Identification of Mycobacterium Species by DNA Microarray Chip Method

Summary

Here, we present a DNA microarray chip method for identifying Mycobacterium species, which will improve diagnostic accuracy and efficiency of related diseases for clinical use.

Abstract

This study presents a DNA microarray chip method designed for the accurate identification of Mycobacterium species. By leveraging asymmetric PCR amplification and hybridization principles, this method targets 17 common mycobacteria, including those in the Mycobacterium tuberculosis complex and various non-tuberculous mycobacteria. It is applicable to a wide range of clinical specimens like sputum, pus, bronchoalveolar lavage fluid, cerebrospinal fluid, and puncture fluid from patients suspected of mycobacterial diseases. The assay involves amplifying target sequences in the Mycobacterium genome through asymmetric PCR, followed by hybridization with the probes on the microarray chip. The unique probe arrangement (repeated 5x in a 12 row x 10 column microarray) and the use of control probes enhance its reliability. Clinical trials with 1,724 samples demonstrated high performance. The method achieved 100% clinical specificity, sensitivity, and overall concordance compared to sequencing results, except for two rare non-tuberculous mycobacterial samples beyond its detection scope. This DNA microarray chip method offers a significant improvement over traditional diagnostic techniques, shortening the diagnosis time and providing a more comprehensive detection, thus having great potential in the diagnosis and management of mycobacterial diseases.

Introduction

Mycobacterium, a genus of bacteria, encompasses a diverse group of organisms with significant implications for human and animal health. Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), has been a long-standing global health scourge. Despite decades of efforts in prevention and treatment, TB remains a major public health threat, responsible for a staggering number of annual deaths and new infections worldwide^1,2. Non-tuberculous mycobacteria (NTM) are also increasingly recognized as important pathogens, especially in specific patient populations and environmental settings.

The accurate identification of Mycobacterium species is the cornerstone of effective disease management^3,4. Traditional diagnostic methods have inherent limitations. Culture-based techniques, which have historically been relied upon, are notoriously time-consuming. The process of isolating and culturing mycobacteria can take weeks to months, during which patients may experience disease progression and potential transmission to others. Moreover, some mycobacterial species are extremely difficult to culture, leading to false-negative results and delayed or incorrect treatment^5,6.

Acid-fast staining, another commonly used method, can identify mycobacteria based on their unique cell wall properties that resist decolorization. However, it lacks the specificity to distinguish between different Mycobacterium species, providing only a broad indication of mycobacterial presence⁷. Molecular methods such as polymerase chain reaction (PCR) have emerged as alternatives to overcome some of these drawbacks. PCR can detect mycobacterial DNA more rapidly than culture methods, enabling a quicker diagnosis. However, conventional PCR assays typically target a limited number of genes and may not offer comprehensive species identification, especially when dealing with closely related Mycobacterium species⁵.

DNA microarray technology has revolutionized the fields of microbiology and diagnostics. It offers the ability to simultaneously analyze multiple genetic targets, providing a more comprehensive and detailed view of the microbial population^8,9. Recent research in this area has further illuminated the potential and importance of advanced molecular techniques for Mycobacterium identification. For instance, a study by Wang et al.¹⁰ demonstrated the enhanced sensitivity and specificity of a modified DNA microarray chip in detecting rare Mycobacterium species in a clinical setting. Their findings emphasized the value of continuous optimization of microarray technology. Another study by Wang and colleagues¹¹focused on the integration of DNA microarray data with clinical outcomes in TB patients. The results provided insight into the prognostic value of accurate Mycobacterium species identification and the potential for personalized medicine.

Testing principle
This kit employs DNA microarray chip technology, combined with asymmetric polymerase chain reaction (PCR) amplification and hybridization principles, for the identification of Mycobacterium species. The kit includes a Mycobacterium species identification chip and asymmetric PCR amplification reagents. The chip is fixed with species-specific probes for the detection and identification of Mycobacterium species. The detection targets include 17 clinically common species or groups, such as the Mycobacterium tuberculosis complex, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium gordonae, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium scrofulaceum, Mycobacterium flavescens, Mycobacterium terrae, Mycobacterium chelonae and Mycobacterium abscessus, Mycobacterium phlei, Mycobacterium nonchromogenicum, Mycobacterium marinum and Mycobacterium ulcerans, Mycobacterium aurum, Mycobacterium szulgai and Mycobacterium malmoense, Mycobacterium xenopi, and Mycobacterium smegmatis.

The detection of these 17 Mycobacterium species is critical for accurate clinical diagnosis and tailored patient management. The Mycobacterium tuberculosis complex (MTBC) is prioritized due to its role in tuberculosis, necessitating immediate isolation and multidrug therapy to curb transmission and address drug resistance. Species within the Mycobacterium avium-intracellulare complex (MAC), such as M. avium and M. intracellulare, predominantly affect immunocompromised individuals (e.g., HIV/AIDS) or those with chronic lung disease, requiring prolonged macrolide-based regimens. Rapidly growing mycobacteria (RGMs) like M. abscessus and M. fortuitum are notorious for antibiotic resistance and often cause severe skin, soft tissue, or pulmonary infections, demanding aggressive combinations of surgery and targeted antibiotics (e.g., amikacin, macrolides). Environmental species such as M. gordonae or M. smegmatis are frequent contaminants but may cause opportunistic infections in specific contexts, requiring careful clinical correlation to avoid unnecessary treatment. Pathogens like M. marinum (associated with aquatic exposure) and M. ulcerans (causing Buruli ulcer) have distinct epidemiological niches, guiding exposure history assessments. Rare but clinically relevant species (e.g., M. szulgai, M. malmoense) mimic tuberculosis and require species-specific regimens. Accurate identification is paramount, as treatment duration, drug selection (e.g., rifampin sensitivity in M. kansasii vs. intrinsic resistance in NTMs), and prognosis vary drastically. Molecular diagnostics and antimicrobial susceptibility testing are essential to optimize outcomes, particularly in immunocompromised populations or complex infections.

First, the target sequences in the Mycobacterium genome are amplified through asymmetric PCR. Then, the amplified products are hybridized with the probes on the chip. If the target Mycobacterium is present in the sample, the amplified products will specifically bind to the corresponding probes on the chip. Finally, by scanning the hybridization signals on the chip, the species of Mycobacterium in the sample can be determined.

Using gene chip micro-arraying technology, specific probes for detecting the aforementioned genes and various control probes are fixed on the substrate. Each detection probe and control probe is repeated 5x, forming a 12 row x 10 column microarray. The probe arrangement is shown in Figure 1. Each chip contains four identical microarrays, and each microarray can detect one sample.

Protocol

This study was approved by the Ethics Committee of Quanzhou First Hospital Affiliated to Fujian Medical University (Approval No. 2024-K035). Informed consent was obtained from all patients prior to the collection of clinical specimens. Patients were informed about the purpose of the study, the procedures involved, and their right to withdraw at any time without affecting their medical care.

1. Sample requirements

NOTE: When collecting samples from patients with clinically suspected tuberculosis and non-tuberculous mycobacteria (NTM) diseases for testing with this kit, follow these steps:

1. Collect samples such as sputum, pus or secretions, bronchoalveolar lavage fluid, puncture fluid (including cerebrospinal fluid, pleural and peritoneal fluid, pericardial fluid, joint fluid, and bile), and urine from the patients.
   1. For sputum samples:
      1. Instruct the patient to rinse the mouth with clean water in the morning and then cough up deep sputum into a sterile sample storage container, seal it, and send it for testing.
      2. Add 1-2x the volume of 4% NaOH, shake, and mix evenly.
      3. After 15-20 min, add an equal volume of pH 6.8 phosphate buffer and mix evenly.
      4. At room temperature, centrifuge at 2,000 × g for 15 min and discard the supernatant. Add 1 mL of 0.9% NaCl via a pipette and mix thoroughly; use for detection.
   2. For pus or secretion samples:
      1. Rinse the surface with sterile normal saline. Collect the pus or secretion with a sterile swab, seal it, and send it for testing.
      2. Add 3x the volume of 4% NaOH and shake it thoroughly to mix well.
      3. Let it stand at room temperature for 20 min (shake it manually 2x during this period), centrifuge at 2,000 × g at room temperature for 15 min, and then discard the supernatant. Add 1 mL of 0.9% NaCl via a pipette and mix it evenly; use for detection.
   3. For bronchoalveolar lavage fluid:
      1. Inject 10-20 mL of normal saline through a protected bronchoalveolar lavage (PBAL) tube.
      2. Collect the sample in a sterile bottle by negative pressure suction and send it for testing.
   4. For cerebrospinal fluid:
      1. Collect at least 2 mL of sample using aseptic technique.
      2. Centrifuge at 2,000 × g for 15 min at room temperature. Discard the supernatant. Add 1 mL of 0.9% sodium chloride solution via a pipette, mix thoroughly, and use for testing.
   5. For urine samples:
      NOTE: It is advisable to use the first full amount of urine in the morning or the middle and late-night urine. The urine can also be collected by catheterization.
      1. Collect the sample in a sterile container and let the collected urine sample stand for 4-5 h.
      2. Centrifuge at 2,000 × g for 15 min at room temperature. Discard the supernatant. Add 1 mL of 0.9% sodium chloride solution via a pipette, mix thoroughly, and use for testing.
2. Sample storage
   NOTE: Samples on solid medium should not be stored frozen, as freezing may affect subsequent operations.
   1. Store samples existing in the liquid medium at -70 °C for no more than 5 years.
   2. Store all samples at 2-8 °C for no more than 2 months.
3. Sample transportation
   1. When transporting the test isolate samples, be sure to use a closed container that meets biosafety requirements under the conditions permitted by regulations.

2. Experiment preparation

1. Preparation of Chip Washing Solutions
   NOTE: Prepare Chip Washing Solution I and Chip Washing Solution II according to the number of chips. The volume should be such that the chips can be completely submerged during washing. Mix them evenly and equilibrate them to room temperature (10-30 °C). The final concentration of SSC is 2x, and the final concentration of SDS is 0.2%.
   1. To prepare Chip Washing Solution I, mix 20x SSC, distilled water (or purified water), and 10% SDS in the ratio of 10:88:2 in sequence to prepare Chip Washing Solution I. For example, when preparing a total volume of 600 mL, first measure 60 mL of 20x SSC and 528 mL of distilled water (or purified water) and place them in a 1 L beaker and mix evenly with a stirring rod. Then, add 12 mL of 10% SDS and mix evenly again.
   2. To prepare Chip Washing Solution II: The final concentration of SSC is 0.2x. Mix 20x SSC and distilled water (or purified water) in a ratio of 1:99 to obtain Chip Washing Solution II. Taking the preparation of a total volume of 600 mL as an example, measure 6 mL of 20x SSC and 594 mL of distilled water (or purified water) and place them in a 1 L beaker and mix evenly.
      NOTE: If the 10% SDS solution develops white flocculent precipitates, heat it at 50 °C, mix thoroughly, and then prepare the microarray washing buffer. If Washing Buffer I develops white flocculent precipitates, heat it at 50°C, mix thoroughly, equilibrate to room temperature before use.
2. Prepare 350 mL of ice-water mixture.
3. Get distilled water (or purified water) ready.

3. Preparation of instruments and materials

1. Instrument selection and setup
   1. Identify and prepare the microarray chip scanner, ensuring it is calibrated and ready for use.
   2. Set up the nucleic acid extraction instrument, checking its functions and loading necessary reagents.
   3. Place the Hybridization box gene microarray chip hybridization box in the appropriate location and confirm its proper working condition.
2. Pipetting tools preparation
   1. Gather micropipettes with specifications of 0.1-10 µL, 2-20 µL, and 20-200 µL, along with their corresponding clean and sterile pipette tips. Ensure the micropipettes are accurately calibrated and the tips are properly fitted.
3. Ancillary equipment arrangement
   1. Prepare a constant temperature water bath and set it to the required temperature (50 °C).
   2. Position the microcentrifuge capable of centrifuging 1.5 mL centrifuge tubes and check its balance and speed settings.
   3. Organize clean and sterile centrifuge tubes (200 µL and 1.5 mL) and optional clean and sterile eight-well strips (200 µL) on the centrifuge tube racks.
4. Sample handling equipment readiness
   1. Place a constant temperature shaker in a convenient location and set the appropriate shaking parameters.
   2. Set aside two containers for washing chips and ensure they are clean and dry.
   3. Arrange slide racks and a centrifuge, and have a container for centrifuging and drying chips at hand.
5. PCR amplifier preparation
   1. Switch on the PCR amplifier and program it according to the experimental protocol, ensuring it is ready to perform amplification reactions.

4. Nucleic acid extraction

NOTE: Carry out the operation in the specimen preparation area and follow these steps:

1. Preparation
   1. Add 80 µL of nucleic acid extraction solution into the nucleic acid extraction tube and add 1 mL of 0.9% NaCl.
2. Collection of isolate samples
   1. To initiate sample collection, examine the source of samples, whether they are from solid medium or liquid medium.
   2. To collect solid medium samples, use a sterile inoculating loop to pick a visible colony from the appropriate solid medium. If the bacteria have grown to cover the entire medium, then pick a corresponding amount of bacterial lawn. Carefully place it into the nucleic acid extraction tube containing the nucleic acid extraction solution, taking care to avoid picking the solid medium as much as possible.
   3. For liquid medium samples, aspirate 10-20 µL of the bacterial suspension with a turbidity equal to or greater than 1 McFarland standard. Inject it into the nucleic acid extraction tube containing the nucleic acid extraction solution.
      NOTE: After sample collection, whether from solid or liquid medium, the samples are ready for the subsequent nucleic acid extraction operation.
3. Nucleic acid extraction
   1. After adding the pretreated specimens or isolate samples from Step 1.1 mentioned above, use the nucleic acid rapid extraction instrument to extract nucleic acids. Place the extracted nucleic acids at -20 °C for temporary storage.
      NOTE: As an indicator of extraction efficiency, the automatic extractor can extract samples with a viral nucleic acid load greater than 100 copies/mL. The temporary storage time should not exceed 2 months.

5. PCR amplification

1. Preparation of PCR reaction system
   1. In the reagent storage and preparation area, prepare 200 µL centrifuge tubes or eight-well strips according to the number of samples to be tested, and mark the sample numbers in advance. Take out the PCR amplification reagents from the kit, let them thaw and melt naturally, gently shake and mix evenly, and then centrifuge quickly to make the reagents gather at the bottom of the tubes.
   2. According to the number of samples, aliquot the PCR amplification reagents into 200 µL centrifuge tubes or eight-well strips at a volume of 18 µL per tube. Tightly close the tube caps and then transfer them to the amplification reaction mixture preparation and amplification area.
   3. Add 2 µL of template DNA (the DNA of the sample to be tested (i.e., the supernatant in the nucleic acid extraction tube), positive control products, or negative control products) in the specimen preparation area. Ensure that the total volume of each PCR reaction system reaches 20 µL.
      NOTE: Use positive control products and negative control products every time during amplification. The positive control product can be used for quality control in the PCR amplification and hybridization processes, and the negative control product can be used to monitor the environment and the operation process.
2. Amplification
   1. Place the centrifuge tubes or eight-well strips containing the reagents in the PCR amplification instrument. Ensure the instrument is preheated to the required temperature. Conduct the PCR amplification reaction according to the following thermal cycling program: initial denaturation at 94 °C for 600 s (1 cycle); 35 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 40 s; a final extension at 72°C for 420 s (1 cycle); hold at 4 °C indefinitely (1 cycle).

6. Chip hybridization

NOTE: Before the PCR amplification reaction ends, perform the following operations:

1. Set the temperature of the constant temperature water bath to 50 °C and preheat it.
2. Preparation of chips
   1. Add 200 µL of distilled water (or purified water, as shown in Figure 2A) to the bottom of the gene microarray chip hybridization box. Place the bracket between the two positioning columns inside the box.
   2. Carefully place the chip with the front side up on the bracket, and cover the chip with the cover slip with its four bosses facing down (as shown in Figure 2B).
      NOTE: Ensure the correct placement direction of the cover slip so that its end aligns with the label at the end of the chip.
3. Preparation, denaturation, and cooling of hybridization reaction mixture
   1. Prepare 200 µL centrifuge tubes or eight-well strips according to the number of samples and label them properly.
   2. Take out the hybridization buffer from the kit and heat it at 50 °C until it is completely melted. After thorough mixing, centrifuge it quickly in a microcentrifuge. Aliquot 9 µL of the hybridization buffer into each prepared 200 µL centrifuge tube or eight-well strip. Then, add 6 µL of the corresponding PCR product to each tube, resulting in a total volume of 15 µL per hybridization reaction mixture.
   3. Heat the hybridization reaction mixture (including the hybridization buffer and the PCR product) to 95 °C (in a PCR instrument or a water bath) and denature it for 5 min. Take out the hybridization reaction mixture that has undergone the denaturation treatment in the previous step, and immediately immerse it in an ice-water mixture for 3 min.
4. Hybridization reaction
   1. Take the hybridization reaction mixture out of the ice bath, pipette it up and down 2x with a micropipette to mix it evenly. After the white flocculent precipitate (if any) disappears, add 13.5 µL of the hybridization reaction mixture through the loading hole of the cover slip (as shown in Figure 2C), quickly cover the hybridization box, and seal it (as shown in Figure 2D). Record the chip number, the position of the microarray, and the corresponding sample number.
      NOTE: Only one sample can be added to each microarray.
   2. Immediately place the sealed hybridization boxes horizontally into the constant temperature water bath that has been preheated to 50 °C. Start the timer for 120 min after all the hybridization boxes have been put in.
      NOTE: Avoid introducing air bubbles during the loading process. In all the steps from the start of loading until the chips are dried, avoid the liquid on the chip microarrays being directly exposed to the air for a long time to prevent the chip background from becoming too high due to drying and interfering with the result interpretation.
5. Washing and drying of chips
   1. After the hybridization reaction is completed, take out the hybridization boxes horizontally, disassemble them, and take out the chips for washing. Immediately place the chips on the slide rack in the container (such as a beaker) filled with Chip Washing Solution I that has been equilibrated to room temperature (10-30 °C). Wash them at a speed of 80-100 rpm on a constant temperature shaker for 3 min at room temperature.
   2. Wash the chips with Chip Washing Solution II equilibrated to room temperature at a speed of 80-100 rpm on a constant temperature shaker for 3 min at room temperature.
   3. Place the chips in a centrifuge and centrifuge at 100 × g at room temperature for 5 minutes. Scan them after drying.
      NOTE: Do not allow the chips to collide or rub against each other.

7. Chip scanning and result interpretation

NOTE: Use the microarray chip scanner and the corresponding software to read signals and interpret results. Also see Table 1 for abnormal results and troubleshooting. Follow these steps to operate (refer to the user manuals of the scanner and the software).

1. Turn on the scanner and the corresponding software, and then click the Laser Control button to let it preheat for 10 min.
2. Input various types of information related to the samples, such as the Sample Number.
3. After the scanner has finished preheating, click the Eject button, and place the chip steadily on the small bracket piece. Gently and horizontally push the chip into the opening of the scanner compartment and click on the Load button.
4. Input the chip number, click to select the detection area (microarray 1-4), and then click Select Sample to choose the corresponding samples for each microarray. Next, click the Start Detection button to conduct chip scanning. The scanning results will be displayed on the screen and automatically saved.
5. After scanning one chip, repeat steps 7.3 and 7.4 to scan the next chip. Follow this procedure until all chips have been scanned.
6. Use the Data Query page to conduct data query and printing operations.
7. After completing all operations, first turn off the laser, then exit the software, and finally turn off the scanner.

Results

Positive results
For the probes of Mycobacterium genus (internal control, IC) and Mycobacterium tuberculosis, their positive result values are determined by the Receiver Operating Characteristic (ROC) method. For the probes used for detecting the other 16 NTM, their positive result values are determined by the percentile method.

Experimental quality control
The detection result of the positive control product should be "Mycobacter...

Discussion

The DNA microarray chip method introduced in this paper can directly detect nucleic acids in clinical specimens (including sputum, pus, bronchoalveolar lavage fluid, cerebrospinal fluid, and puncture fluid) from patients suspected of having tuberculosis and non-tuberculous mycobacteriosis and can significantly shorten the time for confirmed diagnosis. The DNA microarray chip method presented in this study represents a significant advancement in the identification of Mycobacterium species. It offers several advan...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.9% Sodium Chloride Injection (NaCl)	FuZhou HAIWANGFUYAO	H35020289
Applied Biosystems amplification apparatus	Thermo Fisher Scientific	Veriti 96-Well Thermal Cycler
CapitalBio LuxScan10K/B	CapitalBio	LuxScan10K/B
CapitalBio SlideWasher 24	CapitalBio	SlideWasher 24
Centrifuge	Eppendorf	5424R	MaX.speed 15000 min-1
Hybridization buffer
HybSet gene microarray chip and cover
Mycobacterium species identification kit	CapitalBio	301030
Negative controls, positive controls
Nucleic acid extractor	TIANLONG	GeneRotex 96
PCR amplification reagent
pH 6.8 phosphate buffer	ShangHai yuanye Bio-Technology	R20029-10
pipettors and tips	Thermo Scientific	0.1 - 10 μL, 2 - 20 μL, and 20 - 200 μL, 100-1000 μL
qEx-DNA/RNA Nucleic acid extraction or purification kit	TIANLONG	qEx-DNA/RNA
SDS	CapitalBio	441060
Sodium hydroxide(NaOH)	XILONGS  Scientific	XK13-011-00027
SSC	CapitalBio	441050
thermostatic water bath	SURUI	HH-W600