Here, a protocol for detecting Mycobacterium tuberculosis and its drug resistance using real-time fluorescence quantitative PCR (qPCR) technology is presented.
Method Article
Rapid Diagnosis of Mycobacterium tuberculosis Infection and Drug Resistance Based on Real-Time Fluorescence PCR
* These authors contributed equally
In This Article
Summary
Abstract
Tuberculosis (TB) is a major health concern that disproportionately affects vulnerable populations. The emergence and spread of drug-resistant TB pose a serious threat to global public health. Traditional diagnostic methods for Mycobacterium tuberculosis (MTB), such as smear microscopy and culture, have significant limitations and often result in delayed clinical treatment. To address this challenge, an efficient diagnostic scheme based on real-time fluorescent PCR was developed. High-quality MTB nucleic acids can be extracted from test samples, and resistance status can be identified at key resistance sites. Compared to traditional methods, this approach significantly reduces detection time to just a few hours, enabling the determination of infection and resistance status shortly after a patient's initial visit. Detection sensitivity exceeds 85%, and specificity reaches 95%. This non-invasive technology not only minimizes patient discomfort but also supports accurate diagnosis in grassroots medical institutions due to its low cost and high efficiency. Overall, the approach represents a significant improvement in TB diagnostics.
Introduction
Tuberculosis (TB), a chronic infectious disease caused by Mycobacterium tuberculosis (MTB), is among the top ten global causes of death1. Drug-resistant tuberculosis (DR-TB) presents a significant challenge to TB control efforts. The Action Plan for Building TB-Free Communities (2022-2027), issued by the Chinese Center for Disease Control and Prevention (China CDC), emphasizes the need for early drug resistance screening in all bacteriologically confirmed pulmonary TB patients2. It advocates the adoption of novel molecular diagnostic technologies to enhance DR-TB detection capabilities, shorten diagnostic timeli....
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Protocol
This research has been approved by the Ethics Committee of the Fifth Division Hospital of Xinjiang Production and Construction Corps; the ethics number is LLWYH2025001, and there is no ethical conflict. The reagents and the equipment used are listed in the Table of Materials.
1. Sample collection
- Use internationally standardized screw-cap sputum bottles recommended by the World Health Organization (WHO), or sealed plastic boxes and wax paper boxes, to collect sputum samples.
- Label the sputum containers with the patient's name, number (the outpatient sequence number for new patients....
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Results
This study utilized real-time fluorescence PCR and melting curve analysis to test sputum samples from 17 tuberculosis patients. Additionally, 30 patients underwent rifampicin resistance testing, and 20 were tested for resistance to isoniazid and ethambutol. The real-time fluorescence PCR method demonstrated an 88.2% positive detection rate for the Mycobacterium tuberculosis complex.
Resistance.......
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Discussion
The emergence of drug-resistant Mycobacterium tuberculosis complex (MTBC) strains on a global scale has significantly complicated the treatment of tuberculosis (TB) patients. Particularly, multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis pose formidable challenges to TB control12. Rapid detection of drug resistance is crucial for designing appropriate treatment regimens, preventing therapeutic failure, and curtailing the further spread of resistant strains
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Disclosures
The authors declare no conflicts of interest.
Acknowledgements
This study was funded by a special grant from the Corps Tuberculosis Project.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| High speed centrifuge | |||
| Lysis Buffer | Xiamen Zhishan Biotechnology Co., Ltd. | 24110701 | |
| Mycobacterium tuberculosis Ethambutol Resistance Mutation Detection Kit | Xiamen Zhishan Biotechnology Co., Ltd. | 24030801 | |
| Mycobacterium tuberculosis Isoniazid Resistance Mutation Detection Kit | Xiamen Zhishan Biotechnology Co., Ltd. | 24011101 | |
| Mycobacterium tuberculosis Rifampicin Resistance Mutation Detection Ki | Xiamen Zhishan Biotechnology Co., Ltd. | 24031001 | |
| SLAN-96P Real-Time PCR System | Shanghai Hongshi Medical Technology Co., Ltd. | SLAN-96P | |
| Sputum Processing Solution | Xiamen Zhishan Biotechnology Co., Ltd. | 24110701 | |
| TopPette Pipettor |
References
- Salari, N., et al. Global prevalence of drug-resistant tuberculosis: A systematic review and meta-analysis. Infect Dis Poverty. 12 (1), 57(2023).
- Cai, X., Xu, X., He, G., Jiang, X., Wu, L. Drug r....
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