环介导等温扩增技术可视化快速检测表皮葡萄球菌方法的建立

承德医学院学报 ›› 2023, Vol. 40 ›› Issue (6): 456-460.

环介导等温扩增技术可视化快速检测表皮葡萄球菌方法的建立

李冉, 薛晶, 郭文平, 康精萌, 刘金霞^*

承德医学院病原生物学实验中心,河北承德 067000

收稿日期:2022-12-17 出版日期:2023-12-10 发布日期:2023-12-15
通讯作者: *
基金资助:
河北省科技计划项目（ZD2017101）; 河北省教育厅项目（2021GJJG321）; 河北省高教学会重点项目（GJXHZ2021）

Development of Loop-mediated Isothermal Amplification for Rapid and Visual Detection of Staphylococcus epidermidis

LI Ran, XUE Jing, GUO Wen-ping, KANG Jing-meng, LIU Jin-xia^*

Laboratory Center for Pathogen Biology in Chengde Medical University, Chengde, Hebei, 067000, China

Received:2022-12-17 Online:2023-12-10 Published:2023-12-15

摘要/Abstract

摘要： 目的建立可视化环介导等温扩增（LAMP）检测方法,应用于表皮葡萄球菌的快速检测。方法针对表皮葡萄球菌的SesB基因,选择特异性引物,建立环介导等温扩增体系。对反应的可视化方法、体系中的MgSO₄浓度、甜菜碱浓度、反应温度、反应时间进行优化。将优化好的LAMP反应和PCR反应的灵敏度和特异性进行比较。结果环介导等温扩增反应体系中最适MgSO₄终浓度为4mM;最适甜菜碱浓度为0.4M;最适反应温度和时间为63 ℃,反应60 min;最适宜的可视化方法为钙黄绿素显色法且可视化结果与琼脂糖凝胶电泳结果基本一致;LAMP和PCR反应均具有较好的特异性,LAMP反应的灵敏度为10 copies/μL,PCR反应的灵敏度为100 copies/μL。结论 LAMP钙黄绿素可视化检测表皮葡萄球菌感染优于普通PCR,操作简便且不需要昂贵检测设备,有望应用于基层和偏远贫困地区早期诊断。

关键词: 环介导等温扩增技术, 表皮葡萄球菌, SesB基因, 可视化, 快速检测

Abstract: Objective To establish a visual loop-mediated isothermal amplification detection method for rapid detection of Staphylococcus epidermidis. Methods A loop-mediated isothermal amplification system was established by selecting specific primers for SesB gene of Staphylococcus epidermidis. The visualization method of the reaction, the concentration of MgSO₄, betaine in the system and the reaction temperature, time were optimized. The sensitivity and specificity of the optimized LAMP reaction and PCR reaction were compared. Results The optimal MgSO₄ concentration in the loop-mediated isothermal amplification reaction system was 4mM. The optimal betaine concentration was 0.4M. The optimal reaction temperature and time were 63℃ for 60 min. The most suitable visualization method was calcein chromogenic method, and the visualization results were basically consistent with the results of agarose gel electrophoresis. Both LAMP and PCR reactions had good specificity. The sensitivity of LAMP reaction was 10 copies/μL, and the sensitivity of PCR reaction was 100 copies/μL. Conclusion LAMP assay is superior to PCR in the detection of Staphylococcus epidermidis infection. It is more suitable for grass-roots and remote areas due to its simple operation and short reaction time.

Key words: loop-mediated isothermal amplification, Staphylococcus epidermidis, SesB gene, visualization, rapid detection

中图分类号:

R378.91

李冉, 薛晶, 郭文平, 康精萌, 刘金霞. 环介导等温扩增技术可视化快速检测表皮葡萄球菌方法的建立[J]. 承德医学院学报, 2023, 40(6): 456-460.

LI Ran, XUE Jing, GUO Wen-ping, KANG Jing-meng, LIU Jin-xia. Development of Loop-mediated Isothermal Amplification for Rapid and Visual Detection of Staphylococcus epidermidis[J]. Journal of Chengde Medical University, 2023, 40(6): 456-460.

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