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中华口腔医学研究杂志(电子版) ›› 2025, Vol. 19 ›› Issue (02) : 124 -131. doi: 10.3877/cma.j.issn.1674-1366.2025.02.007

综述

基于PfAgo的生物传感器在病原微生物检测中的研究进展
李广大1,2, 王敬夫2, 石晋2, 田磊2,(), 丁明超2   
  1. 1. 佳木斯大学口腔医学院,佳木斯 154002
    2. 口颌系统重建与再生全国重点实验室,国家口腔疾病临床医学研究中心,陕西省口腔疾病临床医学研究中心,空军军医大学口腔医院颌面外科,西安 710032
  • 收稿日期:2024-12-11 出版日期:2025-04-01
  • 通信作者: 田磊
  • 基金资助:
    军事医学与航空医学重大问题科技攻关项目(2023JSYX32)

Research progress of biosensors based on Pyrococcus furiosus Argonaute in detection of pathogenic microorganisms

Guangda Li1,2, Jingfu Wang2, Jin Shi2, Lei Tian2,(), Mingchao Ding2   

  1. 1. Stomatology Collage of Jiamusi University,Jiamusi 154002,China
    2. State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration,National Clinical Research Center for Oral Diseases,Shaanxi Clinical Research Center for Oral Diseases,Department of Oral and Maxillofacial Surgery,School of Stomatology,The Fourth Military Medical University,Xi′an 710032,China
  • Received:2024-12-11 Published:2025-04-01
  • Corresponding author: Lei Tian
引用本文:

李广大, 王敬夫, 石晋, 田磊, 丁明超. 基于PfAgo的生物传感器在病原微生物检测中的研究进展[J/OL]. 中华口腔医学研究杂志(电子版), 2025, 19(02): 124-131.

Guangda Li, Jingfu Wang, Jin Shi, Lei Tian, Mingchao Ding. Research progress of biosensors based on Pyrococcus furiosus Argonaute in detection of pathogenic microorganisms[J/OL]. Chinese Journal of Stomatological Research(Electronic Edition), 2025, 19(02): 124-131.

病原微生物检测是临床感染疾病诊断的常用方法,其高效精准的检测在明确病因和诊断治疗方面尤为重要。随着生物技术和传感器技术的不断发展,智能化和便携化的生物传感器在微生物检测等方面为人类健康作出巨大贡献。目前,学者们发现了一种来源于激烈火球菌(Pyrococcus furiosus)的Argonaute(Ago)蛋白PfAgo,在病原微生物检测方面应用广泛。相较于传统微生物检测技术,PfAgo 酶优势突出,适用于各种复杂环境下的病原微生物快速精准检测。本文简要介绍了PfAgo 酶的发展、原理及优势,阐述了基于PfAgo酶的生物传感器在病原微生物检测等方面的应用案例。综合分析,基于PfAgo酶的生物传感器检测技术具有操作简便、试剂消耗少、检测速度快、假阳性率低和自动化程度高等优势,针对病原微生物检测等方面有着十分广阔的发展空间和应用前景。

The detection of pathogenic microorganisms is a common method for clinical diagnosis of infectious diseases,and its efficient and accurate detection is particularly important in the identification of etiology,diagnosis and treatment.With the continuous development of biotechnology and sensor technology,intelligent and portable biosensors have made great contributions to human health in microbial detection.At present,scholars have discovered an Argonaute protein from Pyrococcus furiosus,which is widely used in the detection of pathogenic microorganisms.Compared with the traditional microbial detection technology,Pyrococcus furiosus Argonaute(PfAgo)enzyme has outstanding advantages and is suitable for rapid and accurate detection of pathogenic microorganisms in various complex environments.This article briefly introduces the development,principle and advantages of PfAgo enzyme,and expounds the application cases of biosensors based on PfAgo enzyme in the detection of pathogenic microorganisms.Comprehensive analysis shows that the biosensor detection technology based on PfAgo enzyme has the advantages of simple operation,low reagent consumption,fast detection speed,low false positive rate and high automation,and has a very broad development space and application prospect for pathogenic microorganism detection.

图1 PfAgo原理反应示意图
图2 基于PfAgo的生物传感器检测平台流程图 该检测流程为:首先,对目标样本进行核酸提取;然后,通过核酸扩增技术对目标基因进行扩增,扩增后的基因激活PfAgo 酶切割荧光探针,产生荧光信号;最后,使用生物传感器或视觉图像检测技术对荧光信号进行快速检测。MRSA为耐甲氧西林金黄色葡萄球菌。
表1 基于PfAgo检测技术与其他检测平台检测性能对比
[1]
Zhang D,Bi H,Liu B,et al.Detection of pathogenic microorganisms by microfluidics based analytical methods[J].Anal Chem,2018,90(9):5512-5520.DOI:10.1021/acs.analchem.8b00399.
[2]
Lee H,Yoon Y.Etiological agents implicated in foodborne illness world wide[J].Food Sci Anim Resour,2021,41(1):1-7.DOI:10.5851/kosfa.2020.e75.
[3]
Torgerson PR,de Silva NR,Fèvre EM,et al.The global burden of foodborne parasitic diseases:An update[J].Trends Parasitol,2014,30(1):20-26.DOI:10.1016/j.pt.2013.11.002.
[4]
Yi Y,Han Y,Cheng X,et al.Three-dimensional surfaceenhanced raman scattering platform with hotspots built by a nanomower for rapid detection of MRSA[J].Anal Chem,2022,94(49):17205-17211.DOI:10.1021/acs.analchem.2c03834.
[5]
Trinh TND,Lee NY.Nucleic acid amplification-based microfluidic approaches for antimicrobial susceptibility testing[J].Analyst,2021,146(10):3101-3113.DOI:10.1039/d1an00180a.
[6]
Opota O,Jaton K,Greub G.Microbial diagnosis of bloodstream infection:Towards molecular diagnosis directly from blood[J].Clin Microbiol Infect,2015,21(4):323-331.DOI:10.1016/j.cmi.2015.02.005.
[7]
Law JW,Ab Mutalib NS,Chan KG,et al.Rapid methods for the detection of foodborne bacterial pathogens: Principles,applications,advantages and limitations[J].Front Microbiol,2015,5:770.DOI:10.3389/fmicb.2014.00770.
[8]
Mazur F,Tjandra AD,Zhou Y,et al.Paper-based sensors for bacteria detection[J].Nat Rev Bioeng,2023,1(3):180-192.DOI:10.1038/s44222-023-00024-w.
[9]
Zhang SX,Li X,Wu JP,et al.Molecular methods for pathogenic bacteria detection and recent advances in wastewater analysis[J].Water,2021,13(24):3551.DOI:10.3390/w13243551.
[10]
Trinh TND,Lee NY.Advances in nucleic acid amplificationbased microfluidic devices for clinical microbial detection[J].Chemosensors,2022,10(4):123.DOI:10.3390/chemosensors 10040123.
[11]
Yao Y,Huang W,Chen J,et al.Flexible and stretchable organic electrochemical transistors for physiological sensing devices[J].Adv Mater,2023,35(35):e2209906.DOI:10.1002/adma.2022 09906.
[12]
He R,Wang L,Wang F,et al. Pyrococcus furiosus Argonautemediated nucleic acid detection[J].Chem Commun(Camb),2019,55(88):13219-13222.DOI:10.1039/c9cc07339f.
[13]
Swarts DC,Hegge JW,Hinojo I,et al.Argonaute of the archaeon Pyrococcus furiosus is a DNA-guided nuclease that targets cognate DNA[J].Nucleic Acids Res,2015,43(10):5120-5129.DOI:10.1093/nar/gkv415.
[14]
Kropocheva EV,Lisitskaya LA,Agapov AA,et al.Prokaryotic argonaute proteins as a tool for biotechnology[J].Mol Biol,2022,56(6):854-873.DOI:10.1134/S0026893322060103.
[15]
Blesa A,César CE,Averhoff B,et al.Noncanonical cell-to-cell DNA transfer in Thermus spp.is insensitive to argonaute -mediated interference[J].J Bacteriol,2015,197(1):138-146.DOI:10.1128/JB.02113-14.
[16]
Liu Q,Guo X,Xun G,et al.Argonaute integrated single-tube PCR system enables supersensitive detection of rare mutations[J].Nucleic Acids Res,2021,49(13):e75.DOI:10.1093/nar/gkab274.
[17]
Alvarado-Ramírez L,Rostro-Alanis M,Rodríguez-Rodríguez J,et al.Enzyme(single and multiple)and nanozyme biosensors:Recent developments and their novel applications in the waterfood-health nexus[J]Biosensors(Basel),2021,11(11):410.DOI:10.3390/bios11110410.
[18]
Chen H,Zhang J,Huang R,et al.The applications of electrochemical immunosensors in the detection of disease biomarkers:A review[J].Molecules,2023,28(8):3605.DOI:10.3390/molecules28083605.
[19]
Melinte G,Hosu O,Cristea C,et al.DNA sensing technology a useful food scanning tool[J].TrAC Trends Anal Chem,2022,154:116679.DOI:10.1016/j.trac.2022.116679.
[20]
Chen C,Wang JS.Optical biosensors:An exhaustive and comprehensive review[J].Analyst,2020,145(5):1605-1628.DOI:10.1039/c9an01998g.
[21]
Singh A,Sharma A,Ahmed A,et al.Recent advances in electrochemical biosensors:Applications,challenges,and future scope[J].Biosensors(Basel),2021,11(9):336.DOI:10.3390/bios11090336.
[22]
Ramanathan K,Danielsson B.Principles and applications of thermal biosensors[J].Biosens Bioelectron,2001,16(6):417-423.DOI:10.1016/S0956-5663(01)00124-5.
[23]
Skládal P.Piezoelectric biosensors:Shedding light on principles and applications[J].Microchim Acta,2024,191(4):184.DOI:10.1007/s00604-024-06257-9.
[24]
Ramesh M,Janani R,Deepa C,et al.Nanotechnology-enabled biosensors: A review of fundamentals,design principles,materials,and applications[J].Biosensors(Basel)2023,13(1):40.DOI:10.3390/bios13010040.
[25]
Qiao JL,Jia JY,Peng WP,et al.Rapid,on-site and yes-or-no detection of Salmonella typhimurium in foods using Argonauteenabled assay[J].Sens Actuator:B.Chem,2024,404:135263.DOI:10.1016/j.snb.2023.135263.
[26]
Yu ZR,Shao Y,Dong YL,et al.LAMP combined with Pyrococcus furiosus Argonaute for the ultrasensitive and highly specific point-of-care test platform for Listeria monocytogenes detection[J].LWT-Food Sci Technol,2024,207:116640.DOI:10.1016/j.lwt.2024.116640.
[27]
Li Y,Kou J,Han X,et al.Argonaute-triggered visual and rebuilding-free foodborne pathogenic bacteria detection[J].J Hazard Mater,2023,454:131485.DOI:10.1016/j.jhazmat.2023.131485.
[28]
Li Y,Tang X,Wang N,et al.Argonaute-DNAzyme tandem biosensing for highly sensitive and simultaneous dual - gene detection of methicillin - resistant Staphylococcus aureus[J].Biosens Bioelectron,2024,244:115758.DOI:10.1016/j.bios.2023.115758.
[29]
Chen W,Zhang J,Wei H,et al.Rapid and sensitive detection of methicillin-resistant Staphylococcus aureus through the RPAPfAgo system[J].Front Microbiol,2024,15:1422574.DOI:10.3389/fmicb.2024.1422574.
[30]
Shi Y,Tan Z,Li W,et al.Enzyme-assisted endogenous guide DNA generation - mediated Pyrococcus furiosus Argonaute for Alicyclobacillus acidoterrestris detection[J].J Agric Food Chem,2024,72(2):1354-1360.DOI:10.1021/acs.jafc.3c07881.
[31]
Shi Y,Tan Z,Wu D,et al. Pyrococcus furiosus argonaute based Alicyclobacillus acidoterrestrsis detection in fruit juice[J].Food Microbiol,2024,120:104475.DOI:10.1016/j.fm.2024.104475.
[32]
Lin L,Luo Q,Li L,et al.Recombinase polymerase amplification combined with Pyrococcus furiosus Argonaute for fast Salmonella spp. testing in food safety[J].Int J Food Microbiol,2024,417:110697.DOI:10.1016/j.ijfoodmicro.2024.110697.
[33]
Guo BY,Zhao Y,Zhou CY,et al.The detection of Salmonella in food based on PCR combined with Pyrococcus furiosus Argonaute[J].Microchem J,2024,206:111311.DOI:10.1016/j.microc.2024.111311.
[34]
Xun GH,Lane ST,Petrov VA,et al.A rapid,accurate,scalable,and portable testing system for COVID-19 diagnosis[J].Nat Commun,2021,12(1):2905.DOI:10.1038/s41467-021-23185-x.
[35]
Zhao CJ,Yang LH,Zhang X,et al.Rapid and sensitive genotyping of SARS-CoV-2 key mutation L452R with an method[J].Anal Chem,2022,94(49):17151-17159.DOI:10.1021/acs.analchem.2c03563.
[36]
Wang F,Yang J,He RY,et al.PfAgo-based detection of SARSCoV-2[J].Biosens Bioelectron,2021,177:112932.DOI:10.1016/j.bios.2020.112932.
[37]
Han R,Wang F,Chen WP,et al.A fast and sensitive one-tube SARS - CoV - 2 detection platform based on RTX - PCR and Pyrococcus furiosus Argonaute[J]Biosensors(Basel),2024,14(5):245.DOI:10.3390/bios14050245.
[38]
Wang LY,He RY,Lv B,et al. Pyrococcus furiosus Argonaute coupled with modified ligase chain reaction for detection of SARSCoV-2 and HPV[J].Talanta,2021,227:122154.DOI:10.1016/j.talanta.2021.122154.
[39]
Chen YH,Zhang XY,Yang X,et al. PfAgo-based zika virus detection[J].Viruses,2024,16(4):539.DOI:10.3390/v1604 0539.
[40]
Chen WR,Qiu LY,Luo T,et al.Novel nucleic acid detection for human parvovirus B19 based on Pyrococcus furiosus argonaute protein[J].Viruses,2023,15(3):595.DOI:10.3390/v15030 595.
[41]
Liao JY,Feng XY,Zhang JX,et al.RT-RPA-PfAgo detection platform for one-tube simultaneous typing diagnosis of human respiratory syncytial virus[J].Front Cell Infect Microbiol,2024,14:1419949.DOI:10.3389/fcimb.2024.1419949.
[42]
He P,Zhou WH,Wei HP,et al.Fast and ultrasensitive detection of monkeypox by a Pyrococcus furiosus argonaute system coupled with a short amplification[J].Viruses,2024,16(3):382.DOI:10.3390/v16030382.
[43]
Zhao Y,Zhang TJ,Zhou CY,et al. Pyrococcus furiosus argonaute based detection assays for porcine deltacoronavirus[J].ACS Synth Biol,2024,13(4):1323-1331.DOI:10.1021/acssynbio.4c00045.
[44]
Zhao Y,Yang M,Zhou CY,et al.Establishment of a simple,sensitive,and specific ASFV detection method based on Pyrococcus furiosus argonaute[J].Biosens Bioelectron,2024,254:116230.DOI:10.1016/j.bios.2024.116230.
[45]
Zhao Y,Zhou CY,Guo BY,et al.Pyrococcus furiosus Argonautemediated porcine epidemic diarrhea virus detection[J].Appl Microbiol Biotechnol,2024,108(1):137.DOI:10.1007/s00253-023-12919-0.
[46]
Liu YQ,Chen LH,Zhang ZX,et al.Development and application of a novel recombinase polymerase amplification - Pyrococcus furiosus argonaute system for rapid detection of goose parvovirus[J].Poult Sci,2024,103(10):104141.DOI:10.1016/j.psj.2024.104141.
[47]
Yu ZR,Shi DM,DongYL,et al. Pyrococcus furiosus argonaute combined with loop-mediated isothermal amplification for rapid,ultrasensitive,and visual detection of fowl adenovirus serotype 4[J].Poult Sci,2024,103(10):103729.DOI:10.1016/j.psj.2024.103729.
[48]
Wang Y,Chen YK,Tang YX,et al.A recombinase polymerase amplification and Pyrococcus furiosus Argonaute combined method for ultra-sensitive detection of white spot syndrome virus in shrimp[J].J Fish Dis,2023,46(12):1357-1365.DOI:10.1111/jfd.13853.
[49]
Liu Y,Xia WQ,Zhao W,et al.RT-RPA-PfAgo System:A rapid,sensitive,and specific multiplex detection method for riceinfecting viruses[J].Biosensors(Basel),2023,13(10):941.DOI:10.3390/bios13100941.
[50]
Xiang YQ,Ke WK,Qin YQ,et al. PfAgo-based dual signal amplification biosensor for rapid and highly sensitive detection of alkaline phosphatase activity[J].Microchim Acta,2024,191(7):439.DOI:10.1007/s00604-024-06516-9.
[51]
Huang ZZ,Wei LY,Zhou YN,et al.Guide DNA dephosphorylation - modulated Pyrococcus furiosus Argonaute fluorescence biosensor for the detection of alkaline phosphatase and aflatoxins B1[J].Biosens Bioelectron,2024,265:116692.DOI:10.1016/j.bios.2024.116692.
[52]
Pang FB,Zhang T,Dai FY,et al.A handheld isothermal fluorescence detector for duplex visualization of aquatic pathogens via enhanced one-pot LAMP-PfAgo assay[J].Biosens Bioelectron,2024,254:116187.DOI:10.1016/j.bios.2024.116187.
[53]
Yang LH,Guo B,Wang Y,et al. Pyrococcus furiosus Argonaute combined with recombinase polymerase amplification for rapid and sensitive detection of Enterocytozoon hepatopenaei[J].J Agric Food Chem,2023,71(1):944-951.DOI:10.1021/acs.jafc.2c06582.
[54]
Zhao YL,Zhang YH,Wu WQ,et al.Rapid and sensitive detection of Mycoplasma synoviae using RPA combined with Pyrococcus furiosus Argonaute[J]Poult Sci,2024,103(3):103244.DOI:10.1016/j.psj.2023.103244.
[55]
Lim JM,Kim HH.Basic principles and clinical applications of CRISPR-based genome editing[J].Yonsei Med J,2022,63(2):105-113.DOI:10.3349/ymj.2022.63.2.105.
[56]
Wu ZY,Yu L,Shi WF,et al.Argonaute protein-based nucleic acid detection technology[J].Front Microbiol,2023,14:1255716.DOI:10.3389/fmicb.2023.1255716.
[57]
Kelley K,Cosman A,Belgrader P,et al.Detection of methicillinresistant Staphylococcus aureus by a duplex droplet digital PCR assay[J].J Clin Microbiol,2013,51(7):2033-2039.DOI:10.1128/JCM.00196-13.
[58]
Zhang HW,Ma LY,Ma LN,et al.Rapid detection of methicillinresistant Staphylococcus aureus in pork using a nucleic acidbased lateral flow immunoassay[J].Int J Food Microbiol,2017,243:64-69.DOI:10.1016/j.ijfoodmicro.2016.12.003.
[59]
Wang HY,Kim S,Kim J,et al.Multiplex real-time PCR assay for rapid detection of methicillin-resistant staphylococci directly from positive blood cultures[J]J Clin Microbiol,2014,52(6):1911-1920.DOI:10.1128/JCM.00389-14.
[60]
Guk K,Keem JO,Hwang SG,et al.A facile,rapid and sensitive detection of MRSA using a CRISPR - mediated DNA FISH method,antibody-like dCas9/sgRNA complex[J].Biosensors Bioelectronics,2017,95:67-71.DOI:10.1016/j.bios.2017.04.016.
[61]
Tao ZZ,Wang BH,Cui Q,et al.A signal-off Cas14a1-based platform for highly specific detection of methicillin - resistant Staphylococcus aureus[J].Anal Chim Acta,2023,1256:341154.DOI:10.1016/j.aca.2023.341154.
[62]
Hu YZ,Qiao YF,Li XQ,et al.Development of an inducible Cas9 nickase and PAM - free Cas12a platform for bacterial diagnostics[J].Talanta,2023,265:124931.DOI:10.1016/j.talanta.2023.124931.
[63]
Duraiswamy S,Agarwalla S,Lok KS,et al.A multiplex Taqman PCR assay for MRSA detection from whole blood[J].PLoS One,2023,18(11):e0294782.DOI:10.1371/journal.pone.0294782.
[64]
Wei LY,Wang ZL,Wang J,et al.Aptamer-based colorimetric detection of methicillin-resistant Staphylococcus aureus by using a CRISPR/Cas12asystemandrecombinasepolymerase amplification[J].Anal Chim Acta,2022,16:1230.DOI:10.1016/j.aca.2022.340357.
[65]
Sahibzada S,Pang S,Hernández-Jover M,et al.Prevalence and antimicrobial resistance of MRSA across different pig age groups in an intensive pig production system in Australia[J].Zoonoses Public Health,2020,67(5):576-586.DOI:10.1111/zph.12721.
[66]
Chen CG,Zhao QY,Guo JW,et al.Identification of methicillinresistant Staphylococcus aureus (MRSA) using simultaneous detection of mecA,nuc,and femB by loop-mediated isothermal amplification(LAMP)[J].Curr Microbiol,2017,74(8):965-971.DOI:10.1007/s00284-017-1274-2.
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