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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2023, Vol. 43 Issue (10): 52-61    DOI: 10.13523/j.cb.2303027
综述     
基于环介导等温扩增的微流控芯片检测技术研究进展*
李林岳1,**,袁嘉康1,**,李任峰1,***(),庞俊增1,秦保亮2,周艳琳3,王自良1,***()
1 河南科技学院动物科技学院 河南省高校动物疫病防控创新团队 新乡 453003
2 新乡市动物疫病预防控制中心 新乡 453003
3 新乡医学院三全学院 新乡 453003
Research Progress of Microfluidic Chip Detection Technology Based on Loop-mediated Isothermal Amplification
LI Lin-yue1,**,YUAN Jia-kang1,**,LI Ren-feng1,***(),PANG Jun-zeng1,QIN Bao-liang2,ZHOU Yan-lin3,WANG Zi-liang1,***()
1 College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Innovative Research Team for Animal Disease Prevention and Control in Universities of Henan Province, Xinxiang 453003, China
2 Animal Disease Prevention and Control Center of Xinxiang City, Xinxiang 453003, China
3 Sanquan College of Xinxiang Medical University, Xinxiang 453003, China
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摘要：

基于环介导等温扩增(loop-mediated isothermal amplification,LAMP)的微流控芯片是一种新型核酸检测方法。该方法能够将核酸提取、等温扩增和反应结果集成在厘米级的芯片上,具有灵敏度高、特异性强、不易污染等特点,并且可以实现“样品-答案”的快速检测,已成为当前检测领域的研究热点。综述LAMP的反应原理、微流控芯片的种类、LAMP微流控芯片在病原微生物和肿瘤检测中的应用,并分析LAMP微流控芯片目前存在的问题及未来发展前景,以进一步拓展对新型检测技术的认识,加快其在现场快速检测中的推广和应用。
关键词： 环介导等温扩增;  微流控芯片;  病原检测    
Abstract:

The microfluidic chip based on loop-mediated isothermal amplification (LAMP) is a novel nucleic acid detection method that integrates nucleic acid extraction, LAMP reaction and signal display on a chip of several centimeters. It has advantages such as high sensitivity, strong specificity, convenient and fast operation, and a detection process not easy to be contaminated and can realize the detection process from sample to answer. It has become a hot spot in the current detection field. In this paper, the principle of LAMP reaction, the types of microfluidic chips and the research progress of LAMP microfluidic chips in the detection of pathogens and tumors are reviewed, and the existing problems and future development prospects of LAMP microfluidic chips are analyzed. This review may help further expand the understanding of new detection technologies and accelerate their application in on-site rapid detection.
Key words: Loop-mediated isothermal amplification (LAMP)    Microfluidic chip    Examination of pathogen
收稿日期: 2023-03-10 出版日期: 2023-11-02
ZTFLH:  Q78  
基金资助: *河南省重点研发与推广专项(科技攻关)(212102110098);河南省重点研发与推广专项(科技攻关)(232102310011)
通讯作者: ***电子信箱:lirenfeng@sina.com;wangziliang66@126.com   
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李林岳
袁嘉康
李任峰
庞俊增
秦保亮
周艳琳
王自良

引用本文:

李林岳, 袁嘉康, 李任峰, 庞俊增, 秦保亮, 周艳琳, 王自良. 基于环介导等温扩增的微流控芯片检测技术研究进展*[J]. 中国生物工程杂志, 2023, 43(10): 52-61.

LI Lin-yue, YUAN Jia-kang, LI Ren-feng, PANG Jun-zeng, QIN Bao-liang, ZHOU Yan-lin, WANG Zi-liang. Research Progress of Microfluidic Chip Detection Technology Based on Loop-mediated Isothermal Amplification. China Biotechnology, 2023, 43(10): 52-61.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2303027    或    https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I10/52

图1  LAMP反应过程
性能指标 玻璃 PDMS PMMA PC 纸基材料
生物相容性 较好 易非特异性吸附 较好 较好 较好
光学特性 较好 较好 较好 紫外吸收较差 检测背景低
导热系数 较好 低 低 低 低
电绝缘性 较好 较好 较好 较好 较好
热稳定性 -80~860℃ -50~200℃ -80~90℃ -60~120℃ 试剂受热易蒸发
材料成本 适中 适中 适中 适中 低
加工难度 较难 适中 适中 适中 容易
表1  微流控芯片常用材料及其性能
图2  焦磷酸镁沉淀生成原理
特点 羟基萘酚蓝 钙黄绿素 SYBR Green I 甲酚红 中性红
补充试剂 无 锰离子 无 无 无
指示物质 镁离子 锰离子、镁离子 双链DNA 氢离子 氢离子
使用浓度 120 μmol/L 50 μmol/L 1 × 120 μmol/L 270 μmol/L
颜色变化 紫罗兰色-天蓝色 橙黄色-草绿色,有荧光 橙色-黄绿素,有荧光 紫红色-黄色 橙黄色-紫红色
反应抑制作用 无 轻微 严重 无 无
加入时间 反应前 反应前 反应后 反应前 反应前
观察难度 颜色对比度低 可见光下颜色对比度低,
需紫外光源		 可见光下颜色对比度低,
需紫外光源		 容易 容易
表2  常见LAMP指示剂的特性
图3  免疫层析试纸法原理
病原和肿瘤 检测速度 灵敏度 检测样本数 检出样本数 符合率/% 多重检测 参考文献
细菌 金黄色葡萄球菌 40 min 30 CFU/反应 - - - 是 [42]
副溶血性弧菌 65 min 10个细胞/反应 - - - 是 [43]
沙门氏菌 65 min 10个细胞/反应 - - - 是 [43]
大肠杆菌 65 min 10个细胞/反应 - - - 是 [43]
病毒 猪流行腹泻病毒 30 min 10拷贝/μL 11 5 100 是 [44]
新型冠状病毒 60 min 0.5拷贝/μL 12 12 100 否 [45]
人类免疫缺陷病毒 50 min 2拷贝/μL 20 20 100 是 [46]
埃博拉病毒 50 min 10拷贝/μL - - - 是 [47]
寨卡病毒 60 min 10拷贝/反应 5 5 100 是 [48]
登革热病毒 60 min 10拷贝/反应 5 5 100 是 [48]
猪瘟病毒 60 min 3.2 × 102拷贝/反应 232 218 94 是 [49]
寄生虫 疟原虫 50 min 100 fg /反应 6 6 100 是 [51]
血吸虫 90 min 2 × 10-2 fg /μL 200 200 100 否 [53]
布氏锥虫 60 min 40拷贝/反应 - - - 否 [54]
肿瘤 骨髓增殖性肿瘤 60 min 1%~3%的突变基因 30 27 90 是 [55]
肺癌 45 min 10拷贝/反应 86 86 - 是 [56]
宫颈癌 90 min 5~50拷贝/μL 41 35 100 是 [57]
前列腺癌 75 min 0.34 fg/μL - - - 否 [58]
表3  LAMP微流控芯片在病原和肿瘤检测中的应用
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