应条件进行优化,并对LAMP反应体系的特异性和灵敏度进行评价。结果表明:使用Chelex-100提取的DNA可以直接进行LAMP扩增反应,显色效果较好;LAMP最适反应条件为扩增温度63 ℃、反应时间60 min、Mg2+浓度6 mmol/L;LAMP对17份非烟草材料和1份烟草材料基因组DNA的扩增显色结果与荧光值的检测结果一致,具有较好的特异性,其最低检测限为102 copies/μL。基于烟草属特异性基因Ntsp151的LAMP检测结果可视化强,且灵敏度高、特异性强,适用于现场抽检。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.
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Key words:
- Nicotiana-specific gene /
- Ntsp151 /
- loop-mediated isothermal amplification /
- visualization /
- genome
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