肺炎支原体实验室诊断方法研究进展

doi:10.3969/j.issn.1673-8640.2026.03.015

摘要/Abstract

摘要：

肺炎支原体（MP）是呼吸道感染主要的病原体之一，由MP引发的社区获得性肺炎（CAP）在儿童和青少年人群中发病率较高。MP实验室检测方法主要包括病原学检测、血清学检测和分子生物学检测，不同方法各有优缺点。MP分离培养耗时长、特异性不高，临床实验室已较少常规开展。目前主要采用MP特异性抗原、抗体和聚合酶链反应（PCR）检测MP 。近年来，环介导等温扩增（LAMP）、成簇规律间隔短回文重复序列/关联蛋白（CRISPR/Cas）技术、纳米生物传感器等新型检测方法在MP诊断领域取得显著进展，给临床提供了更多选择。文章对MP常用检测方法和新型实验室诊断技术的发展现状、优缺点、应用价值进行综述，旨在为MP的临床诊断和应用场景选择提供参考。

关键词: 肺炎支原体, 临床实验室, 诊断, 病原学, 免疫学, 分子生物学, 基因测序, 环介导等温扩增, 成簇规律间隔短回文重复序列, 纳米生物传感器

Abstract:

Mycoplasma pneumoniae（MP）is one of the main pathogens causing respiratory tract infections，with a relatively high incidence of community-acquired pneumonia（CAP）among children and adolescents. Laboratory determination methods for MP mainly include etiological determination，serological determination and molecular biological determination，each with its own advantages and disadvantages. MP isolation and culture is time-consuming and has low specificity，and it is rarely routinely performed in clinical laboratories. Currently，the main methods used are MP-specific antigen，antibody and polymerase chain reaction（PCR）for MP. In recent years，new determination technologies such as loop-mediated isothermal amplification（LAMP），clustered regularly interspaced short palindromic repeat/associated protein（CRISPR/Cas）and nanobiosensors have made significant progress in the field of MP diagnosis，providing more options for clinical practice. This review focuses on the current status，advantages and disadvantages and application value of common MP determination methods and new laboratory diagnostic technologies，aiming to provide references for the clinical diagnosis and application of MP.

Key words: Mycoplasma pneumoniae, Clinical laboratory, Diagnosis, Etiology, Immunology, Molecular biology, Gene sequencing, Loop-mediated isothermal amplification, Clustered regularly interspaced short palindromic repeat, Nanobiosensor

中图分类号:

R375

陈添, 蔡秦真, 陶宇煊, 王军, 商宇, 伍墨, 庹文彬, 袁纯辉, 向贇. 肺炎支原体实验室诊断方法研究进展[J]. 检验医学, 2026, 41(3): 299-306.

CHEN Tian, CAI Qinzhen, TAO Yuxuan, WANG Jun, SHANG Yu, WU Mo, TUO Wenbin, YUAN Chunhui, XIANG Yun. Research progress on laboratory diagnostic methods for Mycoplasma pneumonia[J]. Laboratory Medicine, 2026, 41(3): 299-306.

图/表 2

参考文献 48

[1]	HAN S, FANG Q, WU X, et al. Clinical features associated with macrolides resistance of Mycoplasma pneumoniae pneumonia in children: a single-center analysis[J]. BMC Infect Dis, 2025, 25(1):854. DOI
[2]	MIYASHITA N, KAWAI Y, TANAKA T, et al. Diagnostic sensitivity of a rapid antigen test for the detection of Mycoplasma pneumoniae:comparison with real-time PCR[J]. J Infect Chemother, 2015, 21(6):473-475. DOI URL
[3]	LI W, LIU Y, ZHAO Y, et al. Rapid diagnosis of Mycoplasma pneumoniae in children with pneumonia by an immuno-chromatographic antigen assay[J]. Sci Rep, 2015, 5:15539. DOI
[4]	LIU F C, CHEN P Y, HUANG F L, et al. Do serological tests provide adequate rapid diagnosis of Mycoplasma pneumoniae infection?[J]. Jpn J Infect Dis, 2008, 61(5):397-399. DOI URL
[5]	LIN L J, CHANG F C, CHI H, et al. The diagnostic value of serological studies in pediatric patients with acute Mycoplasma pneumoniae infection[J]. J Microbiol Immunol Infect, 2020, 53(2):351-356. DOI URL
[6]	BUSSON L, VAN DEN WIJNGAERT S, DAHMA H, et al. Evaluation of 10 serological assays for diagnosing Mycoplasma pneumoniae infection[J]. Diagn Microbiol Infect Dis, 2013, 76(2):133-137. DOI URL
[7]	LEE W J, HUANG E Y, TSAI C M, et al. Role of serum Mycoplasma pneumoniae IgA,IgM,and IgG in the diagnosis of Mycoplasma pneumoniae-related pneumonia in school-age children and adolescents[J]. Clin Vaccine Immunol, 2017, 24(1):e00471-16.
[8]	TUO W, GUO X, WU M, et al. Application value of antibody titres and RNA detection in the early prediction of Mycoplasma pneumoniae pneumonia in children: a retrospective study[J]. BMC Infect Dis, 2023, 23(1):220. DOI
[9]	MEYER SAUTEUR P M, SEILER M, TRÜCK J, et al. Diagnosis of Mycoplasma pneumoniae pneumonia with measurement of specific antibody-secreting cells[J]. Am J Respir Crit Care Med, 2019, 200(8):1066-1069. DOI URL
[10]	MATSUI H, SUGIMURA M, INOUE-TSUDA M, et al. Development of an immunochromatographic test for the detection of Mycoplasma pneumoniae GroES antigen[J]. J Microbiol Methods, 2021, 191:106359.
[11]	LOENS K, GOOSSENS H, IEVEN M. Acute respiratory infection due to Mycoplasma pneumoniae:current status of diagnostic methods[J]. Eur J Clin Microbiol Infect Dis, 2010, 29(9):1055-1069. DOI URL
[12]	BLANCO S, FUENZALIDA L, BAS A, et al. Comparison of 2 molecular assays and a serologic test in diagnosing Mycoplasma pneumoniae infection in paediatrics patients[J]. Diagn Microbiol Infect Dis, 2011, 71(4):463-466. DOI URL
[13]	CHAUDHRY R, SHARMA S, JAVED S, et al. Molecular detection of Mycoplasma pneumoniae by quantitative real-time PCR in patients with community acquired pneumonia[J]. Indian J Med Res, 2013, 138(2):244-251.
[14]	KUMAR S, GARG I B, SETHI G R. Serological and molecular detection of Mycoplasma pneumoniae in children with community-acquired lower respiratory tract infections[J]. Diagn Microbiol Infect Dis, 2019, 95(1):5-9. DOI URL
[15]	ZHANG Y, YANG X, QIAN J, et al. Simultaneous detection of Mycoplasma pneumoniae IgG and IgM using dual-label time resolved fluoroimmunoassay[J]. Anal Biochem, 2018, 548:1-6. DOI URL
[16]	CHEN D, WU P, LIU D, et al. Clinical role of M. pneumoniae typing antibody detected by chemiluminescent immunoassay in the diagnosis of Mycoplasma pneumoniae pneumonia in children[J]. Int Immunopharmacol, 2022, 112:109196.
[17]	LI Q L, DONG H T, SUN H M, et al. The diagnostic value of serological tests and real-time polymerase chain reaction in children with acute Mycoplasma pneumoniae infection[J]. Ann Transl Med, 2020, 8(6):386. DOI URL
[18]	DUMKE R, BENITEZ A J, CHALKER V, et al. Multi-center evaluation of one commercial and 12 in-house real-time PCR assays for detection of Mycoplasma pneumoniae[J]. Diagn Microbiol Infect Dis, 2017, 88(2):111-114.
[19]	ZHANG C, ZHANG Q, DU J L, et al. Correlation between the clinical severity,bacterial load,and inflammatory reaction in children with Mycoplasma pneumoniae pneumonia[J]. Curr Med Sci, 2020, 40(5):822-828. DOI
[20]	WANG Y S, ZHOU Y L, BAI G N, et al. Expert consensus on the diagnosis and treatment of macrolide-resistant Mycoplasma pneumoniae pneumonia in children[J]. World J Pediatr, 2024, 20(9):901-914. DOI
[21]	GUO D, HU W, XU B, et al. Allele-specific real-time PCR testing for minor macrolide-resistant Mycoplasma pneumoniae[J]. BMC Infect Dis, 2019, 19(1):616. DOI
[22]	ZHANG Y, CAO L, XU Z, et al. Evaluation of a multiplex PCR assay for detection of respiratory viruses and Mycoplasma pneumoniae in oropharyngeal swab samples from outpatients[J]. J Clin Lab Anal, 2020, 34(1):e23032. DOI URL
[23]	CAI Z H, DAI Y Y, HUANG L Y, et al. Diagnosis of Mycoplasma pneumoniae by loop-mediated isothermal amplification:systematic review and meta-analysis[J]. BMC Infect Dis, 2019, 19(1):173. DOI
[24]	PETRONE B L, WOLFF B J, DELANEY A A, et al. Isothermal detection of Mycoplasma pneumoniae directly from respiratory clinical specimens[J]. J Clin Microbiol, 2015, 53(9):2970-2976. DOI URL
[25]	ZHAO H, YAN C, FENG Y, et al. Absolute quantification of Mycoplasma pneumoniae in infected patients by droplet digital PCR to track disease severity and treatment efficacy[J]. Front Microbiol, 2023, 14:1177273.
[26]	LI W, FANG Y H, SHEN H Q, et al. Evaluation of a real-time method of simultaneous amplification and testing in diagnosis of Mycoplasma pneumoniae infection in children with pneumonia[J]. PLoS One, 2017, 12(5):e0177842. DOI URL
[27]	中华医学会儿科学分会呼吸学组, 国家呼吸系统疾病临床医学研究中心, 中华儿科杂志编辑委员会. 儿童肺炎支原体肺炎诊断与治疗循证指南(2023)[J]. 中华儿科杂志, 2024, 62(12):1137-1144.
[28]	王诗婵, 叶光明, 陈良君, 等. RNA恒温扩增-金探针层析法在肺炎支原体检测中的应用[J]. 武汉大学学报(医学版), 2020, 41(2):260-263.
[29]	TANG M, WANG D, TONG X, et al. Comparison of different detection methods for Mycoplasma pneumoniae infection in children with community-acquired pneumonia[J]. BMC Pediatr, 2021, 21(1):90. DOI
[30]	JIANG X, GUO H, SUN J, et al. Diagnostic value of metagenomic next-generation sequencing for bronchoalveolar lavage diagnostics in patients with lower respiratory tract infections[J]. Diagn Microbiol Infect Dis, 2025, 111(2):116620.
[31]	CHEN S, HOU C, KANG Y, et al. Application of metagenomic next-generation sequencing in the diagnosis and resistome analysis of community-acquired pneumonia pathogens from bronchoalveolar lavage samples[J]. J Appl Microbiol, 2023, 134(6):lxad102.
[32]	LI C, RAO J, WANG X, et al. Navigating the outbreak:a comprehensive analysis of pediatric Mycoplasma pneumoniae pneumonia via targeted next-generation sequencing in Wuhan,2022-2023[J]. Microbiol Spectr, 2025, 13(5):e0246324. DOI URL
[33]	LIN R, XING Z, LIU X, et al. Performance of targeted next-generation sequencing in the detection of respiratory pathogens and antimicrobial resistance genes for children[J]. J Med Microbiol, 2023, 72(11):1771.
[34]	LIN Q, YAO Y, LI X, et al. The application of nanopore targeted sequencing for pathogen diagnosis in bronchoalveolar lavage fluid of patients with pneumonia: a prospective multicenter study[J]. Infect Dis(Lond), 2024, 56(2):128-137.
[35]	DENG Z, HU H, TANG D, et al. Ultrasensitive,specific,and rapid detection of Mycoplasma pneumoniae using the ERA/CRISPR-Cas12a dual system[J]. Front Microbiol, 2022, 13:811768.
[36]	LIU T, LIU Q, CHEN F, et al. An accurate and convenient method for Mycoplasma pneumoniae via one-step LAMP-CRISPR/Cas12b detection platform[J]. Front Cell Infect Microbiol, 2024, 14:1409078.
[37]	LI F, XIAO J, YANG H, et al. Development of a rapid and efficient RPA-CRISPR/Cas12a assay for Mycoplasma pneumoniae detection[J]. Front Microbiol, 2022, 13:858806.
[38]	ZHOU J, XIAO F, FU J, et al. Rapid,ultrasensitive and highly specific diagnosis of Mycoplasma pneumoniae by a CRISPR-based detection platform[J]. Front Cell Infect Microbiol, 2023, 13:1147142.
[39]	JING W, ZHANG T, MIN X, et al. CHAMP:a centrifugal microfluidics-based CRISPR/Cas12b-combined real-time LAMP one-pot method for Mycoplasma pneumoniae infection diagnosis[J]. ACS Omega, 2024, 9(37):38989-38997. DOI URL
[40]	ZHU R, JIANG H, LI C, et al. CRISPR/Cas9-based point-of-care lateral flow biosensor with improved performance for rapid and robust detection of Mycoplasma pneumonia[J]. Anal Chim Acta, 2023, 1257:341175.
[41]	HE J, HU X, WENG X, et al. Efficient,specific and direct detection of double-stranded DNA targets using Cas12f1 nucleases and engineered guide RNAs[J]. Biosens Bioelectron, 2024, 260:116428.
[42]	WANG S, HU Y, DENG Z, et al. CRISPR/Cas12a-enhanced DNA nanomachine for multiple respiratory pathogens detection[J]. Chem Commun(Camb), 2024, 60(99):14814-14817.
[43]	HENDERSON K C, SHEPPARD E S, RIVERA-BETANCOURT O E, et al. The multivariate detection limit for Mycoplasma pneumoniae as determined by nanorod array-surface enhanced Raman spectroscopy and comparison with limit of detection by qPCR[J]. Analyst, 2014, 139(24):6426-6434. DOI URL
[44]	WANG H, MA Z, QIN J, et al. A versatile loop-mediated isothermal amplification microchip platform for Streptococcus pneumoniae and Mycoplasma pneumoniae testing at the point of care[J]. Biosens Bioelectron, 2019, 126:373-380. DOI URL
[45]	LI S, JIANG W, HUANG J, et al. Highly sensitive and specific diagnosis of COVID-19 by reverse transcription multiple cross-displacement amplification-labelled nanoparticles biosensor[J]. Eur Respir J, 2020, 56(6):2002060.
[46]	ZHAO F, NIU L, YAN L, et al. Establishment and application of multiple cross displacement amplification coupled with lateral flow biosensor(MCDA-LFB)for visual and rapid detection of Candida albicans in clinical samples[J]. Front Cell Infect Microbiol, 2019, 9:102. DOI URL
[47]	WANG H, LI Y, TIAN L, et al. A LAMP-based hydrogen ion selective electrochemical sensor for highly sensitive detection of Mycoplasma pneumoniae[J]. Anal Methods, 2024, 16(19):3020-3029. DOI URL
[48]	全国医用临床检验实验室和体外诊断系统标准化技术委员会(SAC/TC 136) GB/Z43281—2023即时检验(POCT)设备监督员和操作员指南[S]. 北京: 中国标准出版社, 2023.

方法	目标	优点	缺点	样本类型	参考文献
ICA	核糖体蛋白L7/L12、	经济、快速、操作简便	不同检测方法之间敏感性差异较大（62.5%~97.4%）、易与其他呼吸道病原体产生交叉反应	咽拭子	[4]
	P1黏附蛋白、			咽拭子/痰	[3]
	GroES分子伴侣蛋白			MP分离株/GroES重组蛋白	[10]
CFT	IgM	成本低、适用范围广、特异性较高（97.0%）	操作复杂、敏感性较低（58.8%）、无法区分抗体类型	血清	[11]
PA	IgM/IgG	快速、操作相对简便、无需专业仪器	易受非特异性凝集干扰、不同抗体滴度条件下的诊断灵敏度和特异性存在差异、主观判读易产生误差	血清	[8,12]
ELISA	IgA/IgM/IgG	经济、特异性较高（74.8%~ 96.0%），可区分抗体类型，可定量分析，通量高	不同品牌试剂盒灵敏度差异大（57.9%~91%）、耗时较长、对仪器性能和操作人员的熟练度要求高	血清	[5,7,13-14]
CLIA	IgM/IgG	自动化检测、检测线性范围广，敏感性（62.1%~74.3%）和特异性（72.9%~87.0%）较高	设备成本高、未被临床大量验证	血清	[15-16]
ELISpot	Mp-IgM-ASC	可以区分携带和感染	灵敏度低、耗时长	全血	[9]

方法	目标	优点	缺点	样本类型	参考文献
ICA	核糖体蛋白L7/L12、	经济、快速、操作简便	不同检测方法之间敏感性差异较大（62.5%~97.4%）、易与其他呼吸道病原体产生交叉反应	咽拭子	[4]
	P1黏附蛋白、			咽拭子/痰	[3]
	GroES分子伴侣蛋白			MP分离株/GroES重组蛋白	[10]
CFT	IgM	成本低、适用范围广、特异性较高（97.0%）	操作复杂、敏感性较低（58.8%）、无法区分抗体类型	血清	[11]
PA	IgM/IgG	快速、操作相对简便、无需专业仪器	易受非特异性凝集干扰、不同抗体滴度条件下的诊断灵敏度和特异性存在差异、主观判读易产生误差	血清	[8,12]
ELISA	IgA/IgM/IgG	经济、特异性较高（74.8%~ 96.0%），可区分抗体类型，可定量分析，通量高	不同品牌试剂盒灵敏度差异大（57.9%~91%）、耗时较长、对仪器性能和操作人员的熟练度要求高	血清	[5,7,13-14]
CLIA	IgM/IgG	自动化检测、检测线性范围广，敏感性（62.1%~74.3%）和特异性（72.9%~87.0%）较高	设备成本高、未被临床大量验证	血清	[15-16]
ELISpot	Mp-IgM-ASC	可以区分携带和感染	灵敏度低、耗时长	全血	[9]

方法类型	信号策略	系统名称	效应蛋白	信号放大方式	目标分子	灵敏度	单碱基分辨率	检测周期	参考文献
CRISPR/Cas+等温扩增	可视化/荧光	ERA^①/CRISPR-Cas12a双系统	LbCas12a	ERA^①	DNA	100 拷贝·mL^-1，1 拷贝·mL^-1	是	30.0 min	[35]
	荧光	LAMP-CRISPR/Cas12b	Cas12b	LAMP	DNA	33.7 拷贝·tesP分离培养耗时较长	是		[36]
	荧光	RPA^②-CRISPR/Cas12a	Cas12a	RPA^②	DNA	2 拷贝·test^-1	是	<1 h	[37]
	荧光	RPA^②-CRISPR	Cas12b	RPA^②	DNA	5 fg	是		[38]
CRISPR/Cas+等温扩增+生物传感器	荧光	CHAMP系统	Cas12b	LAMP	DNA	0.5 拷贝·μL^-1	是	23.5 min	[39]
	可视化	CRISPR/Cas9-LFB	Cas9	RPA^②	DNA	3 拷贝	是	30.0 min	[40]
	荧光	PDTCTR	Cas12f_ge4.1	RPA^②	DNA	10 拷贝·μL^-1	是	50.0 min	[41]
	质谱	CRISPR/ Cas12a增强型DNA纳米机	Cas12a		DNA	38 amol	是		[42]

方法类型	信号策略	系统名称	效应蛋白	信号放大方式	目标分子	灵敏度	单碱基分辨率	检测周期	参考文献
CRISPR/Cas+等温扩增	可视化/荧光	ERA^①/CRISPR-Cas12a双系统	LbCas12a	ERA^①	DNA	100 拷贝·mL^-1，1 拷贝·mL^-1	是	30.0 min	[35]
	荧光	LAMP-CRISPR/Cas12b	Cas12b	LAMP	DNA	33.7 拷贝·tesP分离培养耗时较长	是		[36]
	荧光	RPA^②-CRISPR/Cas12a	Cas12a	RPA^②	DNA	2 拷贝·test^-1	是	<1 h	[37]
	荧光	RPA^②-CRISPR	Cas12b	RPA^②	DNA	5 fg	是		[38]
CRISPR/Cas+等温扩增+生物传感器	荧光	CHAMP系统	Cas12b	LAMP	DNA	0.5 拷贝·μL^-1	是	23.5 min	[39]
	可视化	CRISPR/Cas9-LFB	Cas9	RPA^②	DNA	3 拷贝	是	30.0 min	[40]
	荧光	PDTCTR	Cas12f_ge4.1	RPA^②	DNA	10 拷贝·μL^-1	是	50.0 min	[41]
	质谱	CRISPR/ Cas12a增强型DNA纳米机	Cas12a		DNA	38 amol	是		[42]