LAMP耦合荧光侧向流层析试纸条的空间微生物快速检测技术

韩培; 侯红渠; 樊云龙; 王文甲; 吕雪飞; 张伟; 李晓琼

doi:10.11728/cjss2023.02.211125124

LAMP耦合荧光侧向流层析试纸条的空间微生物快速检测技术

doi: 10.11728/cjss2023.02.211125124

1.
中国科学院空间应用工程与技术中心　北京　100094
2.
北京理工大学生命学院　北京　100081

基金项目: 中国科学院空间应用工程与技术中心所长基金课题项目（Y903191）和国防科技战略先导计划项目（20-ZLXD-21-03-02-002-03）共同资助

详细信息

作者简介:
韩培：E-mail：hp@csu.ac.cn

中图分类号: V524
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- HTML全文浏览量: 20
- PDF下载量: 4
- 被引次数: 0
出版历程
- 收稿日期: 2021-11-24
- 修回日期: 2022-10-11
- 网络出版日期: 2023-04-08

Space Microbial Detection Method Based on Fluorescent LAMP

1.
Center for Space Science and Applied Research, Chinese Academy of Sciences, Beijing 100094
2.
School of Life, Beijing Institute of Technology, Beijing 100081

摘要

摘要: 微生物种类及其含量监测是空间站内微生物控制的重要环节。但是空间环境的微重力条件及对资源的条件限制导致地面常规检测实验难以开展，因此在轨微生物检测主要依靠培养法。基于侧流层析试纸条的生物分子识别检测方法具有不受微重力环境影响的优点，耦合荧光检测方法可以达到较高的检测灵敏度，是在轨微生物检测的潜在方法之一。针对空间环境中对航天员生活环境及仪器仪表设备具有潜在危害的微生物，研究了一种基于环介导等温扩增（LAMP）耦合荧光侧流层析试纸条的微生物核酸鉴别技术。研究结果表明，该技术可实现对大肠杆菌、金黄色葡萄球菌等空间站常见有害微生物的高灵敏、高特异性、快速检测，检测时间小于60 min，灵敏度达到100 copy·μL^–1。
- 空间微生物 /
- 侧流层析试纸条 /
- 环介导等温扩增
Abstract: Relevant studies on the International Space Station have shown that during the long-term operation of the space station, the existence of microorganisms may pose a threat to the long-term operation of the space station and stay of astronauts. Microbial species and content monitoring are important parts of microbial control in the station. Due to resource constraints such as volume and power consumption in space applications, detection methods based on lateral flow chromatography test strips and supporting handheld devices will be more suitable for daily monitoring of space microorganisms, combined with loop-mediated isothermal amplification and up-conversion luminescence characteristic spectra. Nucleic acid identification technology can meet the needs of rapid, sensitive and specific identification of microorganisms. In this research, aiming at the potential harmful microorganisms in the space for the living environment of astronauts and instrumentation equipment, the nucleic acid identification technology based on fluorescent LAMP (Loop-mediated isothermal Amplification) of the lateral flow chromatography test strip was developed. Research results showed that this technology can achieve high sensitivity, high specificity and rapid detection of common harmful microorganisms on the space station such as Escherichia coli and Staphylococcus aureus. The detection time is less than 60 minutes and the sensitivity can reach 100 copy·μL^–1.
- Space microorganism /
- Lateral flow chromatography test strip /
- Loop-mediated isothermal amplification

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图 1 malB及nuc基因的LAMP扩增荧光曲线

Figure 1. LAMP amplification fluorescence curves of malB and nuc genes

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图 2 偶联后不同浓度样品LAMP扩增曲线（a）；偶联后不同浓度样品LAMP扩增产物凝胶电泳成像（b）；偶联后不同浓度样品LAMP扩增产物试纸条上样，采用470 nm波段激发后荧光成像结果（c）

Figure 2. LAMP amplification curves of samples with different concentrations after coupling (a); Gel electrophoresis imaging of LAMP amplification products of samples with different concentrations after coupling (b); Test strip loading of LAMP amplification products of samples with different concentrations after coupling, fluorescence imaging results after excitation at 470 nm (c)

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图 3 荧光检测光学原理

Figure 3. Principle of fluorescence detection

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图 4 荧光检测光学结构

Figure 4. Fluorescence detector optical structure

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图 5 荧光检测光学线性曲线

Figure 5. Fluorescence linear curve for fluorescence detector

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图 6 荧光检测光学试纸条扫描曲线

Figure 6. Fluorescence curve of test strip scanned by fluorescence detector

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