Mycobacterium tuberculosis colorimetric detection method based on DNAWalker driving double-signal amplification

Abstract

The invention discloses a mycobacterium tuberculosis colorimetric detection method based on DNAWalker driving double-signal amplification. The detection principle is as follows: target DNA triggers AP hybridization to form Y-type connection with CP, an Nt.BbvCI restriction enzyme cutting site is exposed, DNAWalker is released through restriction enzyme cutting circulation, and target circulation amplification is realized. Meanwhile, PDANS (palladium-iron-nano particles) is synthesized through Fe < 2 + > / H2O2 Fenton reaction, a pH indicator is loaded through silver nano particle modification (PDANS (at) Ag), and a signal is enhanced. An amplified product is combined with PDANS (at) Ag through a magnetic bead sandwich structure, remarkable color change is caused, and quantitative detection can be performed through a smart phone App (such as' Color Grab ') or a microwell plate reader. The method is easy and convenient to operate, high in sensitivity and suitable for on-site rapid identification of mycobacterium tuberculosis.

Description

Technical Field

[0001] A colorimetric detection method for Mycobacterium tuberculosis based on DNAWalker-driven dual-signal amplification belongs to the field of life science technology. Background Technology

[0002] Mycobacterium tuberculosis, a deadly zoonotic pathogen, causes more than 10 million new infections and 1.3 million deaths worldwide each year. Its complex cell wall structure—rich in mycolic acid (60% of dry weight), with multi-layered cross-links of arabinogalactan and peptidoglycan—constitutes a significant detection barrier. This structural feature leads to: (1) environmental resistance: tolerance to 70% ethanol disinfectant for >5 min, and survival in dried sputum for >8 weeks; (2) detection delay: culture method: dependent on Roche medium. Slant growth requires 3-8 weeks for identification; smear microscopy: Zinis acid-fast staining sensitivity is only 10. 4 With a detection rate of CFU / mL, the false negative rate reaches 40-60%. Although molecular diagnostic technologies (such as GeneXpert MTB / RIF) shorten the detection time to 2 hours, they rely on a stable power supply, cold chain, and professional operators, resulting in a coverage rate of <35% in resource-scarce areas (such as sub-Saharan Africa). Existing colorimetric sensing technologies have limited sensitivity due to their inability to efficiently lyse cell walls and cannot achieve point-of-care testing.

[0003] In recent years, colorimetric biosensing technology has enhanced its applicability in specific scenarios by integrating with smartphones and utilizing visual signals. However, existing systems suffer from limited detection sensitivity (typically with a detection limit of 1-10 nM) due to their single amplification mechanism and signal attenuation caused by nanoparticles. pH-responsive polydopamine nanostructures (PDANS) enhance signal stability through proton-coupled colorimetric reactions, but their sole function is still insufficient for detecting trace pathogens. Similarly, DNA-walking systems utilize nicking endonucleases (such as Nt.BbvCI) to achieve exponential signal amplification through autonomous target cycling, but their clinical application is limited by the complexity of probe design and the lack of a universal detection framework.

[0004] Concurrently, a dual-signal amplification biosensor system was designed. By integrating a programmable DNA walker system with target sequence-independent recognition capabilities and a pH-responsive PDANS@Ag nanocomposite material that acts as a bifunctional signal transducer, a stronger signal enhancement effect than traditional PDANS was achieved. Based on a magnetic bead-based sandwich assembly technology, interference-free signal transduction was realized. This integration method not only achieved femtomolar-level sensitivity but also maintained ease of operation, making it suitable for field applications. This opens up new avenues for Mycobacterium tuberculosis detection and fills the technological gap between laboratory diagnostics and field application needs. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to solve the problem of Mycobacterium tuberculosis cell wall lysis by constructing a colorimetric detection method for Mycobacterium tuberculosis based on DNAWalker-driven dual-signal amplification, thereby enabling timely on-site detection and efficient differential diagnosis of Mycobacterium tuberculosis.

[0006] To achieve the above objectives, the innovative aspects of the technical solution of this invention include: ① integrating low-frequency ultrasound-assisted lysis (40kHz, 5min) to increase cell wall permeability and improve DNA extraction rate; ② designing nucleic acid probes targeting Mycobacterium tuberculosis genes and combining them with the efficient nicking cycle of Nt.BbvCI enzyme to shorten the detection time to 30min.

[0007] A colorimetric detection method for Mycobacterium tuberculosis based on DNAWalker-driven dual-signal amplification includes the following steps:

[0008] (1) Preparation of PDANS@Ag / TP / DP nanoparticles

[0009] 2 mg of PDANS@Ag nanoparticles were ultrasonically dispersed in 900 μL of ultrapure water. Then, 100 μL of 10 μM DP solution was added to the dispersion to achieve a final concentration of 1 μM. After incubating the mixture with shaking at 37 °C for 12 h, the product was centrifuged and washed three times to remove unbound DP. The DP-modified PDANS@Ag nanoparticles (PDANS@Ag / DP) were redispersed in 1 mL of water. 400 μL of 10 mM TP solution was added to the PDANS@Ag / DP solution. The mixture was shaken at 37 °C for 2 h, followed by centrifugation and washing three times (10 min each time) at 4 °C and 10,000 rpm. The final product, PDANS@Ag / TP / DP, was redispersed in 1 mL of water and stored at 4 °C for later use.

[0010] (2) Functional modification of magnetic beads

[0011] 10 μL of streptavidin (10 mg / mL) was coated onto magnetic beads and magnetically washed three times with 1 mL Tris-HCl buffer (50 mM, pH 7.4). The magnetic beads were then further coupled by mixing with the capture probe (CP, 6 μM, 5 μL) in 250 μL Tris-HCl buffer and incubating at 37 °C for 90 min by rotation. The mixture was then magnetically washed three times to remove unbound probe. After blocking with 200 μL BSA solution (1% w / v, dissolved in Tris-HCl) at 37 °C for 1 h, the functionalized magnetic beads were resuspended and stored at 4 °C for later use.

[0012] (3) Enzyme digestion cycle and colorimetric detection

[0013] Take 10 μL of the functionalized magnetic beads obtained in claim 4, perform magnetic separation, and mix with 3 μL of the following reagents: AP (2 μM, 7.5 μL), Nt. BbvCI enzyme (0.5 μL), and NEBuffer (10×, 2 μL). Then add 30 μL of the detection sample, incubate at 37°C with shaking for 1 h, and after magnetic separation and washing, add 25 μL of PDANS@Ag / TP / DP nanoparticles obtained in claim 3 to the magnetic beads, and react at 37°C for 90 min. After washing, add 200 μL of alkaline colorimetric solution (0.1 M NaOH, pH 13.0) to start the colorimetric reaction, and detect by spectrophotometry after 16 s (OD). 450 The generated colorimetric signals can be quantitatively detected using a SpectraMax M5 microplate reader or a smartphone-based analysis method. Attached Figure Description

[0014] Figure 1 This is a schematic diagram illustrating the principle of the invention's detection method.

[0015] Figure 2 Linear relationship between absorbance and different concentrations of Mycobacterium tuberculosis DNA. Detailed Implementation

[0016] The specific embodiments of the present invention will be further described in detail below with reference to examples.

[0017] Example 1. Design of DNA oligonucleotides: DP: 5′-NH2-(CH2)6-GGGGGTCCTGCTGA-3′ Target DNA TB :5′-ATTCGAGAACATTGGGAATC-3′, CP TB :5′-TCCTGGATTCCCAATTCCTCAGCAGGAGGGGG-biotin-3′, AP TB :5′-TGC TGA GGA AAGTTCTCGAAT-3′, Single mismatch CP-bound region: 5′-GAGAATCCGGGAAGGATGAC-3′, Single mismatch AP-bound region: 5′-GAGAATACGGGGACGGATGAC-3′, Double mismatch CP-bound region: 5′-GAGAATCCGGGAAGCATGAC-3′, Double mismatch AP-bound region: 5′-GAGAGTACGGGACGGATGAC-3′, Single mismatches (CP&AP): 5′-GAGAATACGGGAAGGATGAC-3′.

[0018] Example 2. Construction of the detection method

[0019] A colorimetric detection method for Mycobacterium tuberculosis based on DNAWalker-driven dual-signal amplification includes the following steps:

[0020] (1) Preparation of PDANS@Ag / TP / DP nanoparticles

[0021] 2 mg of PDANS@Ag nanoparticles were ultrasonically dispersed in 900 μL of ultrapure water. Then, 100 μL of 10 μM DP solution was added to the dispersion to achieve a final concentration of 1 μM. After incubating the mixture with shaking at 37 °C for 12 h, the product was centrifuged and washed three times to remove unbound DP. The DP-modified PDANS@Ag nanoparticles (PDANS@Ag / DP) were redispersed in 1 mL of water. 400 μL of 10 mM TP solution was added to the PDANS@Ag / DP solution. The mixture was shaken at 37 °C for 2 h, followed by centrifugation and washing three times (10 min each time) at 4 °C and 10,000 rpm. The final product, PDANS@Ag / TP / DP, was redispersed in 1 mL of water and stored at 4 °C for later use.

[0022] (2) Functional modification of magnetic beads

[0023] 10 μL of streptavidin (10 mg / mL) was coated onto magnetic beads and magnetically washed three times with 1 mL Tris-HCl buffer (50 mM, pH 7.4). The magnetic beads were then further coupled by mixing with the capture probe (CP, 6 μM, 5 μL) in 250 μL Tris-HCl buffer and incubating at 37 °C for 90 min by rotation. The mixture was then magnetically washed three times to remove unbound probe. After blocking with 200 μL BSA solution (1% w / v, dissolved in Tris-HCl) at 37 °C for 1 h, the functionalized magnetic beads were resuspended and stored at 4 °C for later use.

[0024] (3) Enzyme digestion cycle and colorimetric detection

[0025] Take 10 μL of the functionalized magnetic beads obtained in claim 4, perform magnetic separation, and mix with 3 μL of the following reagents: AP (2 μM, 7.5 μL), Nt. BbvCI enzyme (0.5 μL), and NEBuffer (10×, 2 μL). The sample is first sonicated at 40 kHz for 5 min, then incubated at 37 °C with shaking for 1 h. After magnetic separation and washing, add 25 μL of PDANS@Ag / TP / DP nanoparticles obtained in claim 3 to the magnetic beads and react at 37 °C for 90 min. After washing, add 200 μL of alkaline colorimetric solution (0.1 M NaOH, pH 13.0) to initiate the colorimetric reaction. Detect the colorimetric reaction using spectrophotometry after 16 s (OD). 450 The generated colorimetric signals can be quantitatively detected using a SpectraMax M5 microplate reader or a smartphone-based analysis method.

[0026] Example 3. Performance Evaluation

[0027] The target DNA was comparatively diluted in ultrapure water to generate concentrations ranging from 10... -6 To establish analytical linearity for the biosensor system, solutions were prepared at 10 nM. Triple measurements were performed for each concentration, and calibration curves were constructed using linear regression analysis. Specificity was assessed by parallel detection of six different DNA variants (1 nM each): perfectly matched target DNA, single-base mismatched DNA at either the CP or AP binding site, double-base mismatched DNA at either the CP or AP binding site, and double single-base mismatched DNA at both the CP and AP binding sites. All experiments included a nuclease-free water blank control.

[0028] Example 4. Actual Sample Detection

[0029] To evaluate the sensor's performance in real samples, milk and eggs were selected as detection matrices. Positive samples were prepared by adding 100 μL of simulated bacterial lysis buffer to 900 μL of milk or egg solution. Negative controls were prepared by replacing the bacterial lysis buffer with an equal volume of buffer solution. Both positive and negative samples were analyzed to evaluate detection performance.

Claims

1. A colorimetric detection method for Mycobacterium tuberculosis based on DNAWalker-driven dual-signal amplification. The detection principle is as follows: The target DNA is the Mycobacterium tuberculosis gene. After pretreatment with 40kHz ultrasound for 5 min, AP hybridization is triggered, forming a Y-link with CP, exposing the Nt.BbvCI restriction site. Through enzyme digestion cycles, the DNAWalker is released, achieving target cyclic amplification. Simultaneously, Fe... 2+ PDANS (palladium-iron nanoparticles) were synthesized via the / H2O2 Fenton reaction, and then modified with silver nanoparticles (PDANS@Ag) to load a pH indicator and enhance the signal. The amplified product binds to PDANS@Ag through a magnetic bead sandwich structure, inducing a significant color change, which can be quantitatively detected by a smartphone app (such as "Color Grab") or a microplate reader. This method is simple to operate, highly sensitive, and suitable for rapid on-site identification of Mycobacterium tuberculosis.

2. The colorimetric detection method for Mycobacterium tuberculosis based on DNAWalker-driven dual-signal amplification according to claim 1, characterized in that... The DNA oligonucleotides described in the detection method have the following sequences: DP: 5′-NH2-(CH2)6-GGGGGTCCTGCTGA-3′, Target DNA TB :5′-ATTCGAGAACATTGGGAATC-3′, CP TB :5′-TCCTGGATTCCCAATTCCTCAGCAGGAGGGGG-biotin-3′, AP TB :5′-TGC TGA GGA AAGTTCTCGAAT-3′, Single mismatch CP-bound region: 5′-GAGAATCCGGGAAGGATGAC-3′, Single mismatch AP-bound region: 5′-GAGAATACGGGGACGGATGAC-3′, Double mismatch CP-bound region: 5′-GAGAATCCGGGAAGCATGAC-3′, Double mismatch AP-bound region: 5′-GAGAGTACGGGACGGATGAC-3′, Single mismatches (CP&AP): 5′-GAGAATACGGGAAGGATGAC-3′.

3. The colorimetric detection method for Mycobacterium tuberculosis based on DNAWalker-driven dual-signal amplification according to claim 1, wherein the preparation steps of PDANS@Ag / TP / DP nanoparticles are as follows: 2 mg of PDANS@Ag nanoparticles are ultrasonically dispersed in 900 μL of ultrapure water. Subsequently, 100 μL of 10 μM DP solution is added to the dispersion to achieve a final concentration of 1 μM. After incubating the mixture at 37°C with shaking for 12 h, the product is centrifuged and washed three times to remove unbound DP. The DP-modified PDANS@Ag nanoparticles (PDANS@Ag / DP) are redispersed in 1 mL of water. 400 μL of 10 mM TP solution is added to the PDANS@Ag / DP solution. The mixture is shaken at 37°C for 2 h, followed by centrifugation and washing three times (10 min each time) at 4°C and 10,000 rpm. The final product, PDANS@Ag / TP / DP, is redispersed in 1 mL of water and stored at 4°C for later use.

4. The colorimetric detection method for Mycobacterium tuberculosis based on DNAWalker-driven dual-signal amplification according to claim 1, wherein the magnetic bead functionalization modification step is as follows: 10 μL of 10 mg / mL streptavidin is coated onto magnetic beads, and the beads are magnetically washed three times with 1 mL Tris-HCl buffer (50 mM, pH 7.4). The magnetic beads are then further mixed with a capture probe (CP, 6 μM, 5 μL) in 250 μL Tris-HCl buffer and incubated at 37°C for 90 min by rotation to couple the beads. The beads are then magnetically washed three times to remove unbound probe. After blocking with 200 μL BSA solution (1% w / v, dissolved in Tris-HCl) at 37°C for 1 h, the functionalized magnetic beads are resuspended and stored at 4°C for later use.

5. A colorimetric detection method for Mycobacterium tuberculosis based on DNAWalker-driven dual-signal amplification according to claim 1, wherein the enzyme digestion cycle and colorimetric detection steps are as follows: Take 10 μL of the functionalized magnetic beads obtained in claim 4, perform magnetic separation, and mix with the following 3 μL reagents: AP (2 μM, 7.5 μL), Nt.BbvCI enzyme (0.5 μL), NEBuffer (10×, 2 μL), then add 30 μL of the detection sample, incubate at 37°C with shaking for 1 h, wash after magnetic separation, add 25 μL of PDANS@Ag / TP / DP nanoparticles obtained in claim 3 to the magnetic beads, and react at 37°C for 90 min. After washing, add 200 μL of alkaline colorimetric solution (0.1M NaOH, pH 13.0) to start the colorimetric reaction, and detect by spectrophotometry after 16 s (OD). 450 The SpectraMax M5 microplate reader or a smartphone-based analysis method can be used to quantitatively detect the generated colorimetric signals, plot a standard curve, and obtain a linear detection equation for different concentrations of Mycobacterium tuberculosis. The absorbance obtained from the detection of samples with unknown concentrations can be substituted into the corresponding linear detection equation to calculate the concentration of the target bacteria.

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