Brucella colorimetric detection method based on DNAWalker driving double-signal amplification

Abstract

The invention develops a Brucella 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 Brucella.

Description

TECHNICAL FIELD

[0001] The application relates to a Brucella colorimetric detection method based on DNA walker-driven double-signal amplification, and belongs to the technical field of life science. BACKGROUND

[0002] Brucella melitensis is an important zoonotic pathogen, and its facultative intracellular parasitic characteristics enable it to evade immune clearance of the host, resulting in a low bacterial load (usually <10 2 The existing technology faces three challenges: (1) low DNA release efficiency caused by intracellular parasitism: traditional lysis methods can only release less than 30% of bacterial DNA, and are easily interfered by host DNA; (2) high serological false positive rate: the rose bengal plate agglutination test cross-reacts with lipopolysaccharide, and the specificity is only 72-85%; (3) insufficient sensitivity of molecular detection: the false negative rate of real-time fluorescent quantitative PCR in low-concentration samples (<10 fM) is as high as 40%. Although some studies have used isothermal amplification technology, complex sample pretreatment is still required, and it is impossible to distinguish between live and dead bacteria.

[0003] In recent years, colorimetric biosensing technology has improved the applicability in specific scenarios by integrating with smartphones and using visual signals. However, the existing systems are limited in detection sensitivity (usually with a detection limit of 1-10 nM) due to a single amplification mechanism and signal attenuation of nanoparticles. pH-responsive polydopamine nanostructures (PDANS) enhance signal stability through proton-coupled color reactions, but it is still difficult to achieve trace pathogen detection by acting alone. Similarly, DNA walking systems use self-targeting cycling of nicking endonucleases (such as Nt.BbvCI) to achieve exponential signal amplification, but the complex probe design and lack of a universal detection framework limit its clinical application.

[0004] Meanwhile, a double-signal amplification biosensing system is designed, which integrates a programmable DNA walker system with target sequence-independent recognition ability and a pH-responsive PDANS@Ag nanocomposite serving as a dual-functional signal converter, achieving stronger signal enhancement than traditional PDANS. The sandwich assembly technology based on magnetic beads realizes interference-free signal transduction. This integrated method not only achieves femtomolar-level sensitivity, but also maintains operational simplicity, which is suitable for field applications, opens up a new way for animal-derived pathogen detection, and fills the technical gap between laboratory diagnosis and field application needs. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application aims to provide a Brucella colorimetric detection method based on DNAWalker-driven double-signal amplification, which realizes high specificity and low false positive on-site detection of Brucella melitensis by optimizing the lysis step and probe design.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the present application is:

[0007] In order to achieve the above-mentioned purpose, the innovation of the present application is: ①Introducing a heat shock pretreatment step (65℃, 10min), which destroys the cell membrane structure of Brucella, and improves the DNA release efficiency; ②Designing specific nucleic acid probes for Brucella genes; ③Combining the pH response signal amplification of PDANS@Ag to realize the specific detection of Brucella.

[0008] A Brucella colorimetric detection method based on DNAWalker-driven double-signal amplification, comprising the following steps:

[0009] (1) Preparation of PDANS@Ag / TP / DP nanoparticles

[0010] 2mg of PDANS@Ag nanoparticles were ultrasonically dispersed in 900μL of ultrapure water. Then, 100μL of 10μM DP solution was added to the dispersion to make the final concentration 1μM. After the mixture was incubated at 37℃ for 12h, the product was centrifuged and washed three times to remove the unbound DP. The DP-modified PDANS@Ag nanoparticles (PDANS@Ag / DP) were redispersed in 1mL of water. 400μL of 10mM TP solution was added to the PDANS@Ag / DP solution. The mixture was shaken at 37℃ for 2h, and then centrifuged and washed three times at 4℃, 10,000rpm (10min each time). The final product PDANS@Ag / TP / DP was redispersed in 1mL of water and stored at 4℃ for standby;

[0011] (2) Magnetic bead functionalization modification

[0012] 10μL of 10mg / mL streptavidin-coated magnetic beads were washed three times with 1mL of Tris-HCl buffer (50mM, pH 7.4) by magnetic separation. Further, the magnetic beads were mixed with the capture probe (CP, 6μM, 5μL) in 250μL of Tris-HCl buffer, incubated at 37℃ for 90min, coupled, and washed three times by magnetic separation to remove the unbound probe. After blocking with 200μL of BSA solution (1%w / v, dissolved in Tris-HCl) at 37℃ for 1h, the functionalized magnetic beads were resuspended and stored at 4℃ for standby.

[0013] (3) Enzymatic cleavage cycle and colorimetric detection

[0014] Take the functionalized magnetic beads 10 μL obtained in claim 4, after magnetic separation, mix with the following 3 μL reagents: AP (2 μM, 7.5 μL), Nt.BbvCI enzyme (0.5 μL), NEBuffer (10x, 2 μL), then add 30 μL detection sample, 37°C, oscillation incubation for 1 h, after magnetic separation and washing, add PDANS@Ag / TP / DP nanoparticles 25 μL obtained in claim 3 to the magnetic beads, 37°C reaction for 90 min. After washing, add 200 μL alkaline color developing solution (0.1M NaOH, pH 13.0) to start the color developing reaction, after 16 s, use spectrophotometry (OD 450 , SpectraMax M5 microplate reader) or smartphone-based analysis method to quantitatively detect the generated colorimetric signal. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The schematic diagram of the principle of the inventive detection method.

[0016] Figure 2 The linear relationship diagram of the detected absorbance and different Brucella melitensi DNA concentrations. DETAILED DESCRIPTION

[0017] The specific embodiments of the present application will be further described in detail below in combination with the examples.

[0018] Example 1. DNA oligonucleotide design DP: 5'-NH2-(CH2)6-GGGGGTCCTGCTGA-3', Target DNA Bmel : 5'-TGC TGA GGA AAGTTCTCGAAT-3', CP Bmel : 5'-TGC TGA GGA AAGTTCTCGAAT-3', AP Bmel : 5'-TGC TGA GGA AAGTTCTCGAAT-3', Single mismatch CP-bound region: 5'-GAGAATCCGGGAAGGATGAC-3', Single mismatch AP-bound region: 5'-GAGAATACGGGACGGATGAC-3', Double mismatch CP-bound region: 5'-GAGAATCCGGGAAGCATGAC-3', Double mismatch AP-bound region: 5'-GAGAGTACGGGACGGATGAC-3', Single mismatches (CP & AP): 5'-GAGAATACGGGAAGGATGAC-3'.

[0019] Example 2. Construction of the detection method

[0020] A Brucella colorimetric detection method based on DNA Walker-driven double-signal amplification, comprising the following steps:

[0021] (1) Preparation of PDANS@Ag / TP / DP nanoparticles

[0022] 2 mg of PDANS@Ag nanoparticles were ultrasonically dispersed in 900 μL of ultrapure water. Subsequently, 100 μL of a 10 μM DP solution was added to the dispersion to achieve a final concentration of 1 μM. After the mixture was incubated 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 a 10 mM TP solution was added to the PDANS@Ag / DP solution. The mixture was shaken at 37°C for 2 h, and then centrifuged and washed three times at 4°C and 10,000 rpm for 10 min each time. The final product PDANS@Ag / TP / DP was redispersed in 1 mL of water and stored at 4°C for later use;

[0023] (2) Magnetic bead functionalization modification

[0024] 10 μL of 10 mg / mL streptavidin-coated magnetic beads were washed three times with 1 mL of Tris-HCl buffer (50 mM, pH 7.4) by magnetic separation. Further, the magnetic beads were mixed with the capture probe (CP, 6 μM, 5 μL) in 250 μL of Tris-HCl buffer, and incubated at 37°C for 90 min by rotation for coupling. The unbound probe was removed by magnetic separation and washing three times. After blocking with 200 μL of a BSA solution (1% w / v in Tris-HCl) at 37°C for 1 h, the functionalized magnetic beads were resuspended and stored at 4°C for later use.

[0025] (3) Enzymatic cleavage cycle and colorimetric detection

[0026] Take the functionalized magnetic beads 10 μL obtained in claim 4, after magnetic separation, mix with the following 3 μL reagents: AP (2 μM, 7.5 μL), Nt.BbvCI enzyme (0.5 μL), NEBuffer (10x, 2 μL), then add 30 μL detection sample, heat shock pretreatment at 65 °C for 10 min, then incubate at 37 °C for 1 h, after magnetic separation and washing, add PDANS@Ag / TP / DP nanoparticles 25 μL obtained in claim 3 to the magnetic beads, react at 37 °C for 90 min. After washing, add 200 μL alkaline color developing solution (0.1 M NaOH, pH 13.0) to start the color developing reaction, and after 16 s, use spectrophotometry (OD 450 , SpectraMax M5 microplate reader) or a smartphone-based analysis method to quantitatively detect the generated colorimetric signal.

[0027] Example 3. Performance evaluation

[0028] The target DNA was diluted in ultrapure water for comparison, generating solutions with a concentration range from 10 -6 nM to 10 nM to establish the analysis linearity of the biosensor system. Each concentration was measured in triplicate, and a calibration curve was constructed by linear regression analysis. Specificity evaluation was performed by parallel detection of six different DNA variants (1 nM each): fully matched target DNA, single-base mismatch DNA at the CP binding site or the AP binding site, double-base mismatch DNA at the CP or AP binding site, and double single-base mismatch DNA at the CP and AP binding sites. All experiments included a blank control with nuclease-free water.

[0029] Example 4. Actual sample detection

[0030] To evaluate the performance of the sensor in real samples, milk and egg were chosen as the detection matrix. Positive samples were prepared by adding 100 μL of simulated bacterial lysate to 900 μL of milk or egg solution. Negative controls were prepared by replacing the bacterial lysate with an equal volume of buffer. Both positive and negative samples were analyzed to evaluate the detection performance.

Claims

1. A colorimetric detection method for Brucella based on DNAWalker-driven dual-signal amplification. The detection principle is as follows: Target DNA of the Brucella invA gene is pretreated with heat shock at 65℃ for 10 min, triggering hybridization between the target DNA and AP, forming a Y-link with CP, exposing the Nt.BbvCI restriction site. Through enzyme cleavage cycles, the DNAWalker is released, achieving target cyclic amplification. Simultaneously, Fe... 2+ PDANS (palladium-iron nanoparticles) were synthesized via the Fenton reaction using H2O2, 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 via 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 Brucella.

2. The method according to claim 1, characterized in that... The heat shock pretreatment was performed in Tris-HCl buffer (pH 8.0) to enhance intracellular DNA release.

3. The Brucella colorimetric detection method 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 Bmel :5′-TGC TGA GGA AAGTCTTCGAAT-3′, CP Bmel :5′-TGC TGA GGA AAGTTCTCGAAT-3′, AP Bmel :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′.

4. The method for colorimetric detection of Brucella 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.

5. The Brucella colorimetric detection method 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.

6. The method for colorimetric detection of Brucella based on DNAWalker-driven dual-signal amplification according to claim 1, wherein the steps of enzyme digestion cycle and colorimetric detection 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 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 the linear detection equation for different concentrations of Brucella melitensis. The absorbance 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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