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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2021, Vol. 41 Issue (2/3): 116-128    DOI: 10.13523/j.cb.2011014
综述     
基于微流控芯片的核酸等温扩增技术研究进展 *
时忠林1,崔俊生2,杨柯2,胡安中2,李亚楠2,刘勇2,邓国庆2,朱灿灿2,**(),朱灵2,**()
1 安徽大学 合肥 230601
2 中国科学院合肥物质科学研究院 安徽光学精密机械研究所 安徽省生物医学光学仪器工程技术研究中心安徽省医用光学诊疗技术与装备工程实验室 合肥 230031
Research Progress in Isothermal Amplification of Nucleic Acid Based on Microfluidic Chip
SHI Zhong-lin1,CUI Jun-sheng2,YANG Ke2,HU An-zhong2,LI Ya-nan2,LIU Yong2,DNEG Guo-qing2,ZHU Can-can2,**(),ZHU Ling2,**()
1 Anhui University, Hefei 230601, China
2 Hefei Institute of Physical Science, Chinese Academy of Sciences,Anhui Institute of Optics and Precision Machinery,Anhui Biomedical Optical Instrument Engineering Technology Research Center,Anhui Medical Optical Diagnosis and Treatment Technology and Equipment Engineering Laboratory,Hefei 230031, China
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摘要:

核酸等温扩增技术是一种在恒温体系内对核酸进行高效扩增的分子扩增技术,它能够在短时间内实现目的基因的指数增长。微流控芯片(microfluidic chip)技术是把研究样品制备、核酸富集、纯化和检测等多个操作步骤集成到一块“微型化”的芯片上,经自动化处理,得出实验结果,即“样品进,结果出”。将核酸等温扩增技术与微流控芯片相结合,不仅可以实现核酸快速扩增,还可以降低对实验器材的依赖。在床边即时诊断、病原体快速筛查中具有广阔的应用前景。综合国内外相关研究报道,综述了各种等温扩增技术原理,以及基于微流控芯片的核酸等温扩增技术应用,展望了集成化微流控芯片的发展趋势和应用前景。

关键词: 微流控芯片核酸等温扩增床边即时诊断    
Abstract:

The isothermal amplification technology of nucleic acid is a molecular amplification technology which can amplify nucleic acid efficiently in a constant temperature system. It can realize exponential growth of target gene in a short time. The microfluidic chip should not be too close to the surface, but should not be too close to the surface. Several steps, such as sample preparation, nucleic acid enrichment, purification and detection, should be integrated onto a “miniaturized”chip, and should be automatically treated to get the experimental result:“sample in, result out”. The combination of the isothermal nucleic acid amplification technology and microfluidic chip can not only realize rapid nucleic acid amplification, but also reduce the dependence on experimental equipment. It has a broad application prospect in bedside instant diagnosis and pathogen rapid screening.The principle of isothermal amplification technology and the application of isothermal amplification technology of nucleic acid based on microfluidic chip are reviewed, and the development trend and application prospect of integrated microfluidic chip are prospeced.

Key words: Microfluidic chip    Isothermal nucleic acid amplification    Bedside instant diagnosis
收稿日期: 2020-11-05 出版日期: 2021-04-08
ZTFLH:  Q52Q819  
基金资助: * 国家自然科学基金(82002189);安徽省自然科学基金()(2008085QF311);安徽省重点研发计划(202004d07020014);中科院合肥研究院“火花”基金资助项目(YZJJ2020QN35)
通讯作者: 朱灿灿,朱灵     E-mail: zhucancan@aiofm.ac.cn;zhul@aiofm.ac.cn
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引用本文:

时忠林,崔俊生,杨柯,胡安中,李亚楠,刘勇,邓国庆,朱灿灿,朱灵. 基于微流控芯片的核酸等温扩增技术研究进展 *[J]. 中国生物工程杂志, 2021, 41(2/3): 116-128.

SHI Zhong-lin,CUI Jun-sheng,YANG Ke,HU An-zhong,LI Ya-nan,LIU Yong,DNEG Guo-qing,ZHU Can-can,ZHU Ling. Research Progress in Isothermal Amplification of Nucleic Acid Based on Microfluidic Chip. China Biotechnology, 2021, 41(2/3): 116-128.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2011014        https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I2/3/116

参数 环介导扩增(LAMP) 链替代扩增(SDA) 重组酶扩增(RPA) 滚环扩增(RCA)
发明年份 2000年 1992年 2006年 1998年
引物数量 4~6 4 2 1
温度(℃) 60~65 37 30~42 37
时间(min) 60~90 120 20 60
产物检测 凝胶电泳、浊度仪、
光学传感器(吸光度)
凝胶电泳、实时荧光 实时荧光 凝胶电泳
多重检测
优势 特异性高、操作简单、
快速
节能、反应速度快 引物设计简单、稳定性好、
干燥的颗粒试剂有助于
现场诊断
特异性好、可进行SNP检测
劣势 引物设计复杂,不能进行
多重扩增
酶选择复杂、扩增长
片段效率较低
不能用于后续反应 引物设计复杂,酶价格高
表1  几种恒温扩增技术的性能比较
图1  LAMP反应原理图[25]
图2  八通道PDMS-玻璃混合微流控芯片和定量分析装置[26]
图3  RCA反应原理图
图4  RPA反应原理图
图5  装有液体试剂容器和冻干试剂的箔片盘照片
图6  HDA反应原理图[18]
图7  SDA反应原理图
图8  MDA原理图
图9  微阵列MDA系统
图10  NASBA原理图
[1] Chang W H, Wang C H, Lin C L, et al. Rapid detection and typing of live bacteria from human joint fluid samples by utilizing an integrated microfluidic system. Biosensors and Bioelectronics, 2015,66:148-154.
pmid: 25460896
[2] Rollo F, Salvi R, Amici A, et al. Polymerase chain reaction fingerprints. Nucleic Acids Research, 1987,15(21):9094-9094.
pmid: 3684589
[3] Mullis K, Faloona F, Scharf S, et al. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symposia on Quantitative Biology, 1986,51:263-273.
doi: 10.1101/sqb.1986.051.01.032 pmid: 3472723
[4] Mullis K B, Faloona F A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods in Enzymology, 1987,155:335-350.
[5] Zhou W C, Wu Y H, Hao P, et al. Highly sensitive and multi-parameter optical detection for whole blood on centrifugal microfluidic chip. Optics and Precision Engineering, 2013,21(11):2821.
[6] Manz A, Graber N, Widmer H M. Miniaturized total chemical analysis systems-a novel concept for chemical sensing. Sensors and Actuators B: Chemical, 1990,1(1-6):244-248.
[7] Zatti M S, Arantes T D, Theodoro R C. Isothermal nucleic acid amplification techniques for detection and identification of pathogenic fungi: A review. Mycoses, 2020,63(10):1006-1020.
doi: 10.1111/myc.13140 pmid: 32648947
[8] Jiang S, Li Y. Principle and application of isothermal amplification technology. Chinese Journal of Laboratory Medicine, 2020,43(5):591-596.
[9] Giuffrida M C, Zanoli L M, D’Agata R, et al. Isothermal circular-strand-displacement polymerization of DNA and microRNA in digital microfluidic devices. Analytical and Bioanalytical Chemistry, 2015,407(6):1533-1543.
[10] Ye X, Li Y, Wang L J, et al. All-in-one microfluidic nucleic acid diagnosis system for multiplex detection of sexually transmitted pathogens directly from genitourinary secretions. Talanta, 2021,221:121462-121462.
pmid: 33076082
[11] 叶璟, 朱慧. 核酸等温扩增产物检测方法的研究进展. 科技通报, 2019,35(3):215-218.
Ye J, Zhu H. Research progress in the detection methods of isothermal nucleic acid amplification amplicons. Bulletin of Science and Technology, 2019,35(3):215-218.
[12] Notomi T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, 2000,28(12):e63.
doi: 10.1093/nar/28.12.e63 pmid: 10871386
[13] Xu G L, Hu L, Zhong H Y, et al. Cross priming amplification: Mechanism and optimization for isothermal DNA amplification. Scientific Reports, 2012,2:246.
doi: 10.1038/srep00246 pmid: 22355758
[14] Feng T, Li S F, Wang S N, et al. Cross priming amplification with nucleic acid test strip analysis of mutton in meat mixtures. Food Chemistry, 2018,245:641-645.
[15] Fire A, Xu S Q. Rolling replication of short DNA circles. Proceedings of the National Academy of Sciences, 1995,92(10):4641-4645.
[16] Lizardi P M, Huang X H, Zhu Z R, et al. Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nature Genetics, 1998,19(3):225-232.
pmid: 9662393
[17] Piepenburg O, Williams C H, Stemple D L, et al. DNA detection using recombination proteins. PLoS Biology, 2006,4(7):e204.
doi: 10.1371/journal.pbio.0040204 pmid: 16756388
[18] Vincent M, Xu Y, Kong H M. Helicase-dependent isothermal DNA amplification. EMBO Reports, 2004,5(8):795-800.
doi: 10.1038/sj.embor.7400200 pmid: 15247927
[19] Walker G T, Little M C, Nadeau J G, et al. Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system. Proceedings of the National Academy of Sciences, 1992,89(1):392-396.
[20] Anonymous. Nucleic acid sequence-based amplification (NASBA) for detection of EF-TU mRNA of mycobacterium tuberculosis. Clinical Biochemistry, 2005,38(9):843-843.
[21] Hill C S. Molecular diagnostic testing for infectious diseases using TMA technology. Expert Review of Molecular Diagnostics, 2001,1(4):445-455.
doi: 10.1586/14737159.1.4.445 pmid: 11901859
[22] Notomi T, Nagamine K, Hase T. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Molecular and Cellular Probes, 2002,16(3):223-229.
[23] Gill P, Ranjbar B, Saber R. Scanning tunneling microscopy of cauliflower-like DNA nanostructures synthesised by loop-mediated isothermal amplification. IET Nanobiotechnology, 2011,5(1):8-13.
doi: 10.1049/iet-nbt.2010.0008 pmid: 21241156
[24] Kumar S T, Krishnan N A, Rajan J S J , et al. Visual loop-mediated isothermal amplification (LAMP) for the rapid diagnosis of Enterocytozoon hepatopenaei (EHP) infection. Parasitology Research, 2018,117(5):1485-1493.
doi: 10.1007/s00436-018-5828-4 pmid: 29550998
[25] Notomi T, Mori Y, Tomita N, et al. Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects. Journal of Microbiology, 2015,53(1):1-5.
[26] Fang X E, Liu Y Y, Kong J L, et al. Loop-mediated isothermal amplification integrated on microfluidic chips for point-of-care quantitative detection of pathogens. Analytical Chemistry, 2010,82(7):3002-3006.
pmid: 20218572
[27] Lee S Y, Huang J G, Chuang T L, et al. Compact optical diagnostic device for isothermal nucleic acids amplification. Sensors and Actuators B: Chemical, 2008,133(2):493-501.
[28] Ou X C, Song Y Y, Zhao B, et al. A multicenter study of cross-priming amplification for tuberculosis diagnosis at peripheral level in China. Tuberculosis, 2014,94(4):428-433.
doi: 10.1016/j.tube.2014.04.006 pmid: 24880705
[29] Wang Y, Wang Y, Ma A J, et al. Rapid and sensitive detection of Listeria monocytogenes by cross-priming amplification of lmo0733 gene. Fems Microbiology Letters, 2014,361(1):43-51.
doi: 10.1111/1574-6968.12610 pmid: 25273275
[30] Liu W Z, Zhou Y, Fan Y D, et al. Development of cross-priming amplification coupled with vertical flow visualization for rapid detection of infectious spleen and kidney necrosis virus (ISKNV) in mandarin fish, Siniperca chuatsi. Journal of Virological Methods, 2018,253:38-42.
[31] Xiang Y, Yan L, Zheng X C, et al. Rapid detection of Pseudomonas aeruginosa by cross priming amplification. Journal of Integrative Agriculture, 2020,19(10):2523-2529.
[32] Chen H, Huang S W, Xu C P, et al. The establishment of rapid detection of Staphylococcus aureus by cross primer amplification technology. Chinese Journal of Health Laboratory Technology, 2019,29(11), 1303-1305.
[33] Murakami T, Sumaoka J, Komiyama M. Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification. Nucleic Acids Research, 2009,37(3):e19.
[34] Lee D C, Yip S P, Lee T M H . Simple and sensitive electrochemical DNA detection of primer generation-rolling circle amplification. Electroanalysis, 2013,25(5):1310-1315.
[35] Xue Q, Lv Y, Cui H, et al. A DNA nanomachine based on rolling circle amplification-bridged two-stage exonuclease III-assisted recycling strategy for label-free multi-amplified biosensing of nucleic acid. Analytica Chimica Acta, 2015,856:103-109.
[36] Murakami T, Sumaoka J, Komiyama M. Sensitive RNA detection by combining three-way junction formation and primer generation-rolling circle amplification. Nucleic Acids Research, 2012,40(3):e22.
[37] Conze T, Shetye A, Tanaka Y, et al. Analysis of genes, transcripts, and proteins via DNA ligation. Annual Review of Analytical Chemistry(Palo Alto, Calif), 2009,2:215-239.
[38] Stougaard M, Juul S, Andersen F F, et al. Strategies for highly sensitive biomarker detection by Rolling Circle Amplification of signals from nucleic acid composed sensors. Integrative Biology, 2011,3(10):982-992.
[39] Larsson C, Koch J, Nygren A, et al. In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes. Nature Methods, 2004,1(3):227-232.
doi: 10.1038/nmeth723 pmid: 15782198
[40] Miller J C, Zhou H P, Kwekel J, et al. Antibody microarray profiling of human prostate cancer sera: antibody screening and identification of potential biomarkers. Proteomics, 2003,3(1):56-63.
doi: 10.1002/pmic.200390009 pmid: 12548634
[41] Nallur G, Luo C H, Fang L H, et al. Signal amplification by rolling circle amplification on DNA microarrays. Nucleic Acids Research, 2001,29(23) : e118.
doi: 10.1093/nar/29.23.e118 pmid: 11726701
[42] 于志超, 陈林军, 赵治国, 等. 牛病毒性腹泻病毒检测技术研究进展. 动物医学进展, 2019,40(1):93-97.
Yu Z C, Chen L J, Zhao Z G, et al. Progress on detection methods of bovine viral diarrhea virus. Progress in Veterinary Medicine, 2019,40(1):93-97.
[43] Kim J Y, Lee J L. Rapid detection of salmonella enterica serovar enteritidis from eggs and chicken meat by real-time recombinase polymerase amplification in comparison with the two-step rea--time PCR. Food Sleribenafety, 2016,36(3) : 402-411.
[44] Fan X X, Li L, Zhao Y G. Clinical validation of two recombinase-based isothermal amplification assays (RPA/RAA) for the rapid detection of African Swine fever virus. Frontiers in Microbiology, 2020,11:1696-1696.
doi: 10.3389/fmicb.2020.01696 pmid: 32793160
[45] Lutz S, Weber P, Focke M, et al. Microfluidic lab-on-a-foil for nucleic acid analysis based on isothermal recombinase polymerase amplification (RPA). Lab on a Chip, 2010,10(7):887-893.
[46] Zhang P, Wu Y, Wang L, et al. Primers with designed terminal sequence improved efficiency of helicase-dependent DNA amplification. Chinese Journal of Biochemistry and Molecular Biology, 2018,34(7):747-753.
[47] Kim H J, Tong Y H, Tang W, et al. A rapid and simple isothermal nucleic acid amplification test for detection of herpes simplex virus types 1 and 2. Journal of Clinical Virology, 2011,50(1):26-30.
[48] Kolm C, Martzy R, Führer M, et al. Detection of a microbial source tracking marker by isothermal helicase-dependent amplification and a nucleic acid lateral-flow strip test. Scientific Reports, 2019,9:1-9.
doi: 10.1038/s41598-018-37186-2 pmid: 30626917
[49] Xu D, Ming X, Gan M, et al. Rapid detection of Cronobacter spp. in powdered infant formula by thermophilic helicase-dependent isothermal amplification combined with silica-coated magnetic particles separation. Journal of Immunological Methods, 2018,462:54-58.
[50] Barreda-García S, Miranda Castro R, De-Los-santos-álvarez N, et al. Helicase-dependent isothermal amplification: a novel tool in the development of molecular-based analytical systems for rapid pathogen detection. Analytical and Bioanalytical Chemistry, 2018,410(3):679-693.
doi: 10.1007/s00216-017-0620-3 pmid: 28932883
[51] Barreda-García S, González-álvarez M J, De-Los-santos-álvarez N , et al. Attomolar quantitation of Mycobacterium tuberculosis by asymmetric helicase-dependent isothermal DNA-amplification and electrochemical detection. Biosensors and Bioelectronics, 2015,68:122-128.
[52] Schwenkbier L, Pollok S, Rudloff A, et al. Non-instrumented DNA isolation, amplification and microarray-based hybridization for a rapid on-site detection of devastating Phytophthora kernoviae. The Analyst, 2015,140(19):6610-6618.
[53] Tang W, Goldmeyer J, Kong H M. Development of a novel one-tube isothermal reverse transcription thermophilic helicase-dependent amplification platform for rapid RNA detection. Journal of Molecular Diagnostics , 2007,9(5):639-644.
[54] Zhang Z H, Xiao L L, Lou Y, et al. Development of a multiplex real-time PCR method for simultaneous detection of Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella spp.in raw shrimp. Food Control, 2015,51:31-36.
[55] Walker G T, Fraiser M S, Schram J L, et al. Strand displacement amplification: an isothermal, in vitro DNA amplification technique. Nucleic Acids Research, 1992,20(7):1691-1696.
[56] He Y Q, Zeng K, Zhang S Q, et al. Visual detection of gene mutations based on isothermal strand-displacement polymerase reaction and lateral flow strip. Biosensors and Bioelectronics, 2012,31(1):310-315.
[57] Fang X X, Zhang H Q, Zhang F, et al. Real-time monitoring of strand-displacement DNA amplification by a contactless electrochemical microsystem using interdigitated electrodes. Lab on a Chip, 2012,12(17):3190-3196.
doi: 10.1039/c2lc40384f pmid: 22773155
[58] Dean F B, Nelson J R, Giesler T L, et al. Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification. Genome Research, 2001,11(6):1095-1099.
[59] de Bourcy C F A, de Vlaminck I, Kanbar J N, et al. A quantitative comparison of single-cell whole genome amplification methods. PLoS One, 2014,9(8):e105585.
pmid: 25136831
[60] Marcy Y, Ishoey T, Lasken R S, et al. Nanoliter reactors improve multiple displacement amplification of genomes from single cells. PLoS Genetics, 2007,3(9):1702-1708.
[61] Gole J, Gore A, Richards A, et al. Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells. Nature Biotechnology, 2013,31(12):1126-1132.
pmid: 24213699
[62] Sidore A M, Lan F, Lim S W, et al. Enhanced sequencing coverage with digital droplet multiple displacement amplification. Nucleic Acids Research, 2016,44(7):e66.
pmid: 26704978
[63] Compton J. Nucleic-acid sequence-based amplification. Nature, 1991,350(6313):91-92.
pmid: 1706072
[64] Mader A, Riehle U, Brandstetter T, et al. Microarray-based amplification and detection of RNA by nucleic acid sequence based amplification. Analytical and Bioanalytical Chemistry, 2010,397(8):3533-3541.
pmid: 20596698
[65] Tillmann R L, Simon A, Müller A, et al. Comparison of in-house PCR, rapid ELISA and NASBA technology for the detection of respiratory syncytial virus in clinical specimen. Journal of Clinical Virology, 2008,41(2):168-169.
doi: 10.1016/j.jcv.2007.10.005 pmid: 18024201
[66] Rodríguez-Lázaro D, D’Agostino M, Pla M, et al. Construction strategy for an internal amplification control for real-time diagnostic assays using nucleic acid sequence-based amplification: development and clinical application. Journal of Clinical Microbiology, 2004,42(12):5832-5836.
[67] Zhao X Y, Dong T, Yang Z C, et al. Compatible immuno-NASBA LOC device for quantitative detection of waterborne pathogens: Design and validation. Lab on a Chip, 2012,12(3):602-612.
[68] Larry T M, Jeffrey M L, Michael G, et al. Sensitive detection of genetic variants of HIV-1 and HCV with an HIV-1/HCV assay based on transcription-mediated amplification. Journal of virological methods, 2002,102(1-2):139-155.
[69] Chang C C, Chen C C, Wei S C, et al. Diagnostic devices for isothermal nucleic acid amplification. Sensors(Basel, Switzerland), 2012,12(6):8319-8337.
[70] Smith A, Matsubara K, Mickelson E, et al. A comparative study of HLA-DRB typing by TMA/HPA versus PCR/SSOP. Human Immunology, 1996,47(1-2):O218-O218.
[71] Giachetti C, Linnen J M, Kolk D P, et al. Highly sensitive multiplex assay for detection of human immunodeficiency virus type 1 and hepatitis C virus RNA. Journal of Clinical Microbiology, 2002,40(7):2408-2419.
pmid: 12089255
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