A click chemistry coupled biosensing method for antibiotic resistance gene detection

Abstract

The application provides an antibiotic resistance gene detection method of click chemistry coupling biosensing, which can realize the recognition and detection of the femtomole level of the resistance gene (ARG) in water. First, the capture DNA is fixed on the streptavidin biosensor surface of a multi-channel bio-interferometer. In the presence of the target ARG, the capture DNA and the signal DNA are hybridized to the adjacent positions of the target ARG, so as to form a “click” structure. Then, the horseradish peroxidase is used to label the biosensor with the “click” structure, and the biosensor is immersed in the diaminobenzidine substrate, so that the insoluble crystal is deposited on the biosensor surface, the thickness of the biosensor tip biological layer is changed, the wavelength shift caused thereby and the linear relationship of the target ARG concentration are obtained, and the resistance gene in the sample to be detected is quantitatively detected. The method realizes the super-sensitivity and high selectivity detection of the resistance gene in the water environment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of new pollutant analysis and detection of environmental protection, and relates to a method for detecting antibiotic resistance genes by coupling click chemistry with biosensing, which is suitable for sensitive and rapid detection of low-concentration antibiotic resistance genes in real water environment. BACKGROUND

[0002] Bacterial infections involving antibiotic resistance are threatening public health. Therefore, effective monitoring of resistance genes is crucial to cope with the crisis of antibiotic resistance. In addition, different types of resistance genes coexist in the same area, leading to the emergence of multi-drug resistant bacteria, making modern antimicrobial drugs ineffective, and even increasing the incidence and mortality of clinical diseases. Traditional methods for detecting resistance genes, such as quantitative polymerase chain reaction and metagenomic sequencing, rely on nucleic acid amplification. However, the strict requirement for intact DNA templates limits 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 in operation, and are not suitable for real-time or online monitoring. Therefore, it is urgent to develop new analytical methods that can efficiently identify resistance genes without amplification, and achieve high-throughput and rapid targeted analysis of this type of pollutants. SUMMARY

[0003] The purpose of the present application is to provide a method for detecting antibiotic resistance genes by coupling click chemistry with biosensing, which can achieve high sensitivity and high selectivity for targeted recognition and detection of resistance genes in complex water environment media. First, the capture DNA is fixed on the streptavidin sensor surface of the multi-channel bio-interferometer to construct an ARG recognition biosensor. In the presence of target ARG, the capture DNA and signal DNA hybridize to the adjacent positions on the target ARG, thereby forming a "click" structure. In order to further amplify the detection signal, the "click" structure biosensor is labeled with horseradish peroxidase and soaked in a diaminobenzidine substrate for signal amplification. After the reaction, the insoluble crystal precipitates on the streptavidin sensor surface, resulting in changes in the thickness of the sensor tip biological layer, which causes a wavelength shift and a linear relationship between the target ARG concentration. The linear relationship is used to quantitatively detect the concentration of resistance genes in the sample to be tested. This method is rapid, sensitive and highly selective.

[0004] The specific technical solutions to achieve the purpose of the present application are:

[0005] Step 1: Two DNA probes were designed for click chemistry-mediated ligation reaction, first, a 13 bp base number of capture DNA and signal DNA were constructed; the capture DNA was labeled with azide N3 at its 3' end and biotin Biotin at its 5' end; the signal DNA was labeled with dibenzocyclooctyl DBCO at its 5' end and biotin at its 3' end; the target ARGs included tetracycline or sulfonamide resistance genes tetA, tetC, tetG, tetM or sul1;

[0006] Step 2: The capture DNA constructed in step 1 was immobilized on the streptavidin sensor surface of a multi-channel bio-interferometer, obtaining a streptavidin sensor with immobilized capture DNA; the concentration of capture DNA was 250-1000 nM, and the immobilization time was 200-500 s;

[0007] Step 3: The streptavidin sensor with immobilized capture DNA constructed in step 2 was soaked in a skimmed milk powder solution to block the unbound avidin sites on the streptavidin sensor; the concentration of skimmed milk powder was 1%-5%, and the blocking time was 60-120 s;

[0008] Step 4: The streptavidin sensor blocked by skimmed milk powder in step 3 was immersed in PBS phosphate buffer solution for equilibration to obtain a stable baseline; the PBS phosphate buffer solution had a pH of 7.0-7.5, and the equilibration time was 60-120 s;

[0009] Step 5: The equilibrated streptavidin sensor in step 4 was soaked in an analysis solution containing different concentrations or types of resistance genes; the target resistance genes included tetA, tetC, tetG, tetM or sul1, and the concentration was 1 fM-1 nM, and the soaking time was 200-500 s;

[0010] Step 6: The streptavidin sensor with bound resistance genes in step 5 was soaked in a buffer solution containing signal DNA; in the presence of target ARG, the capture DNA and signal DNA hybridized to adjacent positions on the target ARG, bringing the N3 and DBCO groups close to form a triazole bond, thereby generating a "click" structure; the concentration of the buffer solution containing signal DNA was 5-10 nM, and the binding time was 200-300 s;

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

[0012] Step 8: The streptavidin sensor labeled with horseradish peroxidase for the "click" structure in step 7 is immersed in the diamino benzidine substrate for signal amplification; the signal amplification time is 300-600s; the insoluble crystals formed after the reaction precipitate on the surface of the streptavidin sensor, causing the thickness of the bio-layer at the tip of the sensor to change, thereby causing a wavelength shift and a linear relationship between the target ARG concentration to quantitatively detect the concentration of the resistance gene in the sample to be tested.

[0013] Advantages:

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

[0015] Figure 1 For the experiment example 1, the response signal of the multi-channel bio-interferometer changes with the ARG concentration and the standard curve graph established when the present application is used to quantitatively detect the ARG concentration in water;

[0016] Figure 2 For the experiment example 2, the anti-interference performance graph of the present application when 1, 3, and full-base mismatch sequences are alone or coexist with the target ARG;

[0017] Figure 3 For the experiment example 3, the repeatability graph of the sensing method established by the present application. DETAILED DESCRIPTION

[0018] The present application will be described in detail below in combination with the drawings and examples.

[0019] Example 1

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

[0021] (1) First, design two kinds of DNA probes with 13 bp of base number, capture DNA: labeled with azide (N3) at its 3' end, and labeled with biotin at its 5' end; signal DNA: labeled with dibenzocyclooctyl (DBCO) at its 5' end, and labeled with biotin at its 3' end, refer to Table 1, which is the capture DNA, signal DNA, target ARG (tetA, tetC, tetG, tetM and sul1) and 1, 3, and full-base mismatch sequences designed in this example;

[0022] (2) The streptavidin sensor of the multi-channel bio-interferometer is pre-wetted in PBS buffer solution for 30 minutes;

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

[0024] (4) The streptavidin sensor in step (3) is soaked in the capture DNA in step (1) to couple avidin and biotin, and a streptavidin sensor loaded with capture DNA is prepared, the concentration of the capture DNA is 500 nM, and the fixation time is 300 seconds;

[0025] (5) The streptavidin sensor loaded with capture DNA in step (4) is soaked in a 5% skimmed milk powder solution to block the excess avidin binding sites on the sensor, and the blocking time is 90 seconds;

[0026] (6) The streptavidin sensor blocked by skimmed milk powder in step (5) is soaked in PBS buffer solution to obtain a stable baseline two;

[0027] (7) The streptavidin sensor in step (6) is soaked in an analysis solution containing different concentrations or types of resistant genes for 300 seconds; the target ARG (tetA, tetC, tetG, tetM or sul1) is detected at a concentration of 1 fM-1 nM;

[0028] (8) The streptavidin sensor after detecting the resistant gene in step (7) is soaked in a buffer solution containing signal DNA, in the presence of the target ARG, the capture DNA and the signal DNA are hybridized to the 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 signal DNA is 10 nM, and the binding time is 300 seconds;

[0029] (9) The streptavidin sensor generating the "click" structure in step (8) is soaked in a buffer containing 1 ng / mg horseradish peroxidase-labeled avidin to label the "click" structure with horseradish peroxidase, and the labeling time is 300 seconds;

[0030] (10) The streptavidin sensor labeled with horseradish peroxidase in step (9) is soaked in a diaminobenzidine substrate for signal amplification, and the amplification time is 600 seconds. After the reaction, the insoluble crystals formed in the bio-sensor surface cause the change of the thickness of the bio-layer at the tip of the sensor, thereby causing the wavelength shift and the linear relationship between the target ARG concentration to quantitatively detect the concentration of the resistant gene in the sample to be measured. Figure 1Figure 1 shows the linear correlation standard curve of tetA, tetC, tetG, tetM and sul1, wherein a, b, c, d, e, f, g represent the concentration of resistance genes of 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM and 1 nM, respectively. Figure 1 It is concluded from Figure 1 that the established resistance gene detection sensor has a detection linear range of 1 fM-1 nM and a detection time of less than 30 minutes.

[0031] Table 1

[0032]

[0033]

[0034] Comparative Example 1

[0035] The quantitative polymerase chain reaction for detecting ARGs is as follows:

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

[0037] (2) Extract the target ARG from the mixed sample, and the extraction time is 1 hour;

[0038] (3) The concentration and purity of the target ARG obtained after extraction are detected by a small molecule nucleic acid detector;

[0039] (4) The target ARG is amplified by polymerase chain reaction, and the amplification condition program starts at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, and then annealing and extension. After amplification, agarose gel electrophoresis is performed, and the single clone colony is sequenced and the gene sequence is compared. After successful comparison, the target ARG is cultured and extracted to determine the presence of the target ARG;

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

[0041] (6) Provide the control unit with information about the contents and locations 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 producing single-stranded DNA. The bound intercalating dye also dissociates, returning to the ground state;

[0043] (8) Sequence-specific primers bind (anneal) to their target ARG, complementary bases are added to the sequence by DNA polymerase, producing a complementary copy (primer extension);

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

[0045] The method for detecting ARG by quantitative polymerase chain reaction in Comparative Example 1 is complex in operation, and requires extraction and purification of the target ARG from the mixed sample; synthesis of forward and reverse primers for the target ARG and amplification of the target ARG; use of expensive dyes and enzymes in the quantitative test process; and a long test process, which takes 3-4 hours to complete the test.

[0046] Experimental Example 1

[0047] The concentration of the resistance gene in the water body is quantified by the present application.

[0048] First, different concentrations of resistance gene solutions were prepared by serial dilution in the binding buffer. Before measurement, 200 μL of PBS buffer solution was added to the first column of holes in the 96 black hole plate to obtain a stable baseline. The second column of holes in the 96 black hole plate was added with 500 nM of capture DNA, which was coupled with the streptavidin sensor surface avidin to achieve the effect of immobilized capture DNA. The third column of holes in the 96 black hole plate was added with 5% skimmed milk powder solution to block the excess binding sites on the sensor.

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

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

[0051] The fourth step is signal amplification. The eighth column of the 96 black well plate is filled with 1 ng / mg horseradish peroxidase-labeled avidin buffer, and the horseradish peroxidase-labeled "click" structure is labeled for 300 seconds. The horseradish peroxidase-labeled "click" structure of streptavidin sensor is immersed in the ninth column of the 96 black well plate for signal amplification, and the amplification time is 600 seconds. The experimental results are tested by a multi-channel bio-interferometer. After the reaction of the horseradish peroxidase-labeled "click" structure of streptavidin sensor in the diaminobenzidine, insoluble crystals are formed on the surface of the biosensor, which causes the thickness of the biosensor tip layer to change, thereby causing a wavelength shift and a linear relationship between the target ARG concentration to quantitatively detect the concentration of the resistance gene in the sample to be tested. Figure 1 The response signal changes and linear correlation standard curves generated by the detection of tetA, tetC, tetG, tetM and sul1 are shown in FIG. A, B, C, D and E, respectively. In the figures, a, b, c, d, e, f and g represent the resistance gene concentrations of 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM and 1 nM, respectively. It can be seen from FIG. A, B, C, D and E that the linear range of the resistance gene detection sensor established is as low as 1 fM-1 nM, and the detection time is less than 30 minutes. Figure 1 The response signal changes and linear correlation standard curves generated by the detection of tetA, tetC, tetG, tetM and sul1 are shown in FIG. A, B, C, D and E, respectively. In the figures, a, b, c, d, e, f and g represent the resistance gene concentrations of 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM and 1 nM, respectively. It can be seen from FIG. A, B, C, D and E that the linear range of the resistance gene detection sensor established is as low as 1 fM-1 nM, and the detection time is less than 30 minutes.

[0052] Experimental Example 2

[0053] The anti-interference performance of the present application in the determination of resistance genes in complex water bodies is adopted.

[0054] First, different concentrations of mismatched resistance genes or solutions coexisting with target resistance genes are prepared by continuous dilution in the binding buffer. Before determination, 200 μL of PBS buffer solution is added to the first column of the 96 black well plate to obtain a stable baseline. The second column of the 96 black well plate is added with 500 nM of the concentration of the capture DNA, and the biotinylated capture DNA is coupled with the streptavidin sensor surface affinity to achieve the effect of immobilized capture DNA. The third column of the 96 black well plate is added with 5% skimmed milk powder solution to block the excess binding sites on the sensor.

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

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

[0057] The fourth step is signal amplification. The eighth column of the 96 black well plate is a buffer solution containing 1 ng / mg horseradish peroxidase-labeled avidin. The horseradish peroxidase-labeled "click" structure is soaked in the ninth column of the diaminobenzidine substrate for signal amplification for 600 seconds. After the reaction, the insoluble crystals formed precipitate on the surface of the biosensor, causing the thickness of the biosensor tip biological layer to change, thereby triggering a wavelength shift and a linear relationship between the target ARG concentration to quantitatively detect the concentration of the resistance gene in the sample to be tested. Figure 2 To determine the anti-interference performance of the 1, 3, and full-base mismatch sequences alone or coexisting with the target ARG according to the present application; Figure 2 The middle ratio represents the ratio of the displacement value signal generated by the mismatch sequence to the displacement value generated by the target sequence. From Figure 2 It is concluded that the present application can distinguish between 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 present application is used to determine the reuse performance of resistance genes in complex water bodies.

[0060] First, different concentrations of resistance gene solutions are prepared by continuous dilution in the binding buffer. Before measurement, 200 μL of PBS buffer solution is added to the first column of the 96 black well plate to obtain a stable baseline. In the second column of the 96 black well plate, 500 nM of capture DNA is added, and the biotinylated capture DNA is coupled with the streptavidin sensor surface affinity, achieving the effect of immobilized capture DNA. In the third column of the 96 black well plate, 5% skimmed milk powder solution is added to block the excess binding sites on the sensor.

[0061] The second step is the binding step. In the fourth column of the 96 black well plate, 200 μL of PBS buffer solution is added to obtain a stable baseline 2. The first row of the fifth column of the 96 black well plate is a blank control group, and 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 for reducing non-specific adsorption. The seventh column is a 50 mM hydrochloric acid solution for regeneration of the multi-channel bio-interferometric sensor probe. Figure 3The figure shows the repeated performance of the present application in detecting the resistance gene. Figure 3 It is concluded that the sensing method established by the present application can detect the resistance gene for multiple times.

Claims

1. A method for detecting antibiotic resistance genes in water environmental media by click chemistry coupled biosensing, characterized in that, The method comprises the following specific steps: Step 1: Design two DNA probes for click chemistry-mediated chain reaction, first construct a 13 bp base number of capture DNA and 13 bp of signal DNA; Capture DNA: labeled with azide N3 at its 3' end, labeled with biotin Biotin at its 5' end; Signal DNA: labeled with dibenzocyclooctyl DBCO at its 5' end, and labeled with biotin at its 3' end; The target resistance gene ARG is selected from the group consisting of tetracycline or sulfonamide resistance genes tetA, tetC, tetG, tetM or sul1; Step 2: The capture DNA constructed in step 1 is fixed on the streptavidin sensor surface of the multi-channel bio-interferometer, and a streptavidin sensor with fixed capture DNA is obtained; The concentration of capture DNA is 250-1000 nM, and the fixing time is 200-500 s; Step 3: The streptavidin sensor with fixed capture DNA constructed in step 2 is soaked in skimmed milk powder solution to block the unbound avidin sites on the streptavidin sensor; The concentration of skimmed milk powder is 1%-5%, and the blocking time is 60-120 s; Step 4: The streptavidin sensor blocked by skimmed milk powder in step 3 is immersed in PBS phosphate buffer solution for balancing to obtain a stable baseline; The PBS phosphate buffer solution has a pH of 7.0-7.5, and the balancing time is 60-120 s; Step 5: The balanced streptavidin sensor in step 4 is soaked in an analysis solution containing different concentrations or types of resistance genes; The resistance genes are selected from tetA, tetC, tetG, tetM or sul1, and the concentration is 1 fM-1 nM, and the soaking time is 200-500 s; Step 6: The streptavidin sensor combined with the resistance genes in step 5 is soaked in a buffer solution containing the signal DNA; In the presence of the target resistance gene, the capture DNA and the signal DNA hybridize to the adjacent positions on the same strand of the target resistance gene, so that the azide N3 and the DBCO group 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 combination time is 200-300 s; Step 7: The streptavidin sensor with "click" structure in step 6 is soaked in a buffer solution containing horseradish peroxidase-labeled avidin to label the "click" structure with horseradish peroxidase; The concentration of the buffer solution containing horseradish peroxidase-labeled avidin is 1-5 ng / mg; The labeling time is 200-300 s; Step 8: The streptavidin sensor with "click" structure labeled with horseradish peroxidase in step 7 is soaked in a diaminobenzidine substrate for signal amplification; The signal amplification time is 300-600 s; After the reaction, insoluble crystals are precipitated on the surface of the streptavidin sensor, causing the thickness of the sensor tip biological layer to change, thereby causing a wavelength shift and a linear relationship between the concentration of the target resistance gene, so as to quantitatively detect the concentration of the resistance gene in the sample.

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