Comparison of clinical application effect between a novel fluorescent probe-based PCR method and three laboratory methods for detecting the Mycobacterium tuberculosis

doi:10.19982/j.issn.1000-6621.20250423

Abstract

Abstract:

Objective: To compare the diagnostic efficacy of the novel PCR-fluorescent probe method (DiagMed qPCR) with smear microscopy, liquid culture, and real-time fluorescent PCR in the detection of pulmonary tuberculosis. Methods: A total of 238 sputum samples from suspected pulmonary tuberculosis patients at Wuhan Pulmonary Hospital from March 20, 2021, to June 1, 2021, were consecutively enrolled. Taking clinical comprehensive diagnosis results as the reference standard, the diagnostic performance of smear microscopy, liquid culture, real-time fluorescent PCR, and DiagMed qPCR was evaluated and compared. Meanwhile, with the results of liquid culture and species identification as references, the correlation coefficient between the threshold cycle number (Ct) and bacterial load in samples from the two nucleic acid testing methods were assessed and compared. Results: Among the 238 patients, 167 cases were diagnosed with pulmonary TB, 1 case with nontuberculous mycobacterial disease, and 70 cases with pulmonary infections caused by fungi, viruses, and other non-tuberculous conditions based on clinical comprehensive diagnosis. Taking the clinical comprehensive diagnosis as the reference, the sensitivities of smear microscopy, liquid culture, real-time fluorescent PCR, and DiagMed qPCR for diagnosing pulmonary tuberculosis were 45.51% (76/167), 47.31% (79/167), 58.68% (98/167), and 58.68% (98/167), respectively; the specificities were 100.00% (71/71), 100.00% (71/71), 98.59% (70/71), and 98.59% (70/71), respectively; the AUC (%) values were 72.75%, 78.74%, 81.14%, and 82.24%, respectively. DiagMed qPCR and real-time fluorescence PCR showed the same positive detection rate for pulmonary TB (58.68%). The correlation coefficient R² between the Ct value of DiagMed qPCR and sputum bacterial content was higher than that of real-time fluorescent PCR assay (smear bacterial quantity grade: 0.213 vs. 0.187; culture-positive growth time: 0.293 vs. 0.290). Conclusion: In the detection of tuberculosis, both the DiagMed qPCR method and the real-time fluorescent PCR assay exhibit high sensitivity and specificity. However, the former demonstrates a stronger negative correlation between Ct values and bacterial load, which can improve detection efficiency.

Key words: Tuberculosis, pulmonary, Polymerase chain reaction, Clinical laboratory techniques, Sensitivity and specificity, Comparative study

CLC Number:

R521

Wu Gang, Zhang Zhengbin, Lu Zhouqin, Wang Xiaojun, Zhou Meilan, Li Yuehua. Comparison of clinical application effect between a novel fluorescent probe-based PCR method and three laboratory methods for detecting the Mycobacterium tuberculosis[J]. Chinese Journal of Antituberculosis, 2026, 48(5): 616-620. doi: 10.19982/j.issn.1000-6621.20250423

Figures/Tables 4

References 12

[1]	World Health Organization. Global tuberculosis report 2025. Geneva: World Health Organization, 2025.
[2]	屈燕, 李涛, 马文斌, 等. 世界卫生组织《2025年全球结核病报告》解读. 结核与肺部疾病杂志, 2025, 6 (6): 613-623. doi:10.19983/j.issn.2096-8493.20250178.
[3]	中华人民共和国国家卫生和计划生育委员会. WS 288—2017 肺结核诊断. 结核与肺部疾病杂志, 2024, 5 (4): 376-378. doi:10.19983/j.issn.2096-8493.2024022.
[4]	Detjen AK, DiNardo AR, Leyden J, et al. Xpert MTB/RIF assay for the diagnosis of pulmonary tuberculosis in children: a systematic review and meta-analysis. Lancet Respir Med, 2015, 3(6): 451-461. doi:10.1016/S2213-2600(15)00095-8. pmid: 25812968
[5]	赵雁林. 结核病实验室质量保证手册. 北京: 人民卫生出版社, 2017: 87.
[6]	中国疾病预防控制中心. 痰涂片镜检标准化操作及质量保证手册. 北京: 人民卫生出版社, 2009.
[7]	何家花, 余成强, 李步荣, 等. TB-DNA、T-SPOT.TB和TB-Ab平行检测在肺结核诊断中的应用价值. 检验医学与临床, 2021, 18(1): 67-69, 72. doi:10.3969/j.issn.1672-9455.2021.01.018.
[8]	陈丽, 孙彦波, 马玉秋, 等. 多重组合检测在基层实验室诊断肺结核的效能研究. 结核与肺部疾病杂志, 2025, 6(2): 204-209. doi:10.19983/j.issn.2096-8493.20250034.
[9]	Byanyima P, Kaswabuli S, Musisi E, et al. Feasibility and Sensitivity of Saliva GeneXpert MTB/RIF Ultra for Tuberculosis Diagnosis in Adults in Uganda. Microbiol Spectr, 2022, 10(5): e0086022. doi:10.1128/spectrum.00860-22.
[10]	李艳红, 李春艳, 陈天芳, 等. 实时荧光定量PCR与痰涂片抗酸染色法诊断结核病的灵敏度、特异度比较. 临床医学研究与实践, 2019, 4(30): 128-130. doi:10.19347/j.cnki.2096-1413.201930055.
[11]	Furin J, Cox H, Pai M. Tuberculosis. Lancet, 2019, 393(10181): 1642-1656. doi:10.1016/S0140-6736(19)30308-3. pmid: 30904262
[12]	欧喜超, 滕冲, 宋媛媛, 等. 新型PCR荧光探针技术用于结核病患者早期诊断的多中心应用评价研究. 中国防痨杂志, 2025, 47(6): 687-693. doi:10.19982/j.issn.1000-6621.20250031.

检测方法	肺结核 (167例)	非肺结核 (71例)	敏感度(%, 95%CI值)	特异度(%, 95%CI值)	阳性预测值 (%,95%CI值)	阴性预测值 (%,95%CI值)	Kappa值	曲线下面积 (%,95%CI值)
涂片镜检(例)			45.51 (37.8~53.4)	100.00 (94.9~100.00)	100.00 (95.30~100.00)	43.83 (36.20~51.80)	0.333 (0.219~0.446)	72.75 (68.97~76.54)
阳性	76	0
阴性	91	71
液体培养(例)			47.31 (39.88~54.85)	100.00 (94.90~100.00)	100.00 (95.45~100.00)	44.65 (37.06~52.53)	0.349 (0.236~0.462)	78.74 (74.98~82.50)
阳性	79	0
阴性	88	71
实时荧光PCR法(例)			58.68 (51.10~65.87)	98.59 (92.61~99.79)	98.99 (94.38~99.83)	50.36 (42.44~58.27)	0.449 (0.343~0.555)	81.14 (77.36~84.92)
阳性	98	1
阴性	69	70
新型PCR荧光探针法(例)			58.68 (51.10~65.87)	98.59 (92.61~99.79)	98.99 (94.38~99.83)	50.36 (42.44~58.27)	0.449 (0.343~0.555)	82.24 (78.42~86.05)
阳性	98	1
阴性	69	70

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