Rapid Diagnosis of Mycobacterium tuberculosis Infection and Drug Resistance Based on Real-Time Fluorescence PCR

Summary

Here, a protocol for detecting Mycobacterium tuberculosis and its drug resistance using real-time fluorescence quantitative PCR (qPCR) technology is presented.

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 timelines, maximize identification of DR-TB cases, ensure standardized treatment and management, and implement necessary infection control measures³. Furthermore, the Notice on Strengthening DR-TB Prevention and Control (No. 4, 2023) mandates the nationwide promotion of advanced diagnostic techniques. It urges timely resistance screening for all bacteriologically confirmed TB patients to achieve universal screening, improve diagnostic standardization, and enhance healthcare accessibility⁴.

DR-TB refers to TB cases that are resistant to at least one anti-TB drug. Current classifications include rifampicin-resistant (RR), monoresistant, polyresistant, multidrug-resistant (MDR), pre-extensively drug-resistant (pre-XDR), and extensively drug-resistant (XDR) TB. A global meta-analysis of DR-TB epidemiology reveals alarming resistance rates: MDR-TB at 11.6%, isoniazid resistance at 15.7%, rifampicin resistance at 9.4%, and monoresistance at 11.8%. These data underscore the gravity and complexity of DR-TB⁵.

Rifampicin and isoniazid are cornerstone drugs for TB treatment. However, the World Health Organization (WHO) notes that while rapid diagnostic technologies for rifampicin resistance are available, analogous tools for isoniazid resistance are lacking. This gap impedes the timely diagnosis of multidrug-resistant TB (MDR-TB). The provisional definition of "rifampicin-resistant TB" reflects this diagnostic limitation, highlighting the insufficient capacity to rapidly detect resistance to other drugs^6,7. WHO estimates that isoniazid-resistant TB cases are twice as prevalent as rifampicin-resistant cases. Exclusive reliance on rifampicin resistance screening risks overlooking isoniazid-monoresistant TB (Hr-TB)⁸. Undetected Hr-TB patients treated with standard regimens face elevated risks of treatment failure, relapse, and progression to MDR-TB⁹. Ethambutol resistance is also concerning. As a critical drug for TB and MDR-TB treatment, China's 2017 DR-TB baseline survey reported ethambutol resistance rates of 4.9% in new cases and 17.2% in retreatment cases¹⁰.

At present, for the detection of drug resistance of residual tuberculosis, artificial culture is still the preferred method in some laboratories, but real-time fluorescent PCR technology is superior to conventional methods in detecting drug resistance of tuberculosis: detection and quantification: real time fluorescent PCR provides accurate quantitative analysis through real-time monitoring of fluorescent signals, while conventional methods lack this accuracy. Traditional experiment methods are time-consuming and labor-intensive, require trained personnel, and are highly subjective. On the contrary, real-time PCR captures fluorescent probes and signals by computer, minimizing human errors. Conventional data analysis methods are more subjective, while real-time PCR achieves accurate and objective analysis through amplification curves, standard curves, and other analysis methods. These advances make real-time fluorescent PCR the preferred method for detecting drug resistance of tuberculosis¹¹.

In summary, drug-resistant tuberculosis (DR-TB) has emerged as a global crisis. Resistance to key drugs -- isoniazid, rifampicin, and ethambutol -- further complicates TB control efforts. Timely and accurate detection of drug resistance is crucial for guiding tailored treatment regimens, improving therapeutic success, and mitigating the DR-TB threat.

To address the diagnostic challenge, this study utilized a kit that can identify Mycobacterium tuberculosis and detect drug-resistant mutations within 3.5 h using sputum samples. This was achieved with the support of real-time fluorescence qPCR. The method is simple, cost-effective, and accurate, making it a valuable tool in the fight against DR-TB.

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

1. 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.
2. Label the sputum containers with the patient's name, number (the outpatient sequence number for new patients or the registration number for follow-up patients), examination items, and container sequence numbers 1, 2, 3 (1 = immediate sputum on the same day, 2 = night sputum, 3 = morning sputum the next day). For patients without sputum, collect samples through aerosol-induced sputum collection.
   NOTE: Staff should evaluate the quality of collected sputum samples. The sample volume should generally be maintained at 3-5 mL. Qualified samples include dry sputum, brown blood-stained sputum, sputum containing a small amount of fresh blood, or mucous sputum. Unqualified samples, such as saliva, should be resubmitted for testing after further guidance.

2. Sputum preprocessing

1. Add 1-2 times the volume of 4% NaOH solution to the sputum container based on the volume of the collected sputum. Shake and mix the contents for 2 min.
2. Place the container in a biosafety cabinet at room temperature for 15 min, shaking it several times during this period to ensure complete liquefaction of the sputum.
   NOTE: Handle NaOH with care due to its corrosive nature. If the sputum is not fully liquefied, increase the shaking time. This step is particularly important throughout the experiment.

3. Reagent preparation (pre-PCR area)

1. Preparation of reagents: Prepare reagents in the designated pre-PCR area. Thaw frozen reagents to room temperature. Vortex for 5-10 s. Centrifuge at 3000 × g for 5-10 s at room temperature.
   NOTE: This reagent is a dual-system (A/B) and must be prepared separately.
2. PCR master mix preparation
   1. Mix A: In a 1.5 mL microcentrifuge tube, combine (n + 3) × 19.6 µL of RIF PCR Mix A and (n + 3) × 0.4 µL of TB enzyme. Vortex for 5-10 s. Centrifuge at 3000 × g for 5-10 s at room temperature (PCR Mix A: 60 µL).
   2. Mix B: In another 1.5 mL microcentrifuge tube, combine (n + 3) × 19.6 µL of RIF PCR Mix B and (n + 3) × 0.4 µL of TB enzyme. Vortex and centrifuge as above (PCR Mix B: 60 µL).
3. Storage and usage: Store the prepared master mixes at ≤−18 °C. If not used immediately, use within 4 h.
   NOTE: The same protocol applies to EMB and INH reagents.
4. Aliquoting PCR master mix: Dispense 20 µL of PCR Mix A and Mix B into separate PCR thin-walled reaction tubes. Transfer aliquoted tubes in sealed bags to the extraction area.

4. Nucleic acid extraction and sample loading (extraction area)

1. Centrifuge 1.2 mL of fully liquefied sputum at 15,058 × g for 5 min at room temperature. Discard the supernatant and retain the pellet. Optional: Centrifuge the entire digested sample if equipment permits.
2. Resuspend the pellet in 1.2 mL saline, vortex, and centrifuge at 15,058 × g for 5 min at room temperature. Repeat this washing step three times.
3. Add 250 µL TB extraction buffer to the pellet. Vortex and perform thermal lysis at 99 °C for 10 min. Cool to room temperature. Centrifuge at 15,058 × g for 1 min at room temperature and collect the supernatant (extracted sample).
   NOTE: For viscous samples, increase the volume of the liquefaction buffer or extend the vortexing time. If samples are solely for DNA extraction, prolong the liquefaction time to ensure complete digestion.
4. Using a micropipette, add 5 µL of extracted sample or negative/positive controls to each PCR thin-walled reaction tube. Seal tubes immediately and label appropriately.
5. Vortex loaded PCR tubes for 5-10 s and centrifuge briefly (5-10 s). Transfer tubes to the PCR amplification area.

5. PCR amplification (amplification area)

NOTE: Programs for rifampicin, ethambutol, and isoniazid resistance detection were set on a Hongshi thermal cycler (Table 1, Table 2, and Table 3). Cycling conditions are included.

1. Place the loaded PCR thin-walled reaction tubes into the thermal cycler for amplification.
2. Access the cycler interface. Click on Experiment Wizard and select the experiment type as Standard Melt Curve (as shown in Figure 1).
3. Click on Plate Editor, horizontally select two adjacent blank wells, and assign the corresponding drug template from the right panel to define one specimen (as shown in Figure 2).
4. Configure negative/positive controls and sample information. Click on Start to initiate the program. The runtime is 2 h and 22 min.
5. Upon completion, transfer the PCR thin-walled reaction tubes (sealed) into a sealed bag. Secure the closure and handle as biohazardous waste.
6. Click on Analyze on the Thermal Cycler to process the data.
   NOTE: The detection limits of this method are as follows: Composite group: 10 bacteria/mL; Rifampicin: 20,000 bacteria/mL; Isoniazid: 10,000 bacteria/mL; Ethambutol: 10,000 bacteria/mL. (The limit of detection (LoD) data are derived from the statistical analysis of 765 clinical cases provided in the reagent instruction manual.)

6. Result interpretation

1. Determine mutations by comparing the melting temperature (Tm) differences between test samples and positive controls.
   ​NOTE: Wild-type (Drug-sensitive): Tm values of the sample match the positive control in all four channels (deviation ≤1 °C). Mutant (Drug-resistant): Tm value in any channel is ≥2 °C lower than the positive control (ΔTm ≥2 °C).

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...

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

Materials

List of materials used in this article

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
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