环介导等温扩增技术的应用及研究进展
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摘要
环介导等温扩增(LAMP)技术自2000年被发明以来以其快速、灵敏、低耗等特点,引起众多研究者的关注。该技术在人、动物、植物、环境等相关微生物的检测、监控等领域得到了广泛研究与应用,其检测灵敏度普遍高于传统PCR 。但是传统LAMP技术对于产物检测存在耗时、易造成污染等问题,以及在野外或医疗点检测时存在判读不便的弊端。就LAMP技术与其他多种产物检测技术结合后的方法如目视LAMP、实时LAMP、微流控LAMP、横向流LAMP及电化学LAMP进行了总结、举例及讨论。目视LAMP具有费用低、适合多环境下使用的特点;实时LAMP、微流控及电化学LAMP法能够有效提高样品检测的准确度,且可减少检测时间;横向流LAMP具有可操作性强、便于判读等特点。根据目前LAMP技术的研究进展及各技术优劣势的分析,预测 LAMP技术未来发展的方向和重点可能为研发手持便携式检测仪。
Loop-mediated isothermal amplification(LAMP) technology has attracted many researchers' attention for its fast, sensitive and low-consumption characteristics since it was developed in 2000. This method has been widely studied and applied in the detection and monitoring of microorganisms related to human, animals, plants and environment. Its detection sensitivity is generally higher than that of traditional PCR. However, the traditional LAMP is time-consuming and easy to cause pollution in the detection of products, and has disadvantages of inconvenient interpretation in the field or point of care. This paper summarized, illustrated and discussed the methods of LAMP technology combining with other product detection technologies. Visual LAMP has the characteristics of low cost and being suitable for multi-environment. Real-time,micro-fluidic and electrochemical LAMP can effectively improve the accuracy of sample detection and reduce detection time. Lateral flow detect LAMP has the characteristics of strong operability and easy interpretation. According to the current research progress of LAMP technologies and the analysis of their advantages and disadvantages, it is predicted that the future development direction and focus of LAMP technology may be the development of hand-held portable detectors.
Loop-mediated isothermal amplification(LAMP) technology has attracted many researchers' attention for its fast, sensitive and low-consumption characteristics since it was developed in 2000. This method has been widely studied and applied in the detection and monitoring of microorganisms related to human, animals, plants and environment. Its detection sensitivity is generally higher than that of traditional PCR. However, the traditional LAMP is time-consuming and easy to cause pollution in the detection of products, and has disadvantages of inconvenient interpretation in the field or point of care. This paper summarized, illustrated and discussed the methods of LAMP technology combining with other product detection technologies. Visual LAMP has the characteristics of low cost and being suitable for multi-environment. Real-time,micro-fluidic and electrochemical LAMP can effectively improve the accuracy of sample detection and reduce detection time. Lateral flow detect LAMP has the characteristics of strong operability and easy interpretation. According to the current research progress of LAMP technologies and the analysis of their advantages and disadvantages, it is predicted that the future development direction and focus of LAMP technology may be the development of hand-held portable detectors.
引文
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[22]游淑珠,王小玉,冯家望,蔡教英,姚丽锋,唐食明,丁琦,符家忍.LAMP实时浊度法快速检测食品中产志贺毒素大肠埃希氏菌[J].食品工业科技,2018,39(22):241-246.YOU S Z,WANG X Y,FENG J W,CAI J Y,YAO L F,TANG S M,DINGQ,FU J R.Rapid Detection of Shiga Toxin-Producing Escherichia coli by LAMP Real-Time Turbidity Method[J].Science and Technology of Food Industry,2018,39(22):241-246.
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[5]TAHZIMA R,FOUCART Y,PEUSENS GBELIEN T,MASSARTS,DE J K.New sensitive and fast detection of little cherry virus 1using loop-mediated isothermal amplification(RT-LAMP)[J].Journal of Virological Methods,2019,265:91-98.doi:10.1016/j.jviromet.2018.12.019.
[6]WANG Y,WANGg Y,LUO L J,LIU D X,LUO X,XU Y M,HU S K,NIU L N,XU J G,YE C Y.Rapid and sensitive detection of Shigella spp.and Salmonella spp.by multiple endonuclease restriction realtime loop-mediated isothermal amplification technique[J].Frontiers in Microbiology,2015,6:1400.doi:10.3389/fmicb.2015.01400.
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[8]ZHOU S C,HAN S,SHI J L,WU J Q,YUAN X Y,CONG S J,XU S J,WU X Y,LI J,WANG J B.Loop-mediated isothermal amplification for detection of porcine circovirus type 2[J].Virology Journal,2011,8:497-501.doi:10.1186/1743-422X-8-497.
[9]SUN X M,JI Y S,LIU X Y,XIANG M,HE G,XIE L,SUO J X,SUO X.Improvement and evaluation of loop-mediated isothermal amplification for rapid detection of Toxoplasma gondii infection in Human blood samples[J].Plos One,2017,12(1):e0169125.doi:10.1371/journal.pone.0169125.
[10]NAKAUCHI M,TAKAYAMA,TAKAHASHI H,TASHIRO M,KAGEYAMA T.Development of a reverse transcription loop-mediated isothermal amplification assay for the rapid diagnosis of avian influenza A(H7N9)virus infection[J].Journal of Virological Methods,2014,204:101-104.doi:10.1016/j.jviromet.2014.03.028.
[11]OLONINIYI O K,KUROSAKI Y,MIYAMOTO H,TAKADA A,YASUDAJ.Rapid detection of all known ebolavirus species by reverse transcriptionloop-mediated isothermal amplification(RT-LAMP)[J].Journal of Virological Methods,2017,246:8-14.doi:10.1016/j.jviromet.2017.03.011.
[12]SHAO Y,C ZHU S M,JIN C C,CHEN F S.Development of multiplex loop-mediated isothermal amplification-RFLP(mLAMP-RFLP)to detect Salmonella spp.and Shigella spp.in milk[J].International Journal of Food Microbiology,2011,148(2):75-79.doi:10.1016/j.ijfoodmicro.2011.05.004.
[13]NKAOUAWA A,SAKO Y,OKAMOTO M,ITO A.Simple identification of human taenia species by multiplex loop-mediated isothermal amplification in combination with dot enzyme-linked immunosorbent assay[J].The American Journal of Tropical Medicine and Hygiene,2016,94(6):1318-1323.doi:10.4269/ajtmh.15-0829.
[14]RAVAN H,YAZDNPARAST R.Development and evaluation of a loop-mediated isothermal amplification method in conjunction with an enzyme-linked immunosorbent assay for specific detection of Salmonella serogroup D[J].Analytica Chimica Acta,2012,733:64-70.doi:10.1016/j.aca.2012.04.034.
[15]AHEMD M E,ELDIGAIL M H,ELAMIN FM,ALI I A,GROBUSCHM P,ARADAIB I E.Development and evaluation of real-time loopmediated isothermal amplification assay for rapid detection of cystic echinococcosis[J].BMC Veterinary Research,2016,12:202.doi:10.1186/s12917-016-0809-2.
[16]CAI T,LOU G Q,YANG J,XU D,MENG Z H.Development and evaluation of real-time loop-mediated isothermal amplification for hepatitis B virus DNA quantification:a new tool for HBV management[J].Journal of Clinical Virology,2008,41(4):270-276.doi:10.1016/j.jcv.2007.11.025.
[17]LIU N W,ZOU D Y,DONG D R,YANG Z,AO D,LIU W,HUANG L Y.Development of a multiplex loop-mediated isothermal amplification method for the simultaneous detection of Salmonella spp.and Vibrio parahaemolyticus[J].Scientific Reports,2017,7:45601.doi:10.1038/srep45601.
[18]CHEN L,JIAO Z Y,LIU D M,LIU X L,XIA Z H,DENG C L,ZHOUT,FAN Z F.One-step reverse transcription loop-mediated isothermal amplification for the detection of Maize chlorotic mottle virus in maize[J].Journal of Virological Methods,2017,240:49-53.doi:10.1016/j.jviromet.2016.11.012.
[19]BESUSCHIO S A,LLANO M M,BENATAR A F,MONNERAT S,CRUZ I,PICADO A,CURTO M L A,KUBOTA Y,WEHRENDT DP,PAVIA P,MORI Y,PUERTA C,NDUNGU J M,SCHIJMAN A G.Analytical sensitivity and specificity of a loop-mediated isothermal amplification(LAMP)kit prototype for detection of Trypanosoma cruzi DNA in human blood samples[J].PLoS Neglected Tropical Diseases,2017,11(7):e0005779.doi:10.1371/journal.pntd.0005779.
[20]IRIN H,NUNTAREE D,WANIDA L.Semi-quantitative visual detection of loop mediated isothermal amplification(LAMP)-generated DNA by distance-based measurement on a paper device[J].Talanta,2017,175:135-142.doi:10.1016/j.talanta.2017.07.019.
[21]MORI Y,KITAO M,TOMITA N,NOTOMI T.Real-time turbidimetry of LAMP reaction for quantifying template DNA[J].Journal of Biochemical and Biophysical Methods,2004,59(2):145-157.doi:10.1016/j.jbbm.2003.12.005.
[22]游淑珠,王小玉,冯家望,蔡教英,姚丽锋,唐食明,丁琦,符家忍.LAMP实时浊度法快速检测食品中产志贺毒素大肠埃希氏菌[J].食品工业科技,2018,39(22):241-246.YOU S Z,WANG X Y,FENG J W,CAI J Y,YAO L F,TANG S M,DINGQ,FU J R.Rapid Detection of Shiga Toxin-Producing Escherichia coli by LAMP Real-Time Turbidity Method[J].Science and Technology of Food Industry,2018,39(22):241-246.
[23]ARUNRUT N,SUEBSING R,WITHYACHUMNARNKUL B,KIATPATHOMCHAI W.Demonstration of a very inexpensive,turbidimetric,real-time,RT-LAMP detection platform using shrimp Laem-Singh virus(LSNV)as a model[J].Plos One,2014,9(9):e108047.doi:10.1371/journal.pone.0108047.
[24]LIN X A,CHEN Y,LU Y Y,YAN J Y,YAN J.Application of a loopmediated isothermal amplification method for the detection of pathogenic Leptospira[J].Diagnostic Microbiology and Infectious Disease,2009,63(3):237-242.doi:10.1016/j.diagmicrobio.2008.10.012.
[25]ZEGN F W,CONG F,LIU X N,LIAN Y X,WU M L,XIAO L,YUANW,HUANG R,MA J Y,GUO P J,LUO M L.Development of a real time loop-mediated isothermal amplification method for detection of Senecavirus A[J].Journal of Microbiological Methods,2018,261:98-103.doi:10.1016/j.jviromet.2018.08.005.
[26]SEYRIG G,STEDTFELD R D,TOURLOUSSE D M,AHMADF,TOWERY K,CUPPLES A M,TIEDJE J M,HASHSHAM S A.Selection of fluorescent DNA dyes for real-time LAMP with portable and simple optics[J].Journal of Microbiological Methods,2015,119:223-227.doi:10.1016/j.mimet.2015.11.004.
[27]TANNER N A,ZHANG Y,EVANS T C J.Simultaneous multiple target detection in real-time loop-mediated isothermal amplification[J].BioTechniques,2012,53(2):81-89.doi:10.2144/0000113902.
[28]BECHERER L,BAKHEIT M,Frischmann S,STINCO S,BORST N,ZENGERLE R,VON STETTEN F.Simplified real-time multiplex detection of loop-mediated isothermal amplification using novel mediator displacement probes with universal reporters[J].Analytical Chemistry,2018,90(7):4741-4748.doi:10.1021/acs.analchem.7b05371.
[29]NAZARENKO I,PIRES R,LOWE B,OBAIDY M,RASHTCHIAN A.Effect of primary and secondary structure of oligodeoxyribonucleotides on the fluorescent properties of conjugated dyes[J].Nucleic Acids Res,2002,30(9):2089-2195.
[30]GADKAR V J,GOLDGARB D M,GANTT S,TILLEY P A G.Realtime detection and monitoring of loop mediated amplification(LAMP)reaction using self-quenching and de-quenching fluorogenic probes[J].Scientific Reports,2018,8(1):5548.doi:10.1038/s41598-018-23930-1.
[31]MANZ A,GRABER N,WIDMER H M.Miniaturized total chemical analysis systems-A novel concept for chemical sensing[J].Sensor and Actuators B:Chemical,1990,1(1-6):244-248.
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