基于Argonaute蛋白的动物病原核酸检测技术及应用研究进展

doi:10.16431/j.cnki.1671-7236.2025.11.035

摘要/Abstract

摘要： Argonaute(Ago)蛋白是广泛存在于生物体内且高度保守的一类蛋白，具有通过结合小的单链核酸引导序列对互补靶标进行特异性识别和切割的特性。基于Ago蛋白具有的特异序列识别及连续切割活性特性，使其成为新一代核酸分子诊断生物传感器的候选工具，为开发快速、便捷、灵敏、特异、多重核酸检测技术提供了一种新选择。近年来，Ago蛋白介导的核酸检测技术受到研究人员的广泛关注，尤其在动物疫病病原检测领域，已建立起多种动物疫病病原的检测方法，展现出良好的应用前景。笔者简要总结了Ago蛋白的结构特征及功能，嗜热栖热菌(Thermus thermophilus)Ago蛋白(TtAgo)和激烈火球菌(Pyrococcus furiosus)Ago蛋白(PfAgo)介导的代表性核酸检测平台及其在动物病原检测的应用，并提出了提高基于Ago蛋白核酸检测技术应用的潜在策略，以期为动物病原核酸检测生物传感器技术的开发提供参考。

关键词: Argonaute蛋白; 核酸检测技术; 动物疫病; 诊断

Abstract: Argonaute (Ago) protein is a highly conserved protein and widely found in organisms.It can specifically recognize and cleave complementary targets by binding short guide single-stranded nucleic acid sequence.Based on specific sequence recognition and transfer cleavage activity characteristics of Ago,it has emerged as a promising candidate for a new generation of nucleic acid molecular diagnostic biosensors.It provides a novel choice for developing of rapid,sensitive,specific and multiplex nucleic acid detection technology.In recent years,it has attracted the attention of researchers especially in the field of animal pathogen detection.A variety of animal pathogen detection methods have been established.It shows a great application prospects.The authors reviewed the structures and biotechnologies of Ago,summarized representative nucleic acid detection platforms mediated by Thermus thermophilus Ago protein (TtAgo) and Pyrococcus furiosus Ago (PfAgo) and their application in animal pathogen detection,prospected the potential strategies to improve the application with the aims of providing new ideas for the development of a new generation of animal pathogen nucleic acid molecular diagnostic biosensors.

Key words: Argonaute protein; nucleic acid detection technology; animal diseases; diagnosis

中图分类号:

冯杰, 黎春秀, 杨旸, 邹波, 付玲玲, 曹乾大. 基于Argonaute蛋白的动物病原核酸检测技术及应用研究进展[J]. 中国畜牧兽医, 2025, 52(11): 5403-5413.

FENG Jie, LI Chunxiu, YANG Yang, ZOU Bo, FU Lingling, CAO Qianda. Progress on Animal Pathogenic Nucleic Acid Detection Technology Based on Argonaute Protein and Its Application[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(11): 5403-5413.

参考文献

[1] ZELNÍK V.Use of nucleic acids amplification methods in Marek’s disease diagnosis and pathogenesis studies[J].Acta Virologica,2021,65(1):27-32.
[2] PANG L,PI X,YANG X,et al.Nucleic acid amplification-based strategy to detect foodborne pathogens in milk:A review[J].Critical Reviews in Food Science and Nutrition,2024,64(16):5398-5413.
[3] ZHANG Q,LIU Q,OPRIESSNIG T,et al.Multiplex gel-based PCR assay for the simultaneous detection of 5 genotypes of Porcine astroviruses[J].Journal of Veterinary Diagnostic Investigation,2023,35(2):132-138.
[4] MENG W,CHEN Z,JIANG Q,et al.A multiplex Real-time fluorescence-based quantitative PCR assay for calf diarrhea viruses[J].Frontiers in Microbiology,2024,14:1327291.
[5] LONG F,CHEN Y,SHI K,et al.Development of a multiplex crystal digital RT-PCR for differential detection of classical,highly pathogenic,and NADC30-like Porcine reproductive and respiratory syndrome virus[J].Animals,2023,13(4):594.
[6] SOROKA M,WASOWICZ B,RYMASZEWSKA A.Loop-mediated isothermal amplification (LAMP):The better sibling of PCR?[J].Cells,2021,10(8):1931.
[7] TAN M,LIAO C,LIANG L,et al.Recent advances in recombinase polymerase amplification:Principle,advantages,disadvantages and applications[J]. Frontiers in Cellular and Infection Microbiology,2022,12:1019071.
[8] 张庆勋，钟震宇，郭青云，等.基于CRISPR-Cas系统的病原体检测研究进展[J].中国畜牧兽医，2022，49(8)：3190-3199.ZHANG Q X,ZHONG Z X,GUO Q Y,et al.Recent advances of pathogens detection based on CRISPR-Cas system[J].China Animal Husbandry & Veterinary Medicine，2022，49(8)：3190-3199.(in Chinese)
[9] ZHAO Y,ZHANG Y,WU W,et al.Rapid and sensitive detection of Mycoplasma synoviae using RPA combined with Pyrococcus furiosus Argonaute[J].Poultry Science,2024,103(3):103244.
[10] ZHOU J,LI Z,OLAJIDE J S,et al.CRISPR/Cas-based nucleic acid detection strategies:Trends and challenges[J].Heliyon,2024,10(4):e26179.
[11] SONG J,HEGGE J W,MAUK M G,et al.Highly specific enrichment of rare nucleic acid fractions using Thermus thermophilus Argonaute with applications in cancer diagnostics[J].Nucleic Acids Research,2020,48(4):e19.
[12] HE R,WANG L,WANG F,et al.Pyrococcus furiosus Argonaute-mediated nucleic acid detection[J]. Chemical Communications,2019,55(88):13219-13222.
[13] BOHMERT K,CAMUS I,BELLINI C,et al.AGO1 defines a novel locus of arabidopsis controlling leaf development[J].The EMBO Journal,1998,17(1):170-180.
[14] LI Y,ZHAO L,WANG J,et al.Argonaute-based nucleic acid detection technology:Advantages,current status,challenges,and perspectives[J].ACS Sensors,2024,9(11):5665-5682.
[15] OLINA A V,KULBACHINSKIY A V,ARAVIN A A,et al.Argonaute proteins and mechanisms of RNA interference in eukaryotes and prokaryotes[J].Biochemistry (Moscow),2018,83:483-497.
[16] FRANK F,SONENBERG N,NAGAR B.Structural basis for 5'-nucleotide base-specific recognition of guide RNA by human AGO2[J].Nature,2010,465(7299):818-822.
[17] MEIJER H A,SMITH E M,BUSHELL M.Regulation of miRNA strand selection:Follow the leader?[J].Biochemical Society Transactions,2014,42(4):1135-1140.
[18] WANG Z,WANG Y,LIU T,et al.Effects of the PIWI/MID domain of Argonaute protein on the association of miRNAi’s seed base with the target[J].RNA,2019,25(5):620-629.
[19] DJURANOVIC S,NAHVI A,GREEN R.miRNA-mediated gene silencing by translational repression followed by mRNA deadenylation and decay[J].Science,2012,336(6078):237-240.
[20] LISITSKAYA L,SHIN Y,AGAPOV A,et al.Programmable RNA targeting by bacterial Argonaute nucleases with unconventional guide binding and cleavage specificity[J].Nature Communications,2022,13(1):4624.
[21] RYAZANSKY S,KULBACHINSKIY A,ARAVIN A A.The expanded universe of prokaryotic Argonaute proteins[J].mBio,2018,9(6):e01935-18.
[22] OLOVNIKOV I,CHAN K,SACHIDANANDAM R,et al.Bacterial Argonaute samples the transcriptome to identify foreign DNA[J].Molecular Cell,2013,51(5):594-605.
[23] KUZMENKO A,OGUIENKO A,ESYUNINA D,et al.DNA targeting and interference by a bacterial Argonaute nuclease[J].Nature,2020,587(7835):632-637.
[24] KOOPAL B,POTOCNIK A,MUTTE S K,et al.Short prokaryotic Argonaute systems trigger cell death upon detection of invading DNA[J]. Cell,2022,185(9):1471-1486.
[25] LU X,XIAO J,WANG L,et al.The nuclease-associated short prokaryotic Argonaute system nonspecifically degrades DNA upon activation by target recognition[J].Nucleic Acids Research,2024,52(2):844-855.
[26] SWARTS D C,MAKAROVA K,WANG Y,et al.The evolutionary journey of Argonaute proteins[J].Nature Structural & Molecular Biology,2014,21(9):743-753.
[27] MAKAROVA K S,WOLF Y I,VAN DER OOST J,et al.Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements[J]. Biology Direct,2009,4:29.
[28] XING J,MA L,CHENG X,et al.Expression and functional analysis of the Argonaute protein of Thermus thermophilus (TtAgo) in E.coli BL21(DE3)[J].Biomolecules,2021,11(4):524.
[29] SWARTS D C,KOEHORST J J,WESTRA E R,et al.Effects of Argonaute on gene expression in Thermus thermophilus[J].PLoS One,2015,10(4):e0124880.
[30] WANG L,XIE X C,LV B,et al.A bacterial Argonaute with efficient DNA and RNA cleavage activity guided by small DNA and RNA[J].Cell Reports,2022,41(4):111533.
[31] SWARTS D C,JORE M M,WESTRA E R,et al.DNA-guided DNA interference by a prokaryotic Argonaute[J].Nature,2014,507(7491):258-261.
[32] SWARTS D C,HEGGE J W,HINOJO I,et al.Argonaute of the archaeon Pyrococcus furiosus is a DNA-guided nuclease that targets cognate DNA[J].Nucleic Acids Research,2015,43(10):5120-5129.
[33] SWARTS D C,SZCZEPANIAK M,SHENG G,et al.Autonomous generation and loading of DNA guides by bacterial Argonaute[J].Molecular Cell,2017,65(6):985-998.
[34] LIN Q,CAO Y,HAN G,et al.Correction to "programmable Clostridium perfringens Argonaute-based,One-pot assay for the multiplex detection of MiRNAs"[J].Analytical Chemistry,2023,95(45):16772-16773.
[35] WANG Z,FENG N,ZHOU Y,et al.Mesophilic Argonaute-mediated polydisperse droplet biosensor for amplification-free,One-pot,and multiplexed nucleic acid detection using deep learning[J].Analytical Chemistry,2024,96(5):2068-2077.
[36] WEN J,HAN M,FENG N,et al.A digital platform for One-pot signal enhanced foodborne pathogen detection based on mesophilic Argonaute-driven polydisperse microdroplet reactors and machine learning[J].Chemical Engineering Journal,2024,482:148845.
[37] LI X,DONG H,GUO X,et al.Mesophilic Argonaute-based isothermal detection of SARS-CoV-2[J]. Frontiers in Microbiology,2022,13:957977.
[38] SUN K,LIU Y,ZHAO W,et al.Prokaryotic Argonaute proteins:A new frontier in point-of-care viral diagnostics[J].International Journal of Molecular Sciences,2023,24(19):14987.
[39] LIN Q,HAN G,FANG X,et al.Programmable analysis of microRNAs by Thermus thermophilus Argonaute-assisted exponential isothermal amplification for multiplex detection (TEAM)[J].Analytical Chemistry,2022,94(32):11290-11297.
[40] FANG J,YUAN C,LUO X,et al.A Thermus thermophilus Argonaute-coupling exponential amplification assay for ultrarapid analysis of circulating tumor DNA[J].Talanta,2024,266:125034.
[41] ZHANG J,CHEN M,JIANG H,et al.Atom-modified gDNA enhances cleavage activity of TtAgo enabling ultra-sensitive nucleic acid testing[J].Advanced Science,2024,11(28):e2403120.
[42] KONG D,ZHANG S,GUO M,et al.Ultra-fast single-nucleotide-variation detection enabled by Argonaute-mediated transistor platform[J].Advanced Materials,2024,36(5):e2307366.
[43] JANG H,SONG J,KIM S,et al.ANCA:Artificial nucleic acid circuit with Argonaute protein for one-step isothermal detection of antibiotic-resistant bacteria[J].Nature Communications,2023,14(1):8033.
[44] WANG N,ZHANG Q,LIU Y,et al.Sample-in-answer-out centrifugal microfluidic chip reaction biosensor powered by Thermus thermophilus Argonaute (TtAgo) for rapid,highly sensitive and multiplexed molecular diagnostics of foodborne bacterial pathogens[J].Chemical Engineering Journal,2024,495:153434.
[45] XUN G,LIU Q,CHONG Y,et al.Argonaute with stepwise endonuclease activity promotes specific and multiplex nucleic acid detection[J].Bioresources and Bioprocessing,2021,8(1):46.
[46] WANG L,HE R,LV B,et al.Pyrococcus furiosus Argonaute coupled with modified ligase chain reaction for detection of SARS-CoV-2 and HPV[J].Talanta,2021,227:122154.
[47] HE R,WANG L,WANG F,et al.Combination of ultrashort PCR and Pyrococcus furiosus Argonaute for DNA detection[J].Analyst,2022,147(1):35-39.
[48] YE X,ZHOU H,GUO X,et al.Argonaute-integrated isothermal amplification for rapid,portable,multiplex detection of SARS-CoV-2 and Influenza viruses[J].Biosensors and Bioelectronics,2022,207:114169.
[49] 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].Biosensors and Bioelectronics,2024,244:115758.
[50] QIAO J,JIA J,PENG W,et al.Rapid,on-site and yes-or-no detection of Salmonella Typhimurium in foods using Argonaute-enabled assay[J].Sensors and Actuators B:Chemical,2024,404:135263.
[51] XUN G,LANE S T,PETROV V A,et al.A rapid,accurate,scalable,and portable testing system for COVID-19 diagnosis[J].Nature Communications,2021,12(1):2905.
[52] YE X,LI Z,FANG Z,et al.Thermal activation of Argonaute nuclease enables one-pot multiplex detection of viruses[J].Sensors and Actuators B:Chemical,2024,409:135587.
[53] SUN K,LIU Y,TANG Q,et al.CADLAB:A contamination-free,Argonaute-enhanced,dual detection of plant viruses via lateral flow assay biosensors system[J].Sensors and Actuators B:Chemical,2025,426:137045.
[54] WANG X,HU X,PAN S,et al.DNA and ATP synergistically triggered Argonaute-mediated sensor for the ultrasensitive detection of viable Salmonella without DNA extraction and amplification[J].Sensors and Actuators B:Chemical,2024,408:135543.
[55] ZHAO Y,YANG M,ZHOU C,et al.Establishment of a simple,sensitive,and specific ASFV detection method based on Pyrococcus furiosus Argonaute[J].Biosensors and Bioelectronics,2024,254:116230.
[56] SHI Y,TAN Z,LI W,et al.Enzyme-assisted endogenous guide DNA generation-mediated Pyrococcus furiosus Argonaute for Alicyclobacillus acidoterrestris detection[J].Journal of Agricultural and Food Chemistry,2024,72(2):1354-1360.
[57] LI Y,KOU J,HAN X,et al.Argonaute-triggered visual and rebuilding-free foodborne pathogenic bacteria detection[J].Journal of Hazardous Materials,2023,454:131485.
[58] LIU Q,GUO X,XUN G,et al.Argonaute integrated single-tube PCR system enables supersensitive detection of rare mutations[J].Nucleic Acids Research,2021,49(13):e75.
[59] 李明儒.基于LAMP-PfAgo技术检测猪胸膜肺炎放线杆菌的方法建立[D].合肥：安徽农业大学，2023.LI M R.Establishment of a method for detection of Actinobacilluspleuropneumoniae based on LAMP-PfAgo technology[D].Hefei:Anhui Agricultural University,2023.(in Chinese)
[60] ZHAO Y,ZHOU C,GUO B,et al.Pyrococcus furiosus Argonaute-mediated Porcine epidemic diarrhea virus detection[J].Applied Microbiology and Biotechnology,2024,108(1):137.
[61] ZHAO Y,ZHANG T,ZHOU C,et al.Pyrococcus furiosus Argonaute based detection assays for Porcine deltacoronavirus[J].ACS Synthetic Biology,2024,13(4):1323-1331.
[62] YU Z,SHI D,DONG Y,et al.Pyrococcus furiosus Argonaute combined with loop-mediated isothermal amplification for rapid,ultrasensitive,and visual detection of Fowl adenovirus serotype 4[J].Poultry Science,2024,103(7):103729.
[63] LIU Y,CHEN L,ZHANG Z,et al.Development and application of a novel recombinase polymerase amplification-Pyrococcus furiosus Argonaute system for rapid detection of Goose parvovirus[J].Poultry Science,2024,103(10):104141.
[64] GUO B,ZHAO Y,ZHOU C,et al.The detection of Salmonella in food based on PCR combined with Pyrococcus furiosus Argonaute[J].Microchemical Journal,2024,206:111311.
[65] 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].International Journal of Food Microbiology,2024,417:110697.
[66] LI Y,LIU Y,ZHANG Q,et al.An Argonaute-mediated bio-barcode bioassay for one-tube and on-site detection of Staphylococcus aureus[J].Sensors and Actuators B:Chemical,2024,410:135713.
[67] YU Z,SHAO Y,DONG Y,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,2024,207:116640.