Antibiotic resistance gene detection method based on click chemistry coupling biosensing

Abstract

The invention provides an antibiotic resistance gene detection method based on click chemistry coupling biosensing, which can realize the fly-mole-level identification and detection of resistance genes (ARG) in water. Firstly, captured DNA is fixed to the surface of a streptavidin biosensor of a multi-channel biological interferometer, and in the presence of a target ARG, the captured DNA and signal DNA are hybridized to the adjacent position of the target ARG, so that a click structure is formed. Afterwards, a biosensor with a click structure is marked with horse radish peroxidase and immersed in a diaminobenzidine substrate, insoluble crystals are formed and deposited on the surface of the biosensor, and the thickness of a biological layer at the tip of the sensor is changed; and quantitatively detecting the resistance gene in the to-be-detected sample according to the linear relation obtained by the induced wavelength shift and the target ARG concentration. According to the method, ultra-sensitive and high-selectivity detection of the resistance genes in the water environment is realized.

Description

Technical Field

[0001] The invention belongs to the field of analysis and detection of new pollutants in environmental protection, and relates to a click chemistry coupled biosensor antibiotic resistance gene detection method, which is suitable for sensitive and rapid detection of low-concentration resistance genes in real water environments. Background Art

[0002] Bacterial infections involving antibiotic resistance are threatening public health. Therefore, effective monitoring of resistance genes is crucial to address the antibiotic resistance crisis. In addition, different types of resistance genes coexist in the same area, leading to the emergence of multidrug-resistant bacteria, rendering modern antimicrobial drugs ineffective, and even increasing the morbidity and mortality of clinical diseases. Traditional resistance gene detection methods, such as quantitative polymerase chain reaction and metagenomic sequencing, rely on nucleic acid amplification. However, the strict requirements of these strategies for intact DNA templates limit their ability to accurately analyze highly degraded ARG fragments, and it is difficult to achieve rapid quantification of free ARGs in complex environmental matrices. In addition, these methods are complex and time-consuming to operate, and are not suitable for real-time or online monitoring. Therefore, it is urgent to develop new analytical detection methods that can efficiently identify resistance genes without amplification and achieve high-throughput rapid targeted analysis of this type of pollutants. Summary of the invention

[0003] The purpose of the present invention is to provide a method for detecting antibiotic resistance genes by click chemistry coupled biosensing, which can achieve highly sensitive and highly selective targeted recognition and detection of resistance genes in complex water environment media. First, the capture DNA is fixed on the surface of the streptavidin sensor of the multi-channel biointerferometer to construct an ARG recognition biosensor. In the presence of the target ARG, the capture DNA and the signal DNA hybridize to adjacent positions on the target ARG, thereby generating a "click" structure. In order to further amplify the detection signal, the "click" structure biosensor is labeled with horseradish peroxidase and immersed in a diaminobenzidine substrate for signal amplification. The insoluble crystals formed after the reaction are precipitated on the surface of the streptavidin sensor, resulting in a change in the thickness of the biolayer at the tip of the sensor. The linear relationship between the wavelength shift caused by this and the target ARG concentration is used to quantitatively detect the resistance gene concentration in the sample to be tested. The method is fast, sensitive and highly selective.

[0004] The specific technical solution for achieving the purpose of the present invention is:

[0005] Step 1: Design two DNA probes to carry out click chemistry-mediated chain reaction. First, construct capture DNA and signal DNA with a base number of 13 bp; capture DNA: labeled with azide N3 at its 3' end and biotin at its 5' end; signal DNA: labeled with dibenzocyclooctyl DBCO at its 5' end and biotin at its 3' end; target ARGs to be detected include tetracycline or sulfonamide resistance genes tetA, tetC, tetG, tetM or sul1;

[0006] Step 2: fix the capture DNA constructed in step 1 on the surface of the streptavidin sensor of the multi-channel biointerferometer to obtain a streptavidin sensor fixed with the capture DNA; the capture DNA concentration is 250-1000nM, and the fixation time is 200-500s;

[0007] Step 3: Soak the streptavidin sensor with the captured DNA fixed thereon constructed in step 2 in a skim milk powder solution to block the avidin sites on the streptavidin sensor that have not yet been bound; the concentration of the skim milk powder is 1%-5%, and the blocking time is 60-120s;

[0008] Step 4: Immerse the streptavidin sensor blocked with skimmed milk powder in step 3 in PBS phosphate buffer solution to obtain a stable baseline; pH of PBS phosphate buffer solution: 7.0-7.5, stabilization time: 60-120s;

[0009] Step 5: Soak the balanced streptavidin sensor in step 4 in an analytical solution containing different concentrations or types of resistance genes; the target resistance genes include tetA, tetC, tetG, tetM or sul1, the concentration is 1fM-1nM, and the soaking time is 200-500s;

[0010] Step 6: Soak the streptavidin sensor bound to the resistance gene in step 5 in a buffer solution containing signal DNA. In the presence of the target ARG, the capture DNA and the signal DNA are hybridized to adjacent positions on the target ARG, so that the N3 and DBCO groups are close to form a triazole bond, thereby generating a "click" structure; the concentration of the buffer solution containing the signal DNA is 5-10nM, and the binding time is 200-300s;

[0011] Step 7: Immerse the streptavidin sensor with the "click" structure generated in step 6 in a buffer containing horseradish peroxidase-labeled avidin to label the "click" structure with horseradish peroxidase; the concentration of the buffer containing horseradish peroxidase-labeled avidin is 1-5 ng / mg; the labeling time is 200-300 s;

[0012] Step 8: Immerse the streptavidin sensor with the "click" structure labeled with horseradish peroxidase in step 7 in a diaminobenzidine substrate for signal amplification; the signal amplification time is 300-600s; the insoluble crystals formed after the reaction are precipitated on the surface of the streptavidin sensor, causing the thickness of the biolayer at the tip of the sensor to change, and the linear relationship between the wavelength shift induced by this and the target ARG concentration is used to quantitatively detect the resistance gene concentration in the sample to be tested.

[0013] Beneficial effects:

[0014] The present invention is based on the working principle of multi-channel biofilm interferometer combined with click chemistry and enzyme-linked immunosorbent assay signal amplification strategy to establish an efficient ARG identification method, and realizes high sensitivity, high selectivity, high throughput, and online monitoring of ARG in complex environmental media. In addition, the present invention uses a small amount of detection sample, has a wide range of applications, a simple and fast experimental process, and can simultaneously detect up to 8 samples in one test, and has a unique multi-channel advantage. This feature makes the present invention an ideal tool for rapid and reliable monitoring of ARG in complex environmental media. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 When the present invention is used to quantify the concentration of ARG in water in Experimental Example 1, the response signal of the multi-channel bio-interferometer changes with the ARG concentration and the established standard curve diagram;

[0016] Figure 2 This is a graph showing the anti-interference performance of the present invention in Experimental Example 2 when determining 1, 3, and all-base mismatched sequences alone or in combination with the target ARG;

[0017] Figure 3 This is a diagram of the repeatability of the sensing method established by the present invention in Experimental Example 3. DETAILED DESCRIPTION

[0018] The present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0019] Example 1

[0020] A click chemistry coupled biosensor antibiotic resistance gene detection method comprises the following steps:

[0021] (1) First, two DNA probes with a base number of 13 bp were designed: capture DNA: labeled with azide (N3) at its 3' end and biotin at its 5' end; signal DNA: labeled with dibenzocyclooctyl (DBCO) at its 5' end and biotin at its 3' end. See Table 1 for the capture DNA, signal DNA, target ARG (tetA, tetC, tetG, tetM and sul1) designed in this example, and 1, 3, and all-base mismatch sequences;

[0022] (2) Pre-wetting the streptavidin sensor of the multi-channel bio-interferometer in PBS buffer solution for 30 minutes;

[0023] (3) Soak the streptavidin sensor in step (2) in PBS buffer solution for 60 seconds to obtain a stable baseline;

[0024] (4) soaking the streptavidin sensor in step (3) in the captured DNA in step (1) to couple avidin and biotin, and preparing a streptavidin sensor loaded with captured DNA, wherein the captured DNA concentration is 500 nM and the fixation time is 300 seconds;

[0025] (5) Soaking the streptavidin sensor loaded with captured DNA in step (4) in a 5% skim milk powder solution to block the excess avidin binding sites on the sensor for 90 seconds;

[0026] (6) Soaking the streptavidin sensor sealed with skimmed milk powder in step (5) in a PBS buffer solution to obtain a stable baseline II;

[0027] (7) immersing the streptavidin sensor in step (6) in an analytical solution containing different concentrations or types of resistance genes for a binding time of 300 seconds; detecting the target ARG (tetA, tetC, tetG, tetM or sul1) at a concentration of 1 fM-1 nM;

[0028] (8) immersing the streptavidin sensor after detecting the resistance gene in step (7) in a buffer solution containing signal DNA, and in the presence of the target ARG, the capture DNA and the signal DNA are hybridized to adjacent positions on the target ARG, so that the N3 and DBCO groups are close to form a triazole bond, thereby generating a "click" structure, the signal DNA concentration is 10 nM, and the binding time is 300 seconds;

[0029] (9) immersing the streptavidin sensor with the "click" structure generated in step (8) in a buffer solution containing 1 ng / mg horseradish peroxidase-labeled avidin, labeling the "click" structure with horseradish peroxidase for 300 seconds;

[0030] (10) The streptavidin sensor with the "click" structure labeled with horseradish peroxidase in step (9) was immersed in a diaminobenzidine substrate for signal amplification for 600 seconds. The insoluble crystals formed after the reaction were precipitated on the surface of the biosensor, causing the thickness of the biolayer at the tip of the sensor to change. The linear relationship between the wavelength shift caused by this and the target ARG concentration was used to quantitatively detect the resistance gene concentration in the sample to be tested. Figure 1A, B, C, D, and E are the response signal changes and linear correlation standard curves generated when the multi-channel bio-interferometer detects tetA, tetC, tetG, tetM, and sul1, respectively. In the figure, a, b, c, d, e, f, and g represent the resistance gene concentrations of 1fM, 10fM, 100fM, 1pM, 10pM, 100pM, and 1nM. Figure 1 It was concluded that the established resistance gene detection sensor has a linear detection range as low as 1fM-1nM and a detection time of less than 30 minutes.

[0031] Table 1

[0032]

[0033]

[0034] Comparative Example 1

[0035] Quantitative polymerase chain reaction was used to detect ARG. The steps are as follows:

[0036] (1) First, design quantitative polymerase chain reaction forward and reverse primers, which are specifically used for flanking amplification of the target region; the primer sequence must be selected to be unique to the target ARG and avoid off-target binding with similar sequences;

[0037] (2) Extracting the target ARG from the mixed sample with an extraction time of 1 h;

[0038] (3) The concentration and purity of the target ARG obtained after extraction were tested using a small molecule nucleic acid analyzer;

[0039] (4) Amplify the target ARG by polymerase chain reaction. The amplification conditions start at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds, and then annealing and extension. After amplification, perform agarose gel electrophoresis, select monoclonal colonies for sequencing, and compare gene sequences. After successful comparison, expand the culture, extract the plasmid, and confirm the presence of the target ARG.

[0040] (5) Quantitative detection: 5 μL 2x M5 HiPer SYBR Premix Es Taq dye, 1 μL H2O, 1 μL target ARG, and 0.3 μL forward primer and 0.3 μL reverse primer were premixed and gently added to a 96-well plate, and then the plate was sealed;

[0041] (6) providing information to the control unit regarding the contents and location of the wells, the dye or probe chemistry used, any standards or controls included, and the cycling conditions;

[0042] (7) The target ARG is heated, causing denaturation and production of single-stranded DNA. The bound intercalating dye also dissociates and returns to the ground state;

[0043] (8) The sequence-specific primer binds to its target ARG (annealing), and complementary bases are added to the sequence by DNA polymerase to produce a complementary copy (primer extension);

[0044] (9) The fluorescence generated is then detected by the instrument and recorded by the computer. The fluorescence signal is proportional to the number of target ARG copies, thereby quantifying the concentration of target ARG in the unknown sample.

[0045] The method of detecting ARG by quantitative polymerase chain reaction in Comparative Example 1 is complicated to operate, and requires extraction and purification of the target ARG from the mixed sample; forward and reverse primers need to be synthesized for the target ARG and the target ARG needs to be amplified; expensive dyes and enzymes are required in the quantitative test process; the entire test process takes a long time, and it takes 3-4 hours to complete the test.

[0046] Experimental Example 1

[0047] The present invention is used to quantify the concentration of resistance genes in water.

[0048] First, prepare different concentrations of resistance gene solutions by serial dilution in binding buffer. Before the measurement, add 200 μL PBS buffer solution to the first column of wells in the 96-well black plate to obtain a stable baseline. Add 500 nM capture DNA to the second column of wells in the 96-well black plate. The biotinylated capture DNA is coupled with the avidin on the surface of the streptavidin sensor to achieve the effect of immobilized capture DNA. Add 5% skim milk powder solution to the third column of wells in the 96-well black plate to block the excess binding sites on the sensor.

[0049] The second step is the combination step. 200 μL PBS buffer solution is added to the fourth column of the 96-well black plate to obtain a stable baseline 2. The first row of the fifth column of the 96-well black plate is the blank control group, which is added with PBS buffer solution. The second to eighth rows are the experimental groups, and gradient dilutions of ARG solution are added, with a concentration of 1fM-1nM. The sixth column of the 96-well black plate is PBS buffer solution, which is used to reduce nonspecific adsorption.

[0050] The third step is the "click" structure formation step. The seventh column of the 96-well black plate is a buffer solution containing 10 nM signal DNA. In the presence of the target ARG, the capture DNA and signal DNA hybridize to adjacent positions on the target ARG, bringing the N3 and DBCO groups close to form a triazole bond, thereby generating a "click" structure.

[0051] The fourth step is signal amplification. The eighth column of the 96-well black plate contains a buffer solution containing 1 ng / mg horseradish peroxidase-labeled avidin. The "click" structure is labeled with horseradish peroxidase for 300 seconds. The streptavidin sensor with the "click" structure labeled with horseradish peroxidase is immersed in diaminobenzidine in the ninth column of the 96-well black plate for signal amplification for 600 seconds. The experimental results are tested using a multi-channel bio-interferometer. The insoluble crystals formed by the "click" structure streptavidin sensor labeled with horseradish peroxidase in diaminobenzidine are precipitated on the surface of the biosensor, resulting in a change in the thickness of the biolayer at the tip of the sensor. The linear relationship between the wavelength shift caused by this and the target ARG concentration is used to quantitatively detect the resistance gene concentration in the sample to be tested. Figure 1 A, B, C, D, and E are the response signal changes and linear correlation standard curves generated when the present invention detects tetA, tetC, tetG, tetM, and sul1, respectively. In the figure, a, b, c, d, e, f, and g represent the resistance gene concentrations of 1fM, 10fM, 100fM, 1pM, 10pM, 100pM, and 1nM. Figure 1 It was concluded that the established resistance gene detection sensor has a linear detection range as low as 1fM-1nM and a detection time of less than 30 minutes.

[0052] Experimental Example 2

[0053] The present invention is used to measure the anti-interference performance of resistance genes in complex water bodies.

[0054] First, different concentrations of mismatch resistance genes or coexistence solutions of mismatch resistance genes and target resistance genes were prepared by serial dilution in binding buffer. Before the measurement, 200 μL of PBS buffer solution was added to the first column of wells of the 96-well black plate to obtain a stable baseline. A 500 nM capture DNA was added to the second column of wells of the 96-well black plate. The biotinylated capture DNA was coupled with the avidin on the surface of the streptavidin sensor to achieve the effect of immobilized capture DNA. A 5% skim milk powder solution was added to the third column of wells of the 96-well black plate to block the excess binding sites on the sensor.

[0055] The second step is the binding step, adding 200 μL of PBS buffer solution to the fourth column of the 96-well black plate to obtain a stable baseline 2. The first row of the fifth column of the 96-well black plate is the blank control group, and PBS buffer solution is added. The second to eighth rows are the experimental groups, and solutions configured with 1, 3 or full-base mismatch sequences or solutions when these mismatch sequences coexist with the target ARG are added respectively. The sixth column is PBS buffer solution, which is used to reduce nonspecific adsorption.

[0056] The third step is the "click" structure formation step. The seventh column of the 96-well black plate is a buffer solution containing 10 nM signal DNA. In the presence of the target ARG, the capture DNA and signal DNA hybridize to adjacent positions on the target ARG, bringing the N3 and DBCO groups close to form a triazole bond, thereby generating a "click" structure.

[0057] The fourth step is signal amplification. The eighth column of the 96-well black plate contains a buffer solution containing 1 ng / mg horseradish peroxidase-labeled avidin. The "click" structure is labeled with horseradish peroxidase for 300 seconds. The streptavidin sensor with the "click" structure labeled with horseradish peroxidase is immersed in the diaminobenzidine substrate in the ninth column for signal amplification for 600 seconds. The insoluble crystals formed after the reaction are precipitated on the surface of the biosensor, causing the thickness of the biolayer at the tip of the sensor to change. The linear relationship between the wavelength shift caused by this and the target ARG concentration is used to quantitatively detect the concentration of resistance genes in the sample to be tested. Figure 2 The present invention determines the anti-interference performance of 1, 3, and all-base mismatch sequences alone or when they coexist with the target ARG; Figure 2 The ratio represents the ratio of the displacement value signal generated by the mismatch sequence to the displacement value generated by the target sequence. Figure 2 It is concluded that the present invention can distinguish 1, 3, and full-base mismatch sequences coexisting with the target ARG (tetA, tetC, tetG, tetM or sul1) and identify the target ARG.

[0058] Experimental Example 3

[0059] The invention is used to measure the reusability of resistance genes in complex water bodies.

[0060] First, prepare different concentrations of resistance gene solutions by serial dilution in binding buffer. Before the measurement, add 200 μL PBS buffer solution to the first column of wells in the 96-well black plate to obtain a stable baseline. Add 500 nM capture DNA to the second column of wells in the 96-well black plate. The biotinylated capture DNA is coupled with the avidin on the surface of the streptavidin sensor to achieve the effect of immobilized capture DNA. Add 5% skim milk powder solution to the third column of wells in the 96-well black plate to block the excess binding sites on the sensor.

[0061] The second step is a combination step, adding 200 μL of PBS buffer solution to the fourth column of the 96-well black plate to obtain a stable baseline 2. The first row of the fifth column of the 96-well black plate is a blank control group, in which PBS buffer solution is added. The second to eighth rows are experimental groups, containing different concentrations or types of resistance genes. The sixth column is a PBS buffer solution, which is used to reduce nonspecific adsorption. The seventh column is a 50 mM hydrochloric acid solution, which is used to regenerate the multi-channel biointerferometer sensor probe. Figure 3This is a repeatability diagram for detecting resistance genes in the present invention. Figure 3 It was concluded that the sensing method established by the present invention can detect resistance genes multiple times.

Claims

1. A method for detecting antibiotic resistance genes by click chemistry coupled biosensing, characterized in that: The method comprises the following specific steps: Step 1: Design two DNA probes to carry out click chemistry-mediated chain reaction. First, construct capture DNA and signal DNA with a base number of 13 bp; capture DNA: labeled with azide N3 at its 3' end and biotin at its 5' end; signal DNA: labeled with dibenzocyclooctyl DBCO at its 5' end and biotin at its 3' end; target ARGs include tetracycline or sulfonamide resistance genes tetA, tetC, tetG, tetM or sul1; Step 2: fix the capture DNA constructed in step 1 on the surface of the streptavidin sensor of the multi-channel biointerferometer to obtain a streptavidin sensor with the capture DNA fixed thereon; the capture DNA concentration is 250-1000 nM, and the fixation time is 200-500 s; Step 3: Immerse the streptavidin sensor with the captured DNA fixed thereon constructed in step 2 in a skim milk powder solution to block the avidin sites on the streptavidin sensor that have not yet been bound; the concentration of the skim milk powder is 1%-5%, and the blocking time is 60-120 s; Step 4: Immerse the streptavidin sensor blocked with skimmed milk powder in step 3 in PBS phosphate buffer solution for equilibrium to obtain a stable baseline; pH of PBS phosphate buffer solution: 7.0-7.5, stabilization time: 60-120 s; Step 5: soaking the balanced streptavidin sensor in step 4 in an analytical solution containing different concentrations or types of resistance genes; the resistance genes include tetA, tetC, tetG, tetM or sul1, with a concentration of 1 fM-1 nM, and the soaking time is 200-500 s; Step 6: Soak the streptavidin sensor bound to the resistance gene in step 5 in a buffer solution containing signal DNA. In the presence of the target resistance gene, the capture DNA and the signal DNA are hybridized to adjacent positions on the target resistance gene, so that the N3 and DBCO groups are close to form a triazole bond, thereby generating a "click" structure; the concentration of the buffer solution containing the signal DNA is 5-10 nM, and the binding time is 200-300 s; Step 7: Soak the streptavidin sensor with the "click" structure generated in step 6 in a buffer containing horseradish peroxidase-labeled avidin to label the "click" structure with horseradish peroxidase; the concentration of the buffer containing horseradish peroxidase-labeled avidin is 1-5 ng / mg; the labeling time is 200-300 s; Step 8: Immerse the streptavidin sensor with the "click" structure labeled with horseradish peroxidase in step 7 in a diaminobenzidine substrate for signal amplification; the signal amplification time is 300-600 s; the insoluble crystals formed after the reaction are precipitated on the surface of the streptavidin sensor, causing the thickness of the biolayer at the tip of the sensor to change, and the linear relationship between the wavelength shift caused by this and the target resistance gene concentration is used to quantitatively detect the resistance gene concentration in the sample to be tested.

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