基于核酸等温扩增的病原微生物微流控检测技术
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摘要
病原微生物的快速检测对疫情的预防控制至关重要。基于PCR的病原微生物核酸检测方法克服了传统病原微生物培养方法耗时长、免疫学检测存在窗口期等问题,已成为目前最主要的病原微生物筛查方法。然而,对精确控温热循环仪的依赖却严重限制了其在资源匮乏地区的应用。虽然基于核酸等温扩增的病原微生物检测方法可摆脱对高精度温控设备的依赖,但仍需要进行样本核酸分离提取、扩增与检测等步骤。近年来,微流控技术与核酸等温扩增技术相结合,诞生了多种病原微生物等温扩增微流控检测技术。该技术通过设计芯片结构、优化进样模式及检测方式,实现了病原微生物核酸提取、扩增与检测一体化,并具备多重检测、定量检测等功能,具有对仪器依赖度小、对操作人员要求不高、样本量需求小和自动化程度高等优点,适合于在多种环境下的病原微生物快速检测。本文从核酸等温扩增原理、进样方式、检测方式等方面对核酸等温扩增病原微生物微流控检测技术进行了综述,以期为病原微生物的快速筛查提供更多的方案思路,提升公共卫生领域对传染性疾病的防控能力。
Rapid detection of pathogenic microorganisms is key to the epidemiologic identification, prevention and control of disease in the field of public health. PCR-based pathogen detection methods have been widely used because they overcome the time-consuming issues that traditional culture-based methods required including the limited window required by immunological detection. However, the requirement on precision temperature-controlled thermal cyclers severely limits their use in resource-limited areas. The detection methods of pathogenic microorganisms based on isothermal amplification of nucleic acids are free of dependence on high-precision temperature control equipment, but requirements for nucleic acids extraction, amplification and detection must be defined. In recent years, a number of alternative methods for pathogenic microorganism detection have been developed by combining microfluidic technology with nucleic acid isothermal amplification technology. By designing the chip structures, optimizing the injection modes, and utilizing multiple detection and quantitative methods, the integration of pathogen nucleic acid extraction, amplification and detection is achieved. The method provides advantages of less instrument dependence, decreased operator requirements,smaller sample size, and higher automation which are suitable for the rapid detection of pathogenic microorganisms in various environments. In this review, we summarize several microfluidic detection methods based on nucleic acid isothermal amplification for pathogens including amplification principles, injection methods and detection methods. These methods provide more capability for the rapid screening of pathogenic microorganisms which enhances the management of infectious diseases in the field of public health.
Rapid detection of pathogenic microorganisms is key to the epidemiologic identification, prevention and control of disease in the field of public health. PCR-based pathogen detection methods have been widely used because they overcome the time-consuming issues that traditional culture-based methods required including the limited window required by immunological detection. However, the requirement on precision temperature-controlled thermal cyclers severely limits their use in resource-limited areas. The detection methods of pathogenic microorganisms based on isothermal amplification of nucleic acids are free of dependence on high-precision temperature control equipment, but requirements for nucleic acids extraction, amplification and detection must be defined. In recent years, a number of alternative methods for pathogenic microorganism detection have been developed by combining microfluidic technology with nucleic acid isothermal amplification technology. By designing the chip structures, optimizing the injection modes, and utilizing multiple detection and quantitative methods, the integration of pathogen nucleic acid extraction, amplification and detection is achieved. The method provides advantages of less instrument dependence, decreased operator requirements,smaller sample size, and higher automation which are suitable for the rapid detection of pathogenic microorganisms in various environments. In this review, we summarize several microfluidic detection methods based on nucleic acid isothermal amplification for pathogens including amplification principles, injection methods and detection methods. These methods provide more capability for the rapid screening of pathogenic microorganisms which enhances the management of infectious diseases in the field of public health.
引文
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[36]Fang X,Chen H,Yu S,Jiang X,Kong J.Predicting viruses accurately by a multiplex microfluidic loop-mediated isothermal amplification chip.Anal Chem,2010,83(3):690-695.
[37]Rane TD,Chen L,Zec HC,Wang TH.Microfluidic continuous flow digital loop-mediated isothermal amplification(LAMP).Lab Chip,2015,15(3):776-782.
[38]Ma YD,Chang WH,Luo K,Wang CH,Liu SY,Yen WH,Lee GB.Digital quantification of DNA via isothermal amplification on a self-driven microfluidic chip featuring hydrophilic film-coated polydimethylsiloxane.Biosens Bioelectron,2017,99:547-554.
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[2]Bu T,Huang Q,Yan L,Huang L,Zhang M,Yang Q,Yang B,Wang J,Zhang D.Ultra technically-simple and sensitive detection for Salmonella enteritidis by immunochromatographic assay based on gold growth.Food Control,2018,84:536-543.
[3]Saiki RK,Scharf S,Faloona F,Mullis KB,Horn GT,Erlich HA,Arnheim N.Enzymatic amplification of betaglobin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.Science,1985,230(4732):1350-1354.
[4]Saingam P,Li B,Yan T.Use of amplicon sequencing to improve sensitivity in PCR-based detection of microbial pathogen in environmental samples.J Microbiol Meth,2018,149:73-79.
[5]Wang M,Yang J,Gai Z,Huo S,Zhu J,Li J,Wang R,Xing S,Shi G,Shi F,Zhang L.Comparison between digital PCR and real-time PCR in detection of Salmonella typhimurium in milk.Int J Food Microbiol,2018,266:251-256.
[6]Chen JW,Shao N,Zhang YC,Zhu YS,Yang LT,Tao SC.Avisual multiplex PCR microchip with easy sample loading.Hereditas(Beijing),2017,39(6):525-534.陈建伟,邵宁,张雨晨,朱元首,杨立桃,陶生策,卢大儒.一种载样简单的多重可视化PCR微芯片.遗传,2017,39(6):525-534.
[7]Martzy R,Kolm C,Krska R,Mach RL,Farnleitner AH,Reischer GH.Challenges and perspectives in the application of isothermal DNA amplification methods for food and water analysis.Anal Bioanal Chem,2019,411(9):1695-1702.
[8]Zhang L,Tian F,Liu C,Feng Q,Ma T,Zhao Z,Li T,Jiang X,Sun J.Hand-powered centrifugal microfluidic platform inspired by the spinning top for sample-to-answer diagnostics of nucleic acids.Lab Chip,2018,18(4):610-619.
[9]Nasseri B,Soleimani N,Rabiee N,Kalbasi A,Karimi M,Hamblin MR.Point-of-care microfluidic devices for pathogen detection.Biosens Bioelectron,2018,117:112-128.
[10]Piepenburg O,Williams C,Stemple D,Armes NA.DNAdetection using recombination proteins.PLoS Biol,2006,4(7):e204.
[11]Ma YD,Luo K,Chang WH,Lee GB.A microfluidic chip capable of generating and trapping emulsion droplets for digital loop-mediated isothermal amplification analysis.Lab Chip,2018,18(2):296-303.
[12]Choi G,Jung JH,Park BH,Oh SJ,Seo JH,Choi JS,Kim do H,Seo TS.A centrifugal direct recombinase polymerase amplification(direct-RPA)microdevice for multiplex and real-time identification of food poisoning bacteria.Lab Chip,2016,16(12):2309-2316.
[13]Chen J,Xu Y,Yan H,Zhu Y,Wang L,Zhang Y,Lu Y,Xing W.Sensitive and rapid detection of pathogenic bacteria from urine samples using multiplex recombinase polymerase amplification.Lab Chip,2018,18(16):2441-2452.
[14]Rohrman BA,Richards-Kortum RR.A paper and plastic device for performing recombinase polymerase amplification of HIV DNA.Lab Chip,2012,12(17):3082-3088.
[15]Zeng J,Zhang S,Tang K,Chen G,Yuan W,Tang Y.3-Dmanipulation of a single nano-droplet on graphene with an electrowetting driving scheme:critical condition and tunability.Nanoscale,2018,10(34):16079-16086.
[16]Kim TH,Park J,Kim CJ,Cho YK.Fully integrated lab-on-a-disc for nucleic acid analysis of food-borne pathogens.Anal Chem,2014,86(8):3841-3848.
[17]Song SW,Kim SD,Oh DY,Lee Y,Lee AC,Jeong Y,Bae HJ,Lee D,Lee S,Kim J,Kwon S.High‐throughput screening:one‐step generation of a drug‐releasing hydrogel microarray‐on‐a‐chip for large‐scale sequential drug combination Screening.Adv Sci,2019,6(3):1801380.
[18]Kuan CP,Chang WS,Yang TC.Use of fluorescent microsphere-based assay for detection of three CucurbitInfecting viruses.Plant Dis,2018,102(11):2324-2329.
[19]Huang H,Jin L,Yang X,Song Q,Zou B,Jiang S,Sun L,Zhou G.An internal amplification control for quantitative nucleic acid analysis using nanoparticle-based dipstick biosensors.Biosens Bioelectron,2013,42:261-266.
[20]Sreejith KR,Ooi CH,Jin J,Dao DV,Nguyen NT.Digital polymerase chain reaction technology-recent advances and future perspectives.Lab Chip,2018,18(24):3717-3732.
[21]Feng S,Davydova EK,Du W,Kreutz JE,Piepenburg O,Ismagilov RF.Digital isothermal quantification of nucleic acids via simultaneous chemical initiation of recombinase polymerase amplification reactions on SlipChip.Anal Chem,2011,83(9):3533-3540.
[22]Notomi T,Okayama H,Masubuchi H,Yonekawa T,Watanabe K,Amino N,Hase T.Loop-mediated isothermal amplification of DNA.Nucleic Acids Res,2000,28(12):E63.
[23]Nagamine K,Hase T,Notomi T.Accelerated reaction by loop-mediated isothermal amplification using loop primers.Mol Cell Probe,2002,16(3):223-229.
[24]Singh R,Singh DP,Savargaonkar D,Singh OP,Bhatt RM,Valecha N.Evaluation of SYBR green I based visual loop-mediated isothermal amplification(LAMP)assay for genus and species-specific diagnosis of malaria in P.vivax and P.falciparum endemic regions.J Vector Dis,2017,54(1):54-60.
[25]Fang X,Liu Y,Kong J,Jiang X.Loop-mediated isothermal amplification integrated on microfluidic chips for point-of-care quantitative detection of pathogens.Anal Chem,2010,82(7):3002-3006.
[26]Arimatsu Y,Kaewkes S,Laha T,Sripa B.Specific diagnosis of opisthorchis viverrini using loop-mediated isothermal amplification(LAMP)targeting parasite microsatellites.Acta Trop,2015,141(Pt B):368-371.
[27]Kaarj K,Akarapipad P,Yoon JY.Simpler,faster,and sensitive zika virus assay using smartphone detection of Loop-mediated Isothermal amplification on paper microfluidic chips.Sci Rep,2018,8(1):12438.
[28]Lu Y,Ma X,Wang J,Sheng N,Dong T,Song Q,Rui J,Zou B,Zhou G.Visualized detection of single-base difference in multiplexed loop-mediated isothermal amplification amplicons by invasive reaction coupled with oligonucleotide probe-modified gold nanoparticles.Biosens Bioelectron,2016,90:388-393.
[29]Park BH,Oh SJ,Jung JH,Choi G,Seo JH,Kim DH,Lee EY,Seo TS.An integrated rotary microfluidic system with DNA extraction,loop-mediated isothermal amplification,and lateral flow strip based detection for point-of-care pathogen diagnostics.Biosens Bioelectron,2016,91:334-340.
[30]Sayad AA,Ibrahim F,Uddin SM,Pei KX,Mohktar MS,Madou M,Thong KL.A microfluidic lab-on-a-disc integrated loop mediated isothermal amplification for foodborne pathogen detection.Sens Actuators B,2016,227:600-609.
[31]Tang R,Yang H,Gong Y,You M,Liu Z,Choi JR,Wen T,Qu Z,Mei Q,Xu F.A fully disposable and integrated paper-based device for nucleic acid extraction,amplification and detection.Lab Chip,2017,17(7):1270-1279.
[32]Hiltunen J,Liedert C,Hiltunen M,Huttunen OH,Hiitola-Kein?nen J,Aikio S,Harjanne M,Kurkinen M,Hakalahti L,Lee LP.Roll-to-roll fabrication of integrated PDMS-paper microfluidics for nucleic acid amplification.Lab Chip,2018,18(11):1552-1559.
[33]Pang B,Fu K,Liu Y,Ding X,Hu J,Wu W,Xu K,Song X,Wang J,Mu Y,Zhao C,Li J.Development of a self-priming PDMS/paper hybrid microfluidic chip using mixed-dye-loaded loop-mediated isothermal amplification assay for multiplex foodborne pathogens detection.Anal Chim Acta,2018,1040:81-89.
[34]Zhi X,Deng M,Yang H,Gao G,Wang K,Fu H,Zhang Y,Chen D,Cui D.A novel HBV genotypes detecting system combined with microfluidic chip,loop-mediated isothermal amplification and GMR sensors.Biosens Bioelectron,2013,54:372-377.
[35]Hsieh K,Patterson AS,Ferguson BS,Plaxco KW,Soh HT.Rapid,sensitive,and quantitative detection of pathogenic dna at the point of care through microfluidic electrochemical quantitative Loop‐Mediated Isothermal amplification.Angew Chem Int Edit,2012,51(20):4896-4900.
[36]Fang X,Chen H,Yu S,Jiang X,Kong J.Predicting viruses accurately by a multiplex microfluidic loop-mediated isothermal amplification chip.Anal Chem,2010,83(3):690-695.
[37]Rane TD,Chen L,Zec HC,Wang TH.Microfluidic continuous flow digital loop-mediated isothermal amplification(LAMP).Lab Chip,2015,15(3):776-782.
[38]Ma YD,Chang WH,Luo K,Wang CH,Liu SY,Yen WH,Lee GB.Digital quantification of DNA via isothermal amplification on a self-driven microfluidic chip featuring hydrophilic film-coated polydimethylsiloxane.Biosens Bioelectron,2017,99:547-554.
[39]Banér J,Nilsson M,Mendel-Hartvig M,Landegren U.Signal amplification of padlock probes by rolling circle replication.Nucleic Acids Res,1998,26(22):5073-5078.
[40]Ali MM,Li F,Zhang Z,Zhang K,Kang DK,Ankrum JA,Le XC,Zhao W.Rolling circle amplification:a versatile tool for chemical biology,materials science and medicine.Chem Soc Rev,2014,43(10):3324-3341.
[41]Zou B,Ma Y,Wu H,Zhou G.Signal amplification by rolling circle amplification on universal flaps yielded from target-specific invasive reaction.Analyst,2011,137(3):729-734.
[42]Jiang Y,Shan Z,Cao X.A simple dendrimer-aptamer based microfluidic platform for E.coli O157:H7 detection and signal intensification by rolling circle amplification.Sens Actuators B,2017,251:976-984.
[43]Compton J.Nucleic acid sequence-based amplification.Nature,1991,350(6313):91-92.
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