Loop-mediated isothermal amplification(LAMP)is an efficient, simple, highly specific nucleic acid amplification technology without thermal cycling equipment.As LAMP still has great potential for- DOI:10.3969/j.issn.1000-1565.2021.05.013环介导等温扩增技术研究进展石磊1,王曼2,时国强3,董振国3,刘昱2(1.暨南大学 食品安全与营养研究院,广东 广州 510000;2.河北农业大学 生命科学学院,河北 保定 071003; 3. 河北三狮生物科技有限公司 研发部,河北 石家庄 050000)石磊 博士,教授,博士生导师,1985年毕业于河北大学生物系并获学士学位,现任暨南大学食品安全与营养研究院院长,河北农业大学生命科学学院特聘兼职教授;国家重大人才工程入选者,国家重点研发计划首席科学家,肉食品安全生产技术国家重点实验室主任,第一届国家食品安全标准审评委员会委员,中国食源性微生物检测技术创新战略联盟副理事长,广东省免疫学会副理事长,广州双螺旋基因技术有限公司董事长;主要从事病原微生物快速诊断方法、临床微生物耐药基因解析及耐药性基因诊断研究;在SCI收录期刊上发表论文100余篇,申请专利26项,其中发明专利25项,实用新型专利1项;先后主持“国家重点研发计划”、“国家973计划项目”、“国家海洋经济创新发展区域示范专项”等10余项国家级项目;2018年获中国人民解放军军队科技进步二等奖,2019年获第四届中国水产学会范蠡科学技术奖技术推广奖、优秀成果奖.摘 要:环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术是一种高效、简便、高特异性、无需热循环设备的核酸扩增技术. 由于LAMP依然具备巨大的改造潜力,所以近些年来,一直有研究改进其技术以期达到更好的扩增检测效果. 本文对LAMP的基本原理、应用领域范围以及技术改良方向进行了综述,为今后该技术的改良与发展提供参考. 关键词:环介导等温扩增(LAMP);技术改良;发展方向中图分类号:Q7 文献标志码:A 文章编号:1000-1565(2021)05-0565-07Research progress of loop-mediated isothermal amplification technologySHI Lei1, WANG Man2, SHI Guoqiang3, DONG Zhenguo3, LIU Yu2(1. Institute of Food Safety and Nutrition,Jinan University, Guangzhou 510000, China; 2. College of Life Science, Agricultural University of Hebei, Baoding 071003, China; 3.Research and Development Department,Hebei Sanshi Biological Technology Co., LTD, Shijiazhuang 050000, China)Abstract: Loop-mediated isothermal amplification(LAMP)is an efficient, simple, highly specific nucleic acid amplification technology without thermal cycling equipment.As LAMP still has great potential for- 收稿日期:2021-06-16 基金项目:河北省创新能力提升计划项目(205A7601D);石家庄市高层次科技创新创业人才项目(05202001) 第一作者:石磊(1961—),男,河北保定人,暨南大学教授,博士生导师,主要从事病原微生物核酸分子检测等研究. E-mail:leishi88@139.com 通信作者:刘昱(1990—),男,山东临沂人,河北农业大学在读博士研究生,主要从事病原微生物核酸分子检测的研究.E-mail:1085869940@qq.com第5期石磊等:环介导等温扩增技术的研究进展modification, many studies have been conducted in recent years to improve its technology in order to achieve better amplification detection results.In this paper, the basic principle, application scope and technical improvement direction of LAMP were reviewed, to provide references for the improvement and development of LAMP technology in the future.
[1] LIU M, LUO Y, TAO R, et al. Sensitive and rapid detection of genetic modified soybean(roundup ready)by loop-mediated isothermal amplification[J]. Biosci Biotechnol Biochem, 2009, 73(11): 2365-2369.DOI:10.1271/bbb.80723.
[2] WRIGHT C F, BURTON H. The use of cell-free fetal nucleic acids in maternal blood for non-invasive prenatal diagnosis[J]. Hum Reprod Update, 2009, 15(1): 139-151. DOI:10.1093/humupd/dmn047.
[3] 张立, 黄春华, 黄河,等. LAMP法性别鉴定在胚胎工程技术中的应用[J]. 现代畜牧兽医, 2006(6):9-11.DOI:10.3969/j.issn.1672-9692.2006.06.006.
[4] BALNE P K, BASU S, SHARMA S. Loop-mediated isothermal amplification for rapid diagnosis of tubercular uveitis[J]. JAMA Ophthalmol, 2015, 133(2): 225-226. DOI:10.1001/jamaophthalmol.2014.4257.
[5] SCHEEL C M, ZHOU Y T, THEODORO R C, et al. Development of a loop-mediated isothermal amplification method for detection of Histoplasma capsulatum DNA in clinical samples[J]. J Clin Microbiol, 2014, 52(2): 483-488. DOI:10.1128/JCM.02739-13.
[6] 初亚男,成思佳,梁超,等.可视化等温扩增法快速筛查并分型禽流感病毒[J].中华实用诊断与治疗杂志, 2011, 25(7): 670-673.
[7] CHENG S J, CHEN Z Y, CHU Y N, et al. Sensitive detection of influenza A(H1N1)virus by isothermal amplification in a single tube[J]. Chin J Anal Chem, 2011, 39(3): 335-340. DOI:10.1016/S1872-2040(10)60424-0.
[8] HARA-KUDO Y, KONISHI N, OHTSUKA K, et al. Detection of Verotoxigenic Escherichia coli O157 and O26 in food by plating methods and LAMP method: a collaborative study[J]. Int J Food Microbiol, 2008, 122(1/2): 156-161. DOI:10.1016/j.ijfoodmicro.2007.11.078.
[9] NOTOMI T, OKAYAMA H, MASUBUCHI H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Res, 2000, 28(12): e63. DOI:10.1093/nar/28.12.e63.
[10] NAGAMINE K, HASE T, NOTOMI T. Accelerated reaction by loop-mediated isothermal amplification using loop primers[J]. Mol Cell Probes, 2002, 16(3): 223-229. DOI:10.1006/mcpr.2002.0415.
[11] 张曼,刘宝林,高志贤.环介导等温扩增技术的研究进展[J].食品安全质量检测学报, 2019, 10(18): 6124-6130. DOI:10.19812/j.cnki.jfsq11-5956/ts.2019.18.030..
[12] 何琳.环介导等温扩增技术快速检测水产动物病原的研究[D].杭州:浙江大学, 2012: 1-93..
[13] 张蕾,张海予,魏海燕,等.环介导等温扩增方法在食品中沙门菌检测的应用和评价[J].中国食品卫生杂志, 2013, 25(6): 520-524. DOI:10.13590/j.cjfh.2013.06.020.
[14] FERRARA M, LOGRIECO A F, MORETTI A, et al. A loop-mediated isothermal amplification(LAMP)assay for rapid detection of fumonisin producing Aspergillus species[J]. Food Microbiol, 2020, 90: 103469. DOI:10.1016/j.fm.2020.103469.
[15] 吕观,常彦磊,石磊.免疫磁珠-环介导等温扩增快速检测牛肉中的鼠伤寒沙门氏菌与金黄色葡萄球菌[J].肉类研究, 2019, 33(7): 42-48. DOI:10.7506/rlyj1001-8123-20190612-124.
[16] CHEN L, FAN X Z, WANG Q, et al. A novel RT-LAMP assay for rapid and simple detection of classical swine fever virus[J]. Virol Sin, 2010, 25(1): 59-64. DOI:10.1007/s12250-010-3043-2.
[17] 谷晓红, 谭晴晴, 刘艳艳, 等. 利用环介导等温扩增技术快速检测饮用水中的大肠杆菌[J]. 山东农业科学, 2017, 49(1): 130-135. DOI:10.14083/j.issn.1001-4942.2017.1.028.
[18] HTUN Z M, ROTCHANAPREEDA T, RUJIRAWAT T, et al. Loop-mediated isothermal amplification(LAMP)for identification of pythium insidiosum[J]. Int J Infect Dis, 2020, 101: 149-159. DOI:10.1016/j.ijid.2020.09.1430.
[19] NICZYPORUK J S, KOZDRUN' W, CZEKAJ H, et al. Detection of fowl adenovirus D strains in wild birds in Poland by Loop-Mediated Isothermal Amplification(LAMP)[J]. BMC Vet Res, 2020, 16(1): 58. DOI:10.1186/s12917-020-2271-4.
[20] YU Z R, ZHANG D, YANG K K, et al. A simple and rapid diagnostic method to detect new goose astrovirus using reverse-transcription loop-mediated isothermal amplification[J]. 3 Biotech, 2019, 10(1): 1-6. DOI:10.1007/s13205-019-2006-z.
[21] 陈涛, 周琳, 周杰, 等. 环介导等温扩增法快速检测结核分枝杆菌的临床应用评估[J]. 中国防痨杂志, 2012, 34(7): 413-418.
[22] TAKAYAMA I, NAKAUCHI M, TAKAHASHI H, et al. Development of real-time fluorescent reverse transcription loop-mediated isothermal amplification assay with quenching primer for influenza virus and respiratory syncytial virus[J]. J Virol Methods, 2019, 267: 53-58. DOI:10.1016/j.jviromet.2019.02.010.
[23] LE THI N, IKUYO T, NGUYEN GIA B, et al. A clinic-based direct real-time fluorescent reverse transcription loop-mediated isothermal amplification assay for influenza virus[J]. J Virol Methods, 2020, 277: 113801. DOI:10.1016/j.jviromet.2019.113801.
[24] GARCIA-VENZOR A, RUEDA-ZARAZUA B, MARQUEZ-GARCIA E, et al. SARS-CoV-2 direct detection without RNA isolation with loop-mediated isothermal amplification(LAMP)and CRISPR-cas12[J]. Front Med(Lausanne), 2021, 8: 627679. DOI:10.3389/fmed.2021.627679.
[25] GADKAR V J, GOLDFARB D M, GANTT S, et al. Real-time detection and monitoring of loop mediated amplification(LAMP)reaction using self-quenching and de-quenching fluorogenic probes[J]. Sci Rep, 2018, 8(1): 5548. DOI:10.1038/s41598-018-23930-1.
[26] ELBEAINO T, INCERTI O, DAKROUB H, et al. Development of an FTP-LAMP assay based on TaqMan real-time PCR and LAMP for the specific detection of Xylella fastidiosa De Donno and mulberry strains in both plants and insect vectors[J]. J Microbiol Methods, 2020, 175: 105992. DOI:10.1016/j.mimet.2020.105992.
[27] SHI C, SHANG F J, PAN M, et al. The isothermal amplification detection of double-stranded DNA based on a double-stranded fluorescence probe[J]. Biosens Bioelectron, 2016, 80: 54-58. DOI:10.1016/j.bios.2016.01.039.
[28] DING X, YIN K, LI Z Y, et al. Cleavable hairpin beacon-enhanced fluorescence detection of nucleic acid isothermal amplification and smartphone-based readout[J]. Sci Rep, 2020, 10: 18819. DOI:10.1038/s41598-020-75795-y.
[29] KIM H R, LIM D R, CHAE H G, et al. Advanced target-specific probe-based real-time loop-mediated isothermal amplification assay for the rapid and specific detection of porcine circovirus 3[J]. Transbound Emerg Dis, 2020, 67(6): 2336-2344. DOI:10.1111/tbed.13671.
[30] KUBOTA R, ALVAREZ A M, SU W W, et al. FRET-based assimilating probe for sequence-specific real-time monitoring of loop-mediated isothermal amplification(LAMP)[J]. Biol Eng Trans, 2011, 4(2): 81-100. DOI:10.13031/2013.38509.
[31] MARTINEAU R L, MURRAY S A, CI S, et al. Improved performance of loop-mediated isothermal amplification assays via swarm priming[J]. Anal Chem, 2017, 89(1): 625-632. DOI:10.1021/acs.analchem.6b02578.
[32] TANGKANCHANAPAS P, HÖFTE M, DE JONGHE K. Reverse transcription loop-mediated isothermal amplification(RT-LAMP)designed for fast and sensitive on-site detection of Pepper chat fruit viroid(PCFVd)[J]. J Virol Methods, 2018, 259: 81-91. DOI:10.1016/j.jviromet.2018.06.003.
[33] SEYRIG G, STEDTFELD R D, TOURLOUSSE D M, et al. Selection of fluorescent DNA dyes for real-time LAMP with portable and simple optics[J]. J Microbiol Methods, 2015, 119: 223-227. DOI:10.1016/j.mimet.2015.11.004.
[34] 吴亮,夏雯,阴晴,等.LC-MS/MS法同时测定欧前胡素与异欧前胡素血药浓度[J].临床检验杂志, 2020(1):7-10.
[35] FRISCH L M, MANN M A, MAREK D N, et al. Development and optimization of a loop-mediated isothermal amplification(LAMP)assay for the species-specific detection of Penicillium expansum[J]. Food Microbiol, 2021, 95: 103681. DOI:10.1016/j.fm.2020.103681.
[36] ZHOU Y, WAN Z, YANG S, et al. A mismatch-tolerant reverse transcription loop-mediated isothermal amplification method and its application on simultaneous detection of all four serotype of dengue viruses[J]. Front Microbiol, 2019, 10: 1056. DOI:10.3389/fmicb.2019.01056.
[37] MARCINIAK J, KUMMEL A, ESENER S, et al. Coupled rolling circle amplification loop-mediated amplification for rapid detection of short DNA sequences[J]. Biotechniques, 2008, 45(3): 275-280. DOI:10.2144/000112910.
[38] CHEN C, LU J, LONG B, et al. Detection of Mycobacterium kansasii using a combination of loop-mediated isothermal amplification(LAMP)and lateral flow biosensors[J]. Int Microbiol, 2021, 24(1): 75-82. DOI:10.1007/s10123-020-00143-z.
[39] FU Y X, ZHOU X Y, DUAN X L, et al. A LAMP-based ratiometric electrochemical sensing for ultrasensitive detection of Group B Streptococci with improved stability and accuracy[J]. Sensor Actuat B: Chem, 2020, 321: 128502. DOI:10.1016/j.snb.2020.128502.
[40] SHARMA S, KABIR M A, ASGHAR W. Lab-on-a-chip zika detection with reverse transcription loop-mediated isothermal amplification-based assay for point-of-care settings[J]. Arch Pathol Lab Med, 2020, 144(11): 1335-1343. DOI:10.5858/arpa.2019-0667-oa.
[41] SIDDIQUE M P, JANG W J, LEE J M, et al. Detection of vibrio anguillarum and vibrio alginolyticus by singleplex and duplex loop-mediated isothermal amplification(LAMP)assays targeted to groEL and fklB genes[J]. Int Microbiol, 2019, 22(4): 501-509. DOI:10.1007/s10123-019-00079-z.
[42] LIN F, LIU L, HAO G J, et al. The development and application of a duplex reverse transcription loop-mediated isothermal amplification assay combined with a lateral flow dipstick method for Macrobrachium rosenbergii nodavirus and extra small virus isolated in China[J]. Mol Cell Probes, 2018, 40: 1-7. DOI:10.1016/j.mcp.2018.05.001.
[43] KIM M J, KIM H J, KIM H Y. Direct triplex loop-mediated isothermal amplification assay for the point-of-care molecular detection of Salmonella genus, subspecies I, and serovar Typhimurium[J]. Food Control, 2021, 120: 107504. DOI:10.1016/j.foodcont.2020.107504.
[44] 杨粤,付博宇,张蕴哲,等.环介导等温扩增检测技术的应用与方法改进[J].食品安全质量检测学报, 2016, 7(4): 1526-1530. DOI:10.19812/j.cnki.jfsq11-5956/ts.2016.04.034. (
