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

作　　者：张轲 杨金初 赵旭 童治军[3] 李萌 徐永明 张展 孙九喆 ZHANG Ke;YANG Jinchu;ZHAO Xu;TONG Zhijun;LI Meng;XU Yongming;ZHANG Zhan;SUN Jiuzhe(Yunnan Tobacco Quality Inspection&Supervision Station,Kunming 650106,China;Technology Center,China Tobacco Henan Industrial Co.,Ltd.,Zhengzhou 450000,China;Yunnan Academy of Tobacco Agriculture Science/Key Laboratory of Tobacco Biotechnological Breeding/National Tobacco Genetic Engineering Research Center,Kunming 650021,China;College of Food and Bioengineering,Zhengzhou University of Light Industry,Zhengzhou 450001,China)

机构地区：[1]云南省烟草质量监督检测站,云南昆明650106 [2]河南中烟工业有限责任公司技术中心,河南郑州450000 [3]云南省烟草农业科学研究院/烟草行业烟草生物技术育种重点实验室/国家烟草基因工程研究中心,云南昆明650021 [4]郑州轻工业大学食品与生物工程学院,河南郑州450001

出　　处：《轻工学报》2023年第2期110-117,共8页Journal of Light Industry

基　　金：云南省烟草专卖局(公司)重点项目(2020530000241034);河南中烟工业有限责任公司科技计划项目(ZW2015012)。

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、Mg^(2+)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10^(2) 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。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,Mg^(2+)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 10^(2) copies/μL.The LAMP detection based on Nicotiana-specific gene Ntsp151 was result-visualized,highly sensitive,and specific,which was suitable for field inspection.

关 键 词：烟草属特异性基因 Ntsp151 环介导等温扩增 可视化 基因组 

分 类 号：TS47[农业科学—烟草工业] S338