病原微生物的核酸检测分析研究进展

doi:10.19894/j.issn.1000-0518.210099

摘要/Abstract

摘要： 2019年12月,全球爆发新型冠状病毒肺炎疫情(COVID-19),使生物安全面临着巨大危机。病原微生物的快速、准确、低成本检测是保障生物安全的重要组成元素之一,是疫情预防、控制和诊断的关键。本综述介绍了核酸测序技术、等温扩增技术和基因编辑技术(CRISPR/Cas)于便携式一体化设备中的应用,为研发“低成本,高效率,结果可视化”集成式检测方法提供了可能性,旨在为病原微生物的有效防控提供保障。

关键词: 核酸, 快速检测, 病原微生物, 新型冠状病毒, 基因编辑技术

Abstract: In December 2019, the global outbreak of Corona Virus Disease 2019 (COVID-19)made biosafety an attractive and crucial development direction globally. Rapid, accurate and low-cost detection of pathogenic microorganisms is one of the important issue to ensure biosafety, and is the key to epidemic prevention, control and diagnosis. This review elaborately introduces the applications of biosensors based on nucleic acid sequencing, isothermal amplification and gene editing (CRISPR/Cas) in integrated portable devices. The development of these technologies offers the possibility of developing “low-cost, high-efficiency and result-visualization” integrated detection methods. It is expected to provide technology guarantees for the effective prevention and control of pathogenic microorganisms.

Key words: Nucleic acid, Rapid detection, Pathogenic microorganisms, SARS-CoV-2, CRISPR/Cas

中图分类号:

O656

李子玥, 杨同仁, 杨歌, 黄渊余. 病原微生物的核酸检测分析研究进展[J]. 应用化学, 2021, 38(5): 592-604.

LI Zi-Yue, YANG Tong-Ren, YANG Ge, HUANG Yuan-Yu. Research Progresses of Nucleic Acid Based Detection of Pathogenic Microorganisms[J]. Chinese Journal of Applied Chemistry, 2021, 38(5): 592-604.

参考文献

[1] 辛亮, 张兰威. 核酸-微流控芯片检测食品病原微生物的研究进展[J]. 食品科学, 2020, 41(23): 266-272.
XIN L, ZHANG L W. Recent progress in nucleic acid-microfluidic chips used for detection of foodborne pathogens: a review[J]. Food Sci, 2020, 41(23): 266-272.
[2] 董金颖. 共聚焦显微拉曼光谱技术在病原微生物快速检测中的研究[D]. 大连: 大连医科大学, 2018.
DONG J Y. Study on rapid detection of pathogenic microorganisms by confocal Raman microspectroscopy[D]. Dalian: Dalian Medical University, 2018.
[3] 搜狐网. 2003年非典全球各国感染及死亡人数[OL]. [2020-03-09]. https://www.sohu.com/a/378545139_120526932.
SUHO. Number of SARS infections and deaths worldwide in 2003[OL]. [2020-03-09]. https://www.sohu.com/a/378545139_120526932.
[4] DELANNOY S, BEUTIN L, BURGOS Y, et al. Specific detection of enteroaggregative hemorrhagic Escherichia coli O104:H4 strains by use of the CRISPR locus as a target for a diagnostic real-time PCR[J]. J Clin Microbiol, 2012, 50(11): 3485-3492.
[5] KAMORUDEEN R T, ADEDOKUN K A, OLARINMOYE A O. Ebola outbreak in West Africa, 2014-2016: Epidemic timeline, differential diagnoses, determining factors, and lessons for future response[J]. J Infect Public Heal, 2020, 13(7): 956-962.
[6] 网易新闻网. 肺炎疫情实时动态播报[OL]. [2021-04-06]. https://wp.m.163.com/163/page/news/virus_report/index.html.
Netease News. Real time and dynamic report of pneumonia[OL]. [2021-04-06]. https://wp.m.163.com/163/page/news/virus_report/index.html.
[7] XIAO Y, LI Z, WANG X, et al. Comparison of three TaqMan real-time reverse transcription-PCR assays in detecting SARS-CoV-2[J]. J Virol Methods, 2021, 288: 114030.
[8] SHEN M, ZHOU Y, YE J, et al. Recent advances and perspectives of nucleic acid detection for coronavirus[J]. J Pharm Anal, 2020, 10(2): 97-101.
[9] DAO THI V L, HERBST K, BOERNER K, et al. A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples[J]. Sci Transl Med, 2020, 12(556): eabc7075.
[10] CRONE M A, PRIESTMAN M, CIECHONSKA M, et al. A role for biofoundries in rapid development and validation of automated SARS-CoV-2 clinical diagnostics[J]. Nat Commun, 2020, 11(1): 4464.
[11] CAMPEAU P M, KASPERAVICIUTE D, LU J T, et al. The genetic basis of DOORS syndrome: an exome-sequencing study[J]. Lancet Neurol, 2014, 13(1): 44-58.
[12] GU W, MILLER S, CHIU C Y. Clinical metagenomic next-generation sequencing for pathogen detection[J]. Annu Rev Pathol-Mech, 2019, 14:319-338.
[13] KAJI N, OKAMOTO Y, TOKESHI M, et al. Nanopillar, nanoball, and nanofibers for highly efficient analysis of biomolecules[J]. Chem Soc Rev, 2010, 39(3): 948-956.
[14] KIM D, LEE J Y, YANG J S, et al. The architecture of SARS-CoV-2 transcriptome[J]. Cell, 2020, 181(4): 914-921.e10.
[15] EID J, FEHR A, GRAY J, et al. Real-time DNA sequencing from single polymerase molecules[J]. Science, 2009, 323(5910): 133-138.
[16] LEVENE M J, KORLACH J, TURNER S W, et al. Zero-mode waveguides for single-molecule analysis at high concentrations[J]. Science, 2003, 299(5607): 682-686.
[17] LARKIN J, FOQUET M, TURNER S W, et al. Reversible positioning of single molecules inside zero-mode waveguides[J]. Nano Lett, 2014, 14(10): 6023-6039.
[18] JAIN M, KOREN S, MIGA K H, et al. Nanopore sequencing and assembly of a human genome with ultra-long reads[J]. Nat Biotechnol, 2018, 36(4): 338-345.
[19] KILIANSKI A, ROTH P A, LIEM A T, et al. Use of unamplified RNA/cDNA-hybrid nanopore sequencing for rapid detection and characterization of RNA viruses[J]. Emerg Infect Dis, 2016, 22(8): 1448-1451.
[20] RUSSELL J A, CAMPOS B, STONE J, et al. Unbiased strain-typing of arbovirus directly from mosquitoes using nanopore sequencing: a field-forward biosurveillance protocol[J]. Sci Rep-UK, 2018, 8(1): 5417.
[21] WANG S, ZHAO Z, HAQUE F, et al. Engineering of protein nanopores for sequencing, chemical or protein sensing and disease diagnosis[J]. Curr Opin Biotech, 2018, 51:80-89.
[22] OH S, LEE M K, CHI S W. Single-molecule-based detection of conserved influenza a virus RNA promoter using a protein nanopore[J]. ACS Sens, 2019, 4(11): 2849-2853.
[23] GARALDE D R, SNELL E A, JACHIMOWICZ D, et al. Highly parallel direct RNA sequencing on an array of nanopores[J]. Nat Methods, 2018, 15(3): 201-206.
[24] CRESSIOT B, GREIVE S J, MOJTABAVI M, et al. Thermostable virus portal proteins as reprogrammable adapters for solid-state nanopore sensors[J]. Nat Commun, 2018, 9(1): 4652.
[25] BAFNA J A, SONI G V. Fabrication of low noise borosilicate glass nanopores for single molecule sensing[J]. PloS One, 2016, 11(6): e0157399.
[26] GAROLI D, YAMAZAKI H, MACCAFERRI N, et al. Plasmonic nanopores for single-molecule detection and manipulation: toward sequencing applications[J]. Nano Lett, 2019, 19(11): 7553-62.
[27] CHEN C, LI Y, KERMAN S, et al. High spatial resolution nanoslit SERS for single-molecule nucleobase sensing[J]. Nat Commun, 2018, 9(1): 1733.
[28] AHANGAR L E, MEHRGARDI M A. Amplified detection of hepatitis B virus using an electrochemical DNA biosensor on a nanoporous gold platform[J]. Bioelectrochemistry, 2017, 117:83-88.
[29] TOLDR A, O′SULLIVAN C K, CAMP S M. Detecting Harmful algal blooms with isothermal molecular strategies[J]. Trends Biotechnol, 2019, 37(12): 1278-1281.
[30] TOLDR A, O′SULLIVAN C K, DIOG NE J, et al. Detecting harmful algal blooms with nucleic acid amplification-based biotechnological tools[J]. Sci Total Environ, 2020, 749:141605.
[31] NOTOMI T, OKAYAMA H, MASUBUCHI H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Res, 2000, 28(12): e63.
[32] NAWATTANAPAIBOON K, KIATPATHOMCHAI W, SANTANIRAND P, et al. SPR-DNA array for detection of methicillin-resistant staphylococcus aureus (MRSA) in combination with loop-mediated isothermal amplification[J]. Biosens Bioelectron, 2015, 74:335-340.
[33] CHEN W, YU H, SUN F, et al. Mobile Platform for multiplexed detection and differentiation of disease-specific nucleic acid sequences, using microfluidic loop-mediated isothermal amplification and smartphone detection[J]. Anal Chem, 2017, 89(21): 11219-11226.
[34] BARNES L, HEITHOFF D M, MAHAN S P, et al. Smartphone-based pathogen diagnosis in urinary sepsis patients [J]. Ebiomedicine, 2018, 36:73-82.
[35] PIEPENBURG O, WILLIAMS C H, STEMPLE D L, et al. DNA detection using recombination proteins[J]. PloS Biol, 2006, 4(7): e204.
[36] DEL R O J S, SVOBODOVA M, BUSTOS P, et al. Electrochemical detection of Piscirickettsia salmonis genomic DNA from salmon samples using solid-phase recombinase polymerase amplification[J]. Anal Bioanal Chem, 2016, 408(30): 8611-8620.
[37] DEL R O J S, LOBATO I M, MAYBORODA O, et al. Enhanced solid-phase recombinase polymerase amplification and electrochemical detection[J]. Anal Bioanal Chem, 2017, 409(12): 3261-3269.
[38] TIAN J, CHU H, ZHANG Y, et al. TiO₂ Nanoparticle-enhanced linker recombinant strand displacement amplification (LRSDA) for universal label-free visual bioassays[J]. ACS Appl Mater Interfaces, 2019, 11(50): 46504-46514.
[39] REID M S, LE X C, ZHANG H. Exponential isothermal amplification of nucleic acids and assays for proteins, cells, small molecules, and enzyme activities: an EXPAR example[J]. Angew Chem Int Ed, 2018, 57(37): 11856-11866.
[40] CAI R, ZHANG Z, CHEN H, et al. A versatile signal-on electrochemical biosensor for Staphylococcus aureus based on triple-helix molecular switch[J]. Sens Actuators B, 2021, 326: 128842.
[41] GUO Q, YANG X, WANG K, et al. Sensitive fluorescence detection of nucleic acids based on isothermal circular strand-displacement polymerization reaction[J]. Nucleic Acids Res, 2009, 37(3): e20.
[42] MURUGAN K, BABU K, SUNDARESAN R, et al. The revolution continues: newly discovered systems expand the CRISPR-Cas toolkit[J]. Mol Cell, 2017, 68(1): 15-25.
[43] JINEK M, CHYLINSKI K, FONFARA I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J]. Science, 2012, 337(6096): 816-821.
[44] ABUDAYYEH O O, GOOTENBERG J S, KONERMANN S, et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector[J]. Science, 2016, 353(6299): 5573.
[45] ZHANG H, LI Z, DACZKOWSKI C M, et al. Structural basis for the inhibition of CRISPR-Cas12a by anti-CRISPR proteins[J]. Cell Host Microbe, 2019, 25(6): 815-826.
[46] PARDEE K, GREEN A A, TAKAHASHI M K, et al. Rapid, low-cost detection of Zika virus using programmable biomolecular components[J]. Cell, 2016, 165(5): 1255-1266.
[47] HUANG M, ZHOU X, WANG H, et al. Clustered regularly interspaced short palindromic repeats/Cas9 triggered isothermal amplification for site-specific nucleic acid detection[J]. Anal Chem, 2018, 90(3): 2193-2200.
[48] BATISTA A C, PACHECO L G C. Detecting pathogens with Zinc-Finger, TALE and CRISPR-based programmable nucleic acid binding proteins[J]. J Microbiol Meth, 2018, 152:98-104.
[49] ZHANG Y, QIAN L, WEI W, et al. Paired design of dCas9 as a systematic platform for the detection of featured nucleic acid sequences in pathogenic strains[J]. ACS Synth Biol, 2017, 6(2): 211-216.
[50] QIU X Y, ZHU L Y, ZHU C S, et al. Highly effective and low-cost microRNA detection with CRISPR-Cas9[J]. ACS Synth Biol, 2018, 7(3): 807-813.
[51] YANG W, RESTREPO-PEREZ L, BENGTSON M, et al. Detection of CRISPR-dCas9 on DNA with solid-state nanopores[J]. Nano Lett, 2018, 18(10): 6469-6474.
[52] LI S Y, CHENG Q X, LIU J K, et al. CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA[J]. Cell Res, 2018, 28(4): 491-493.
[53] CHEN J S, MA E, HARRINGTON L B, et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity[J]. Science, 2018, 360(6387): 436-439.
[54] LI L, LI S, WU N, et al. HOLMESv2: a CRISPR-Cas12b-assisted platform for nucleic acid detection and DNA methylation quantitation[J]. ACS Synth Biol, 2019, 8(10): 2228-2237.
[55] DAI Y, SOMOZA R A, WANG L, et al. Exploring the trans-cleavage activity of CRISPR-Cas12a (cpf1) for the development of a universal electrochemical biosensor[J]. Angew Chem Int Ed, 2019, 58(48): 17399-173405.
[56] SHMAKOV S, ABUDAYYEH O O, MAKAROVA K S, et al. Discovery and functional characterization of diverse class 2 CRISPR-Cas systems[J]. Mol Cell, 2015, 60(3): 385-397.
[57] GOOTENBERG J S, ABUDAYYEH O O, LEE J W, et al. Nucleic acid detection with CRISPR-Cas13a/C2c2[J]. Science, 2017, 356(6336): 438-442.
[58] ABUDAYYEH O O, GOOTENBERG J S, ESSLETZBICHLER P, et al. RNA targeting with CRISPR- Cas13[J]. Nature, 2017, 550(7675): 280-284.
[59] LI Y, LI S, WANG J, et al. CRISPR/Cas systems towards next-generation biosensing[J]. Trends Biotechnol, 2019, 37(7): 730-743.
[60] GOOTENBERG J S, ABUDAYYEH O O, KELLNER M J, et al. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6[J]. Science, 2018, 360(6387): 439-444.
[61] KAZLAUSKIENE M, KOSTIUK G, VENCLOVAS Cˇ, et al. A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems[J]. Science, 2017, 357(6351): 605-609.
[62] MYHRVOLD C, FREIJE C A, GOOTENBERG J S, et al. Field-deployable viral diagnostics using CRISPR-Cas13[J]. Science, 2018, 360(6387): 444-448.
[63] 盛楠, 马雪萍, 逄淑云, 等. 新型冠状病毒SARS-CoV-2核酸检测技术平台的研究进展[J]. 分析化学, 2020, 48(10):1279-1287.
SHENG N, MA X P, PANG S Y, et al. Research progress of nucleic acid detection technology platforms for new coronavirus SARS-CoV-2[J]. Chinese J Anal Chem, 2020, 48(10):1279-1287
[64] ZHANG J, LI K, ZHENG L, et al. Improving detection efficiency of SARS-CoV-2 nucleic acid testing[J]. Front Cell Infect Microbiol, 2020, 10:558472.
[65] ALFARAJ S H, AL-TAWFIQ J A, MEMISH Z A. Middle East respiratory syndrome coronavirus intermittent positive cases: implications for infection control[J]. Am J Infect Control, 2019, 47(3): 290-293.
[66] MEREDITH L W, HAMILTON W L, WARNE B, et al. Rapid implementation of SARS-CoV-2 sequencing to investigate cases of health-care associated COVID-19: a prospective genomic surveillance study[J]. Lancet Infect Dis, 2020, 20(11): 1263-1271.
[67] WOO C H, JANG S, SHIN G, et al. Sensitive fluorescence detection of SARS-CoV-2 RNA in clinical samples via one-pot isothermal ligation and transcription[J]. Nat Biomed Eng, 2020, 4(12): 1168-1179.
[68] MOITRA P, ALAFEEF M, DIGHE K, et al. Selective Naked-eye detection of SARS-CoV-2 mediated by N gene targeted antisense oligonucleotide capped plasmonic nanoparticles[J]. ACS Nano, 2020, 14(6): 7617-7627.
[69] XIONG E, JIANG L, TIAN T, et al. Simultaneous dual-gene diagnosis of SARS-CoV-2 based on CRISPR/Cas9-mediated lateral flow assay[J]. Angew Chem Int Ed, 2021,60(10): 5307-5315.
[70] ARIZTI-SANZ J, FREIJE C A, STANTON A C, et al. Streamlined inactivation, amplification, and Cas13-based detection of SARS-CoV-2[J]. Nat Commun, 2020, 11(1): 5921.
[71] BROUGHTON J P, DENG X, YU G, et al. CRISPR-Cas12-based detection of SARS-CoV-2[J]. Nat Biotechnol, 2020, 38(7): 870-874.
[72] DING X, YIN K, LI Z, et al. Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay[J]. Nat Commun, 2020, 11(1): 4711.