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急性呼吸道感染是全球范围内传染性疾病发病和死亡的主要原因之一，其主要病原体包括病毒、细菌、真菌、衣原体和支原体等。由于不同病原体导致的呼吸道感染常呈现相似的临床症状，仅依靠临床表现难以实现准确鉴别诊断。因此，建立快速、准确的呼吸道病原体检测技术对于临床及时治疗、疫情防控及传播阻断至关重要。目前，实时荧光PCR因其具备快速、准确、单管封闭的优势成为应用较为广泛的检测方法。但是，该方法的单管检测通量受限于PCR仪荧光通道数目，一管通常只能同步检测4～6种病原体。随着多色探针熔解曲线分析技术的出现，这一技术克服了荧光通道的限制，在保留荧光PCR技术优势的同时，通过多荧光通道和熔解温度（Melting temperature, Tm）值的差异分析，即可实现单管检测覆盖整个症候群的病原谱。因此本文系统综述熔解曲线技术在呼吸道病原体检测中的研究进展。
Abstract：Acute respiratory tract infections are among the leading causes of morbidity and mortality from infectious diseases worldwide. These infections are primarily caused by viruses, bacteria, fungi, Chlamydia, and Mycoplasma. Because infections caused by different pathogens often present with similar clinical symptoms, accurate differential diagnosis based solely on clinical manifestations is challenging. Therefore,the establishment of rapid and precise identification of respiratory pathogens is critical for timely clinical intervention and for preventing outbreaks and further transmission. Currently, real-time fluorescence PCR is one of the most widely used diagnostic techniques, offering high sensitivity, specificity, and a closed-tube format. However, its multiplexing capacity is limited by the number of available fluorescence detection channels, typically allowing the simultaneous detection of only 4-6 pathogens per reaction. The advent of multicolor probe-based melting curve analysis has overcome this limitation. This technique retains the benefits of real-time PCR while expanding the number of detectable targets in a single tube by utilizing multiple fluorescence channels and distinct melting temperatures(Tm). As a result, it enables syndrome-based detection in a single reaction. This review summarizes recent advances in the application of melting curve analysis for the detection of respiratory pathogens.
[1]Cillóniz C,Pericàs JM,Rojas JR,et al.Severe infections due to respiratory viruses[J].Semin Respir Crit Care Med,2022,43(1):60-74.DOI:10.1055/s-0041-1740982.
[2]Di Gennaro F,Gualano G,Palmieri F.Antibiotics and infectious respiratory diseases[J].Antibiotics(Basel),2022,11(7):859.DOI:10.3390/antibiotics11070859.
[3]Detelich JF,Kempker JA.Respiratory infections[J].Clin Chest Med,2023,44(3):509-517.DOI:10.1016/j.ccm.2023.03.007.
[4]马鹏程,陈愉.呼吸道病原体检测方法研究进展[J].中国感染与化疗杂志,2024,24(1):118-122.DOI:10.16718/j.1009-7708.2024.01.020.
[5]王红.非典型呼吸道病原体检测方法研究进展[J].内科,2015,10(4):560-562.DOI:10.16121/j.cnki.cn45-1347/r.2015.04.49.
[6]Walter JM,Wunderink RG.Testing for respiratory viruses in adults with severe lower respiratory infection[J].Chest,2018,154(5):1213-1222.DOI:10.1016/j.chest.2018.06.003.
[7]张春雷.病原微生物检测技术研究进展[J].生物技术进展,2024,14(02):189-195.DOI:10.19586/j.2095-2341.2023.0120.
[8]柳婷婷,徐靖淋,郭子槊,等.人类呼吸道病毒核酸检测技术研究进展[J].病毒学报,2022,38(04):965-973.DOI:10.13242/j.cnki.bingduxuebao.004157.
[9]梁子英,刘芳.实时荧光定量PCR技术及其应用研究进展[J].现代农业科技,2020(6):1-3,8.
[10]张玉林,曹彬.呼吸道感染的病原学检测及其临床应用[J].中华结核和呼吸杂志,2024,47(05):497-500.DOI:10.3760/cma.j.cn112147-20240227-00109.
[11]杨潇雨,牛培华,田晓灵,等.基于RT-qPCR方法对新冠病毒4种基因型鉴定的可行性研究[J].病毒学报,2024,40(2):266-273.DOI:10.13242/j.cnki.bingduxuebao.004478.
[12]Jang M,Kim S,Song J,et al.Rapid and simple detection of influenza virus via isothermal amplification lateral flow assay[J].Anal Bioanal Chem,2022,414(16):4685-4696.DOI:10.1007/s00216-022-04090-8.
[13]杨森,李桂梅,滕新栋.等温扩增技术在呼吸道病毒检测方面的研究进展[J].中国人兽共患病学报,2024,40(3):264-269.DOI:10.3969/j.issn.1002-2694.2024.00.033
[14]沈方园,葛萧,张晓宇,等.重组酶聚合酶扩增技术的研究进展[J].微生物学通报,2024,51(5):1495-1511.DOI:10.13344/j.microbiol.china.230558.
[15]施奕,徐昌平,余蓓蓓,等.重组酶聚合酶扩增技术研究进展[J].病毒学报,2020,36(03):522-532.DOI:10.13242/j.cnki.bingduxuebao.003708.
[16]陈静,牛培华,谭文杰.呼吸道感染样本病毒宏基因组学技术及其应用进展[J].病毒学报,2020,36(6):1193-1200.DOI:10.13242/j.cnki.bingduxuebao.003772.
[17]Xue L,Zhu J,Lei K,et al.Evaluation of the FPMC respiratory panel for detection of respiratory tract pathogens in nasopharyngeal swab and sputum specimens[J].Virol J,2024,21(1):156.DOI:10.1186/s12985-024-02430-x.
[18]张换敬.熔解曲线分析技术用于基因型的研究[D].厦门大学,2011.DOI:http://dspace.xmu.edu.cn:8080/dspace/handle/2288/47395.
[19]Ririe KM,Rasmussen RP,Wittwer CT.Product differentiation by analysis of DNA melting curves during the polymerase chain reaction[J].Anal Biochem,1997,245(2):154-160.DOI:10.1006/abio.1996.9916.
[20]Wittwer CT,Reed GH,Gundry CN,et al.Highresolution genotyping by amplicon melting analysis using LCGreen[J].Clin Chem,2003,49(6):853-860.DOI:10.1373/49.6.853.
[21]Wittwer CT,Hemmert AC,Kent JO,et al.DNA melting analysis[J].Mol Aspects Med,2024,97:101268.DOI:10.1016/j.mam.2024.101268.
[22]张亚旭,刘紫烟.荧光PCR扩增相关技术[J].中国生物化学与分子生物学报,2021,37(7):890-899.DOI:10.13865/j.cnki.cjbmb.2021.01.1480.
[23]Druml B,Cichna-Markl M.High resolution melting(HRM)analysis of DNA—its role and potential in food analysis[J].Food Chem,2014,158:245-254.DOI:10.1016/j.foodchem.2014.02.111.
[24]Erali M,Voelkerding KV,Wittwer CT.High resolution melting applications for clinical laboratory medicine[J].Exp Mol Pathol,2008,85(1):50-58.DOI:10.1016/j.yexmp.2008.03.012.
[25]Eischeid AC.SYTO dyes and EvaGreen outperform SYBR Green in real-time PCR[J].BMC Res Notes,2011,4:263.DOI:10.1186/1756-0500-4-263.
[26]顾建强.高分辨熔解曲线基因分型技术及其在水稻分子育种中的应用[J].福建农业科技,2019,50(10):67-70.DOI:10.13651/j.cnki.fjnykj.2019.10.014.
[27]Liu Z,Wang F,Yuan L,et al.Development of a SYBR-Green I quantitative PCR assay for the detection and genotyping of different hantaviruses[J].Int J Mol Med,2016,38(3):951-960.DOI:10.3892/ijmm.2016.2678.
[28]Tsakogiannis D,Papacharalampous M,Toska E,et al.Duplex Real-time PCR assay and SYBR green I melting curve analysis for molecular identification of HPV genotypes 16,18,31,35,51 and 66[J].Mol Cell Probes,2015,29(1):13-18.DOI:10.1016/j.mcp.2014.09.003.
[29]Zaghloul H,El Morsi AA,Soweha HE,et al.A simple real-time polymerase chain reaction assay using SYBR Green for hepatitis C virus genotyping[J].Arch Virol,2017,162(1):57-61.DOI:10.1007/s00705-016-3059-5.
[30]Wittwer CT.High-resolution DNA melting analysis:advancements and limitations[J].Hum Mutat,2009,30(6):857-859.DOI:10.1002/humu.20951.
[31]邱婷,毛国斌,徐秦峰.核酸扩增实时检测荧光染料的研究进展[J].分析科学学报,2023,39(4):479-486.DOI:10.13526/j.issn.1006-6144.2023.04.017.
[32]胡婷婷,张云龙,邹秉杰.单管闭管多重PCR检测技术的发展[J/OL].中国生物化学与分子生物学报,1-19[2025-05-13].https://doi.org/10.13865/j.cnki.cjbmb.2025.04.1101.
[33]高扬,董泽丰,雅雪蓉,等.病原体多重PCR检测技术研究进展[J].江苏预防医学,2024,35(2):243-247.DOI:10.13668/j.issn.1006-9070.2024.02.030.
[34]钟泽澄,王进,张师音.多重PCR技术研究进展[J].生物工程学报,2020,36(2):171-179.DOI:10.13345/j.cjb.190206.
[35]Herrmann MG,Dobrowolski SF,Wittwer CT.Rapid beta-globin genotyping by multiplexing probe melting temperature and color[J].Clin Chem,2000,46(3):425-428.
[36]Murugesan G,Aboudola S,Szpurka H,et al.Identification of the JAK2 V617F mutation in chronic myeloproliferative disorders using FRET probes and melting curve analysis[J].Am J Clin Pathol,2006,125(4):625-633.DOI:10.1309/TK0X-L917-XK2VLRPQ.
[37]高子珩,邹譞,周宜,等.荧光核酸适配体:核酸酶学分析新机遇[J].生物化学与生物物理进展,2023,50(05):1042-1068.DOI:10.16476/j.pibb.2023.0128.
[38]Navarro E,Serrano-Heras G,Casta?o MJ,et al.Realtime PCR detection chemistry[J].Clin Chim Acta,2015,439:231-250.DOI:10.1016/j.cca.2014.10.017.
[39]谢烨,张思娜,陈代安,等.高灵敏核酸探针在生化分析中的应用研究进展[J].分析试验室,2022,41(04):449-457.DOI:10.13595/j.cnki.issn1000-0720.2021.042601.
[40]梁梦洁,李永鑫,张新.甲型流感病毒分子诊断技术的研究进展[J].国际检验医学杂志,2024,45(13):1640-1644,1650.
[41]张若楠,于雷,王钊,等.多重荧光定量PCR在动物检测中的应用[J].山东畜牧兽医,2020,41(4):75-78.
[42]倪萍,徐娟,胡海涛,等.荧光定量PCR熔解曲线法检测真菌核酸的临床应用价值评估[J].临床检验杂志,2024,42(09):641-647.DOI:10.13602/j.cnki.jcls.2024.09.01.
[43]Wan Z,Zhang YN,He Z,et al.A melting curve-based multiplex RT-qPCR assay for simultaneous detection of four human coronaviruses[J].Int J Mol Sci,2016,17(11):1880.DOI:10.3390/ijms17111880.
[44]薛晓宁,王江凤,张娟,等.熔解曲线法检测和鉴别呼吸道合胞病毒亚型[J].中国国境卫生检疫杂志,2023,46(6):527-529,535.DOI:10.16408/j.1004-9770.2023.06.005.
[45]Gao P,Fan Y,Kong X,et al.A novel assay based on DNA melting temperature for multiplexed identification of SARS-CoV-2 and influenza A/B viruses[J].Front Microbiol,2023,14:1249085.DOI:10.3389/fmicb.2023.1249085.
[46]Ong DSY,Koeleman JGM,Vaessen N,et al.Rapid screening method for the detection of SARS-CoV-2variants of concern[J].J Clin Virol,2021,141:104903.DOI:10.1016/j.jcv.2021.104903.
[47]Tavares ER,de Lima TF,Bartolomeu-Gon?alves G,et al.Development of a melting-curve-based multiplex real-time PCR assay for the simultaneous detection of viruses causing respiratory infection[J].Microorganisms,2023,11(11):2692.DOI:10.3390/microorganism s11112692.
[48]Liu P,Lu L,Xu M,et al.A novel multiplex PCR for virus detection by melting curve analysis[J].J Virol Methods,2018,262:56-60.DOI:10.1016/j.jviromet.2018.09.010.
[49]Liao S,Wang L,Ji X,et al.Simultaneous detection of 15 respiratory pathogens with a fluorescence probe melting curve analysis-based multiplex real-time PCR assay[J].Int J Mol Epidemiol Genet,2019,10(2):29-37.
[50]Hu L,Han B,Tong Q,et al.Detection of eight respiratory bacterial pathogens based on multiplex realtime PCR with fluorescence melting curve analysis[J].Can J Infect Dis Med Microbiol,2020,2020:2697230.DOI:10.1155/2020/2697230.
[51]Tan D,Han J,Sun Q,et al.Application of multiplex fluorescence polymerase chain reaction for detecting pathogenic bacteria in sputum samples from patients with lower respiratory tract infection[J].Infect Drug Resist,2023,16:6999-7005.DOI:10.2147/IDR.S431425.
[52]Ghorbani J,Hashemi FB,Jabalameli F,et al.Multiplex detection of five common respiratory pathogens from broncho alveolar lavages using high resolution melting curve analysis[J].BMC Microbiol,2022,22(1):141.DOI:10.1186/s12866-022-02558-2.
[53]Dung TTN,Phat VV,Vinh C,et al.Development and validation of multiplex real-time PCR for simultaneous detection of six bacterial pathogens causing lower respiratory tract infections and antimicrobial resistance genes[J].BMC Infect Dis,2024,24(1):164.DOI:10.118 6/s12879-024-09028-2.
[54]刘丽琴,张海燕,王捷.高分辨率熔解曲线分析技术及其应用进展[J].现代生物医学进展,2011(S2):3.DOI:10.3969/j.issn.1672-8270.2010.08.022.
[55]潘登.高分辨率熔解曲线分析技术(HRM)系统的建立及其在基因多态性和突变检测方面的应用[D].复旦大学,2013.DOI:10.7666/d.Y2701928.
[56]He P,Wang H,Luo J,et al.A real-time PCR with melting curve analysis for molecular typing of Vibrio parahaemolyticus[J].Curr Microbiol,2018,75(9):1206-1213.DOI:10.1007/s00284-018-1511-3.
[57]Yu Y,Zhang J,Zhan Y,et al.A novel method for detecting nine hotspot mutations of deafness genes in one tube[J].Sci Rep,2024,14(1):454.DOI:10.1038/s41598-023-50928-1.
[58]Hou X,Geng Y,Dai R,et al.Rapid identification of four Fusarium spp.complex by high-resolution melting curve analysis and their antifungal susceptibility profiles[J].Mycopathologia,2022,187(4):345-354.DOI:10.1007/s11046-022-00635-8.
[59]Ju M,Bai X,Zhang T,et al.Mutation spectrum of COL1A1/COL1A2 screening by high-resolution melting analysis of Chinese patients with osteogenesis imperfecta[J].J Bone Miner Metab,2020,38(2):188-197.DOI:10.1007/s00774-019-01039-3.
[60]张卫萍,阿地来·吐尔逊,冯巍,等.HRM技术在非人源性法医物证检测中的应用[J].中国法医学杂志,2022(3):243-245,250.DOI:10.13618/j.issn.1001-5728.2022.03.006.
基本信息:
DOI：10.13242/j.cnki.bingduxuebao.250113
中图分类号:R446.5;R56
引用信息:
[1]郑金凤,韩桃利,陈昱达,等.熔解曲线技术在呼吸道病原体快速检测的研究进展[J].病毒学报,2025,41(05):1565-1572.DOI:10.13242/j.cnki.bingduxuebao.250113.
基金信息:
首都卫生发展科研专项项目(项目号：首发2022-1G-4231),题目：基于污水流行病学监测预警新冠肺炎疫情暴发的技术与应用研究~~
2025-08-15
2025-08-15
2025-08-15