环介导等温扩增技术在呼吸道病原体检测中的应用
Application of Loop-Mediated Isothermal Amplification Technology in the Detection of Respiratory Pathogens
DOI: 10.12677/acm.2025.15113084, PDF,    科研立项经费支持
作者: 张嘉睿, 王心如, 姜 爽, 杨雨婷, 章婷婷, 张 妍, 孟凡达*：山东第一医科大学(山东省医学科学院)临床与基础医学院(基础医学研究所)，山东 济南
关键词: 环介导等温扩增技术；呼吸道病毒；基因检测；Loop-Mediated Isothermal Amplification； Respiratory Virus； Genetic Detection
摘要: 早期检测对于人体呼吸道疾病的预防和控制有着重要作用。环介导等温扩增技术(LAMP)具有高特异性、高灵敏度的特点，在30 min内即可完成检测反应且成本低廉，因此广泛应用于呼吸道疾病的早期诊断中。多重LAMP (mLAMP)、逆转录LAMP (RT-LAMP)等的出现促进了该领域的范式改变，使LAMP的应用范围进一步扩展至RNA病毒、单管多病原体筛查和即时检测等。本文系统梳理了呼吸道病毒的病原特征，重点介绍了LAMP在人类呼吸道病毒识别中的应用，并探讨了引物设计原理、多种结果判读方法以及微流控集成技术等。最后，我们总结了LAMP技术从实验室研究向一线诊断转化所面临的实际问题和关键障碍，如标准化、临床验证和监管审批等。
Abstract: Early detection plays a pivotal role in the prevention and control of human respiratory diseases. Loop-mediated isothermal amplification (LAMP) is characterized by high specificity and sensitivity, completes the reaction within 30 min at low cost, and is therefore widely used for early diagnosis of respiratory infections. The emergence of multiplex LAMP (mLAMP), reverse-transcription LAMP (RT-LAMP), and related variants has shifted the paradigm, extending LAMP to RNA viruses, single-tube multi-pathogen screening, and point-of-care testing. Here we systematically outline the pathogenic features of respiratory viruses, highlight the applications of LAMP in identifying human respiratory viruses, and address primer-design principles, diverse read-out strategies, and microfluidic integration. Finally, we summarize the practical challenges and key barriers, such as standardization, clinical validation, and regulatory approval, that must be cleared before LAMP can transition from laboratory curiosity to front-line diagnostics.
文章引用：张嘉睿, 王心如, 姜爽, 杨雨婷, 章婷婷, 张妍, 孟凡达. 环介导等温扩增技术在呼吸道病原体检测中的应用[J]. 临床医学进展, 2025, 15(11): 188-200. https://doi.org/10.12677/acm.2025.15113084

参考文献

[1] Johnson, G.R., Morawska, L., Ristovski, Z.D., Hargreaves, M., Mengersen, K., Chao, C.Y.H., et al. (2011) Modality of Human Expired Aerosol Size Distributions. Journal of Aerosol Science, 42, 839-851. [Google Scholar] [CrossRef] 
[2] Monto, A.S. (2004) Occurrence of Respiratory Virus: Time, Place and Person. Pediatric Infectious Disease Journal, 23, S58-S64. [Google Scholar] [CrossRef] [PubMed]
[3] 黄斐, 郭玮. 呼吸道病毒快速检测的临床应用现状与挑战[J]. 中国临床医学, 2024, 31(5): 826-831.
[4] Notomi, T., Okayama, H., Masubuchi, H., et al. (2000) Loop-Mediated Isothermal Amplification of DNA. Nucleic Acids Research, 28, e63. [Google Scholar] [CrossRef] [PubMed]
[5] 游淑珠, 王小玉, 冯家望, 等. 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.
[6] 刘培海, 白庆华, 王凯, 等. 环介导等温扩增技术及扩增产物分析方法的比较[J]. 食品研究与开发, 2024, 45(11): 209-218.
[7] Tanner, N.A., Zhang, Y. and Evans, T.C. (2015) Visual Detection of Isothermal Nucleic Acid Amplification Using pH-Sensitive Dyes. BioTechniques, 58, 59-68. [Google Scholar] [CrossRef] [PubMed]
[8] Adams, E.R., Schoone, G.J., Ageed, A.F., Safi, S.E. and Schallig, H.D.F.H. (2010) Development of a Reverse Transcriptase Loop-Mediated Isothermal Amplification (LAMP) Assay for the Sensitive Detection of Leishmania Parasites in Clinical Samples. American Journal of Tropical Medicine and Hygiene, 82, 591-596. [Google Scholar] [CrossRef] [PubMed]
[9] Neshani, A., Zare, H., Sadeghian, H., Safdari, H., Riahi-Zanjani, B. and Aryan, E. (2023) A Comparative Study on Visual Detection of Mycobacterium Tuberculosis by Closed Tube Loop-Mediated Isothermal Amplification: Shedding Light on the Use of Eriochrome Black T. Diagnostics, 13, Article No. 155. [Google Scholar] [CrossRef] [PubMed]
[10] Song, Q., Zhu, R., Sun, Y., Zhao, L., Wang, F., Deng, J., et al. (2014) Identification of Human Metapneumovirus Genotypes a and B from Clinical Specimens by Reverse Transcription Loop-Mediated Isothermal Amplification. Journal of Virological Methods, 196, 133-138. [Google Scholar] [CrossRef] [PubMed]
[11] Thoraneenitiyan, N., Choopara, I., Nuanualsuwan, S., Kokpol, S. and Somboonna, N. (2022) Rapid Visual Candidatus Liberibacter Asiaticus Detection (Citrus Greening Disease) Using Simple Alkaline Heat DNA Lysis Followed by Loop-Mediated Isothermal Amplification Coupled Hydroxynaphthol Blue (AL-LAMP-HNB) for Potential Local Use. PLOS ONE, 17, e0276740. [Google Scholar] [CrossRef] [PubMed]
[12] Nawattanapaiboon, K., Pasomsub, E., Prombun, P., Wongbunmak, A., Jenjitwanich, A., Mahasupachai, P., et al. (2021) Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) as a Visual Diagnostic Platform for the Detection of the Emerging Coronavirus SARS-CoV-2. The Analyst, 146, 471-477. [Google Scholar] [CrossRef] [PubMed]
[13] Lai, M.Y., Ooi, C.H., Jaimin, J.J. and Lau, Y.L. (2020) Evaluation of Warmstart Colorimetric Loop-Mediated Isothermal Amplification Assay for Diagnosis of Malaria. The American Journal of Tropical Medicine and Hygiene, 102, 1370-1372. [Google Scholar] [CrossRef] [PubMed]
[14] Saifuddin, S.A., Rashid, R., Nor Azmi, N.J. and Mohamad, S. (2024) Colorimetric Strategies Applicable for Loop-Mediated Isothermal Amplification. Journal of Microbiological Methods, 223, Article ID: 106981. [Google Scholar] [CrossRef] [PubMed]
[15] Chen, H., Li, Y., Zhang, T., Bi, Y., Shu, M., Zhong, C., et al. (2021) A Novel Real-Time Loop-Mediated Isothermal Amplification Combined with Immunomagnetic Beads Separation and Ethidium Bromide Monoazide Treatment for Rapid and Ultrasensitive Detection of Viable Escherichia coli O157:H7 in Milk. Food Analytical Methods, 14, 944-956. [Google Scholar] [CrossRef] 
[16] Krapoth, T.C., Henle, G.S., Avdyli, M., Bektić, B., Schwarzkopf, K.M., Bešić, L., et al. (2024) Wanted: Dead or Alive Cells with Propidium Iodide Staining in Liver Tissue. International Journal of Molecular Sciences, 25, Article No. 13521. [Google Scholar] [CrossRef] [PubMed]
[17] Gachugia, J., Chebore, W., Otieno, K., Ngugi, C.W., Godana, A. and Kariuki, S. (2020) Evaluation of the Colorimetric Malachite Green Loop-Mediated Isothermal Amplification (MG-LAMP) Assay for the Detection of Malaria Species at Two Different Health Facilities in a Malaria Endemic Area of Western Kenya. Malaria Journal, 19, Article No. 329. [Google Scholar] [CrossRef] [PubMed]
[18] Soroka, M., Wasowicz, B. and Rymaszewska, A. (2021) Loop-Mediated Isothermal Amplification (LAMP): The Better Sibling of PCR? Cells, 10, 1931. [Google Scholar] [CrossRef] [PubMed]
[19] 尹小毛, 希伦, 王治伟. 应用环介导等温扩增技术快速检测血培养阳性瓶中的七种常见病原菌[J]. 广州医科大学学报, 2021, 49(2): 89-93.
[20] 王一波, 孙泓希, 史普想, 等. 环介导等温扩增技术在农业病害检测中的应用综述[J]. 江苏农业科学, 2024, 52(7): 17-24.
[21] 张杨, 郭培全, 岳营, 等. 一起季节性H3N2流感引起的学校聚集疫情全基因特征分析[J]. 中国口岸科学技术, 2022, 4(11): 47-53.
[22] 苏翠, 杨凯翔, 王君, 马萍, 刘新利, 王学良. 2011-2021年铜川市流行性感冒的流行趋势及病原学特点[J]. 医学动物防制, 2024, 40(2): 142-146.
[23] 刘瑞, 闫敏敏, 魏嵘, 张进, 汪卓赟, 郑凌. 12例人感染H7N9禽流感病例临床特征分析[J]. 医学动物防制, 2023, 39(10): 971-975.
[24] Wibawa, H., Wibowo, P.E., Supriyadi, A., Lestari, L., Silaban, J., Fuadi, A.A., et al. (2024) Highly Pathogenic Avian Influenza A(H5N1) Virus Clade 2.3.4.4b in Domestic Ducks, Indonesia, 2022. Emerging Infectious Diseases, 30, 586-590. [Google Scholar] [CrossRef] [PubMed]
[25] Plaza, P.I., Gamarra-Toledo, V., Euguí, J.R. and Lambertucci, S.A. (2024) Recent Changes in Patterns of Mammal Infection with Highly Pathogenic Avian Influenza A (H5N1) Virus Worldwide. Emerging Infectious Diseases, 30, 444-452. [Google Scholar] [CrossRef] [PubMed]
[26] Liu, X., Lin, X., Hong, H., Wang, J., Tao, Y., Huai, Y., et al. (2024) Polysaccharide from Atractylodes Macrocephala Koidz Binding with Zinc Oxide Nanoparticles as a Novel Mucosal Immune Adjuvant for H9N2 Inactivated Vaccine. International Journal of Molecular Sciences, 25, Article No. 2132. [Google Scholar] [CrossRef] [PubMed]
[27] Hohensee, L., Scheibner, D., Schäfer, A., Shelton, H., Mettenleiter, T.C., Breithaupt, A., et al. (2024) The Role of PB1-F2 in Adaptation of High Pathogenicity Avian Influenza Virus H7N7 in Chickens. Veterinary Research, 55, Article No. 5. [Google Scholar] [CrossRef] [PubMed]
[28] Yang, L., Fan, M., Wang, Y., Sun, X. and Zhu, H. (2024) Effect of Avian Influenza Scare on Transmission of Zoonotic Avian Influenza: A Case Study of Influenza a (H7N9). Mathematical Biosciences, 367, Article ID: 109125. [Google Scholar] [CrossRef] [PubMed]
[29] Lyashko, A.V., Timofeeva, T.A., Rudneva, I.A., Lomakina, N.F., Treshchalina, A.A., Gambaryan, A.S., et al. (2023) Antigenic Architecture of the H7N2 Influenza Virus Hemagglutinin Belonging to the North American Lineage. International Journal of Molecular Sciences, 25, Article No. 212. [Google Scholar] [CrossRef] [PubMed]
[30] 杨海玉, 朱伯林, 许佳, 胡道来, 杭佳, 张小梅. A型流感病毒环介导等温扩增快速检测体系的建立与验证[J]. 医学动物防制, 2025, 41(8): 830-834.
[31] Ahn, S.J., Baek, Y.H., Lloren, K.K.S., Choi, W., Jeong, J.H., Antigua, K.J.C., et al. (2020) Correction to: Rapid and Simple Colorimetric Detection of Multiple Influenza Viruses Infecting Humans Using a Reverse Transcriptional Loopmediated Isothermal Amplification (RT-LAMP) Diagnostic Platform. BMC Infectious Diseases, 20, Article No. 965. [Google Scholar] [CrossRef] [PubMed]
[32] Hatano, B., Goto, M., Fukumoto, H., Obara, T., Maki, T., Suzuki, G., et al. (2011) Mobile and Accurate Detection System for Infection by the 2009 Pandemic Influenza a (H1N1) Virus with a Pocket-Warmer Reverse-Transcriptase Loop-Mediated Isothermal Amplification. Journal of Medical Virology, 83, 568-573. [Google Scholar] [CrossRef] [PubMed]
[33] Zhang, S., Luo, Y., Huang, D., Fan, H., Lu, Q., Wo, Y., et al. (2015) Fatal Pneumonia Cases Caused by Human Adenovirus 55 in Immunocompetent Adults. Infectious Diseases, 48, 40-47. [Google Scholar] [CrossRef] [PubMed]
[34] Matthes‐Martin, S., Feuchtinger, T., Shaw, P.J., Engelhard, D., Hirsch, H.H., Cordonnier, C., et al. (2012) European Guidelines for Diagnosis and Treatment of Adenovirus Infection in Leukemia and Stem Cell Transplantation: Summary of Ecil‐4 (2011). Transplant Infectious Disease, 14, 555-563. [Google Scholar] [CrossRef] [PubMed]
[35] Koryukov, M.A., Oscorbin, I.P., Novikova, L.M., Gordukova, M.A., Turina, I.E., Galeeva, E.V., et al. (2024) A Novel Multiplex LAMP Assay for the Detection of Respiratory Human Adenoviruses. International Journal of Molecular Sciences, 25, Article No. 7215. [Google Scholar] [CrossRef] [PubMed]
[36] Shuryaeva, A.K., Malova, T.V., Tolokonceva, A.A., Karceka, S.A., Gordukova, M.A., Davydova, E.E., et al. (2022) Development and Application of LAMP Assays for the Detection of Enteric Adenoviruses in Feces. Microbiology Spectrum, 10, e00516-22. [Google Scholar] [CrossRef] [PubMed]
[37] Hong, L., Li, J., Lv, J., Chao, S., Xu, Y., Zou, D., et al. (2021) Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay for Clinical Diagnosis of Respiratory Human Adenoviruses Emergent in China. Diagnostic Microbiology and Infectious Disease, 101, Article ID: 115401. [Google Scholar] [CrossRef] [PubMed]
[38] Cui, J., Li, F. and Shi, Z. (2018) Origin and Evolution of Pathogenic Coronaviruses. Nature Reviews Microbiology, 17, 181-192. [Google Scholar] [CrossRef] [PubMed]
[39] Shen, M., Zhou, Y., Ye, J., Abdullah AL-maskri, A.A., Kang, Y., Zeng, S., et al. (2020) Recent Advances and Perspectives of Nucleic Acid Detection for Coronavirus. Journal of Pharmaceutical Analysis, 10, 97-101. [Google Scholar] [CrossRef] [PubMed]
[40] Kashir, J. and Yaqinuddin, A. (2020) Loop Mediated Isothermal Amplification (LAMP) Assays as a Rapid Diagnostic for Covid-19. Medical Hypotheses, 141, Article ID: 109786. [Google Scholar] [CrossRef] [PubMed]
[41] Dao Thi, V.L., Herbst, K., Boerner, K., Meurer, M., Kremer, L.P., Kirrmaier, D., et al. (2020) A Colorimetric RT-LAMP Assay and LAMP-Sequencing for Detecting Sars-Cov-2 RNA in Clinical Samples. Science Translational Medicine, 12, Article No. 556. [Google Scholar] [CrossRef] [PubMed]
[42] El-Tholoth, M., Mauk, M.G., Anis, E. and Bau, H.H. (2020) A Closed-Tube, Single-Step, Real Time, Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Infectious Bronchitis Virus Detection in Chickens. Journal of Virological Methods, 284, Article ID: 113940. [Google Scholar] [CrossRef] [PubMed]
[43] Lalli, M.A., Langmade, J.S., Chen, X., Fronick, C.C., Sawyer, C.S., Burcea, L.C., et al. (2020) Rapid and Extraction-Free Detection of Sars-Cov-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification. Clinical Chemistry, 67, 415-424. [Google Scholar] [CrossRef] [PubMed]
[44] Carstens, E.B. and Ball, L.A. (2009) Ratification Vote on Taxonomic Proposals to the International Committee on Taxonomy of Viruses (2008). Archives of Virology, 154, 1181-1188. [Google Scholar] [CrossRef] [PubMed]
[45] Khetsuriani, N., Lu, X., Teague, W.G., Kazerouni, N., Anderson, L.J. and Erdman, D.D. (2008) Novel Human Rhinoviruses and Exacerbation of Asthma in Children. Emerging Infectious Diseases, 14, 1793-1796. [Google Scholar] [CrossRef] [PubMed]
[46] Miller, E.K., Khuri-Bulos, N., Williams, J.V., Shehabi, A.A., Faouri, S., Al Jundi, I., et al. (2009) Human Rhinovirus C Associated with Wheezing in Hospitalised Children in the Middle East. Journal of Clinical Virology, 46, 85-89. [Google Scholar] [CrossRef] [PubMed]
[47] Lamson, D., Renwick, N., Kapoor, V., Liu, Z., Palacios, G., Ju, J., et al. (2006) Masstag Polymerase‐Chain‐Reaction Detection of Respiratory Pathogens, Including a New Rhinovirus Genotype, That Caused Influenza‐Like Illness in New York State during 2004-2005. The Journal of Infectious Diseases, 194, 1398-1402. [Google Scholar] [CrossRef] [PubMed]
[48] Wong, Y., Tee, K.K. and Chee, H. (2024) Diagnostic Potential of Rhinovirus C Using Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP). International Microbiology, 28, 1291-1303. [Google Scholar] [CrossRef] [PubMed]
[49] Wang, X., Zhang, Q., Zhang, F., Ma, F., Zheng, W., Zhao, Z., et al. (2012) Visual Detection of the Human Metapneumovirus Using Reverse Transcription Loop-Mediated Isothermal Amplification with Hydroxynaphthol Blue Dye. Virology Journal, 9, Article No. 138. [Google Scholar] [CrossRef] [PubMed]
[50] Lau, Y.L., Abdullah, M.L. and Lai, M.Y. (2025) Rapid Detection of Zoonotic Malaria Using Room-Temperature Stable and Ready-to-Use Colorimetric LAMP Reagents. Acta Tropica, 267, Article ID: 107678. [Google Scholar] [CrossRef] [PubMed]
[51] Lee, P., Wong, Y., Othman, S. and Chee, H. (2021) Room-Temperature Stable Loop-Mediated Isothermal Amplification (LAMP) Reagents to Detect Leptospiral DNA. Asian Biomedicine, 15, 183-189. [Google Scholar] [CrossRef] [PubMed]
[52] Divakar, S., Dhanalakshmi, H., Sandeep, N., Isloor, S., Rashmi, R., Ananda, K., et al. (2024) Dry LAMP: A Point of Care Diagnostics for Diagnosis of Bovine Tropical Theileriosis. Journal of Vector Borne Diseases, 61, 357-363. [Google Scholar] [CrossRef] [PubMed]
[53] 崔玉玲, 于浩, 张哲, 等. 冻干技术在法医DNA检验中的研究进展及应用[J]. 中国法医学杂志, 2025, 40(3): 343-347.
[54] Shi, L.L., Pang, Z.R., Yu, J.H., et al. (2025) Development of a Portable Multi-Step Microfluidic Device for Point-of-Care Nucleic Acid Diagnostics. Analytica Chimica Acta, 1336, Article ID: 343518. [Google Scholar] [CrossRef] [PubMed]
[55] Tang, D., Li, P., Qi, S., Wu, Q., Yu, R., Liu, M., et al. (2025) Point-of-Care Analysis for Foodborne Pathogens in Food Samples Based on a Fully Enclosed Microfluidic Chip Cartridge. Lab on a Chip, 25, 3537-3548. [Google Scholar] [CrossRef] [PubMed]
[56] Lin, Y., Hung, Y., Chang, W. and Chiou, C. (2024) Integrated Droplet-Based Digital Loop-Mediated Isothermal Amplification Microfluidic Chip with Droplet Generation, Incubation, and Continuous Fluorescence Detection. Biosensors, 14, Article No. 334. [Google Scholar] [CrossRef] [PubMed]
[57] Hodgson, J., Zuckerman, M. and Smith, M. (2007) Development of a Novel Internal Control for a Real-Time PCR for HSV DNA Types 1 and 2. Journal of Clinical Virology, 38, 217-220. [Google Scholar] [CrossRef] [PubMed]
[58] Burggraf, S. and Olgemöller, B. (2004) Simple Technique for Internal Control of Real-Time Amplification Assays. Clinical Chemistry, 50, 819-825. [Google Scholar] [CrossRef] [PubMed]
[59] Li, Y., Ma, L., Duan, S., Li, M. and Chen, J. (2020) Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Streptococcus Pneumoniae Isolates in Clinical Sputum Samples. Indian Journal of Pharmaceutical Sciences, 82, 64-68. [Google Scholar] [CrossRef] 
[60] Serra-Casas, E., Manrique, P., Ding, X.C., Carrasco-Escobar, G., Alava, F., Gave, A., et al. (2017) Loop-Mediated Isothermal DNA Amplification for Asymptomatic Malaria Detection in Challenging Field Settings: Technical Performance and Pilot Implementation in the Peruvian Amazon. PLOS ONE, 12, e0185742. [Google Scholar] [CrossRef] [PubMed]
[61] Han, H.J., Jung, S.J., Oh, M.J. and Kim, D.H. (2011) Rapid and Sensitive Detection of Streptococcus iniae by Loop-Mediated Isothermal Amplification (LAMP). Journal of Fish Diseases, 34, 395-398. [Google Scholar] [CrossRef] [PubMed]
[62] Yao, Y.P., Li, S.S., Cao, J.Q., et al. (2018) Development of Small Molecule Biosensors by Coupling the Recognition of the Bacterial Allosteric Transcription Factor with Isothermal Strand Displacement Amplification. Chemical Communications, 54, 4774-4777. [Google Scholar] [CrossRef] [PubMed]
[63] Yang, Y.Q., Liu, R.Y., Xie, H.Q., et al. (2013) Advances in Nanopore Sequencing Technology. Journal of Nanoscience and Nanotechnology, 13, 4521-4538. [Google Scholar] [CrossRef] [PubMed]
[64] Imai, K., Tarumoto, N., Misawa, K., Runtuwene, L.R., Sakai, J., Hayashida, K., et al. (2017) A Novel Diagnostic Method for Malaria Using Loop-Mediated Isothermal Amplification (LAMP) and Minion™ Nanopore Sequencer. BMC Infectious Diseases, 17, Article No. 621. [Google Scholar] [CrossRef] [PubMed]
[65] Imai, K., Tarumoto, N., Runtuwene, L.R., Sakai, J., Hayashida, K., Eshita, Y., et al. (2018) An Innovative Diagnostic Technology for the Codon Mutation C580Y in Kelch13 of Plasmodium falciparum with Minion Nanopore Sequencer. Malaria Journal, 17, Article No. 217. [Google Scholar] [CrossRef] [PubMed]
[66] Tang, Z., Choi, G., Nouri, R. and Guan, W. (2019) Loop-Mediated Isothermal Amplification-Coupled Glass Nanopore Counting toward Sensitive and Specific Nucleic Acid Testing. Nano Letters, 19, 7927-7934. [Google Scholar] [CrossRef] [PubMed]

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