Multi-antibiotic visual detection test strip based on strand displacement-surface proximity hybridization and preparation method and application thereof

Abstract

The present invention belongs to the field of antibiotic detection, and relates to a visual test strip, and in particular to a multi-antibiotic visual test strip based on chain displacement-surface proximity hybridization, and its preparation method and application. The test strip adopts a circular design, with the spot hole at the center of the circle, and can be divided into multiple detection areas according to the type of antibiotics to be detected. The chain displacement probe and the aptamer are pre-annealed and hybridized to form a complex, and the sample extract is added together with the signal probe labeled with optical nanomaterial carbon dots, mixed, and dropped into the central sample area of ​​the test strip. In the presence of a target, the chain displacement probe is competed off and continues to diffuse outward along the test strip along with the extract and migrates to the detection line. The brightness of the detection band in this detection scheme is proportional to the concentration of the target, which is a signal-on detection mode. This method is economical and rapid, has good specificity and high accuracy, and is expected to be used for the rapid detection market demand of oxytetracycline and chloramphenicol.

Description

Technical Field

[0001] The invention belongs to the field of antibiotic detection, and relates to a visualized detection test strip, in particular to a multi-antibiotic visualized detection test strip based on strand displacement-surface proximity hybridization, and a preparation method and application thereof. Background Art

[0002] If people consume water and meat containing antibiotics in low doses for a long time, it will cause the body to develop drug resistance, induce resistance genes, and ultimately endanger human health.

[0003] As a type of single-stranded oligonucleotide, aptamers undergo corresponding changes in spatial conformation before and after specific interactions with target molecules. Compared with antibodies, they have the advantages of being screened in vitro, having good thermal stability, and being easy to chemically synthesize and modify. They can even distinguish target molecules with single substituent differences that monoclonal antibodies cannot achieve. Various detection methods and technologies based on aptamers have shown advantages such as rapidity, sensitivity, convenience, and low cost. They have shown broad application prospects in biomedical applications, but their applications in rapid antibiotic testing are relatively limited. In 2008, Niazi et al. screened a tetracycline-specific DNA aptamer. In 2010, Professor Zhang Juankun developed an electrochemical sensor based on this aptamer for the detection of tetracycline in milk, with a detection limit of 1 ng·mL -1 , the detection time is 5 min. In 2010, Kim et al. obtained the nucleic acid aptamer of oxytetracycline and developed a light scattering agglutination analysis method based on nucleic acid aptamer-polystyrene microspheres for the rapid detection of oxytetracycline. The entire detection process only takes 3 min, and the detection limit can reach 100 ppb. On this basis, in 2014, Kim et al. developed an enzyme-linked nucleic acid aptamer biosensor technology for the detection of oxytetracycline in feed. The IC value of this method is about 59.7 ng·mL -1In 2012, Plehvar et al. developed a label-free electrochemical nucleic acid aptamer biosensor technology for the detection of chloramphenicol in milk, with a detection limit of 1.6 nM. In the previous patent CN106680346A, our research group studied an electrochemical detection method and application of AFB1 based on chain displacement amplification and surface proximity hybridization reaction. This method has complicated detection steps and requires the introduction of electroactive probes on the surface of screen-printed electrodes to generate corresponding electrical signals, detect the electrical signals, and then substitute them into the standard curve for calculation to obtain the detection results. According to the research on agricultural product quality and safety risk assessment for many years, although this method has high accuracy, it is obviously not able to realize detection in a timely, rapid, and anytime and anywhere manner during grassroots inspection and supervision. Therefore, in order to realize on-site rapid screening and analysis of a large number of samples, our research group intends to develop a qualitative, quantitative / semi-quantitative visual test paper biosensor technology to save time and cost for laboratory confirmation analysis, and then make the following explorations. Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes a multi-antibiotic visual detection test strip based on strand displacement-surface proximity hybridization and a preparation method and application thereof.

[0005] The technical solution of the present invention is achieved in this way:

[0006] The preparation method of a multi-antibiotic visual detection test strip based on strand displacement-surface proximity hybridization comprises the following steps:

[0007] (1) Design aptamers, strand displacement probes, signal probes, detection probes, and quality control probes for oxytetracycline and chloramphenicol, respectively;

[0008] (2) preparing carbon dots CD1 and reacting with the signal probe to obtain the carbon dots labeled signal probe;

[0009] (3) Fix the detection probe and quality control probe on the detection line and quality control line on the NC membrane respectively.

[0010] Furthermore, the sequence of the oxytetracycline aptamer in step (1) is 5'-bio-(CH 2 ) 6 -GATCGGGTGTGGTTGGCGTAAAGGGAGCATCGG-3', the sequence of the strand displacement probe is 5'-CCGATGCT CCCTTTA -3', the sequence of the signal probe is 5'-NH 2 -(CH 2 ) 12 -TTTTTTTTTTAGCATCGGCCGTTAG-3', the sequence of the detection probe is 5'-bio-(CH 2) 6 -TTTTTTTTTTATGCTCT TAAAGGG -3' and the sequence of the quality control probe is 5'-bio- (CH 2 ) 6 -TTTTTTTTTTGATGCTCCCTT-3'.

[0011] Furthermore, the aptamer sequence of chloramphenicol in step (1) is 5'-bio-(CH 2 ) 6 -ACTTCAGTGAGTTGTCCCACGGTCGGCGAGTCGGTGGTAG-3', the sequence of the strand displacement probe is 5'-CTACCACCGAC TCGCCGACCG -3', the sequence of the signal probe is 5'-NH 2 -(CH 2 ) 12 -TTTTTTTTTTGTCGGTGGTAGTAGCCGT-3', the sequence of the detection probe is 5'-bio-(CH 2 ) 6 -TTTTTTTTTTATGCTCT CGGTCGGCGA -3' and the sequence of the quality control probe is 5'-bio-(CH 2 ) 6 -TTTTTTTTTTCCACCGAC TCGCCGA -3'.

[0012] Furthermore, the preparation method of the carbon dot CD1 in step (2) is as follows: 1.26 g of citric acid and 1608 μL of EDA are dissolved in 30 mL of double distilled water, mixed evenly and transferred to a hydrothermal reactor, reacted at 180° C. for 5 h, and the obtained product was purified by dialysis to obtain the carbon dot CD1.

[0013] Furthermore, the preparation method of the carbon dot-labeled signal probe in step (2) is as follows: 5 mg of carbon dot CD1 is dissolved in 7.5 mL of 0.1 M, pH 7.2 Tris-HCl buffer, 3 mg of EDC and 4.5 mg of NHS are added to react for 30 min, and then 50 µL of detection probe / quality control probe is added to react for 12 h, followed by purification by dialyzing with a dialysis membrane to obtain a carbon dot-labeled signal probe.

[0014] Furthermore, the fixation mechanism of the carbon dot-labeled signal probe in step (3) is to use EDC and NHS as cross-linking agents to fix the amino-modified signal probe to the carbon dots containing carboxyl groups on the surface, thereby forming an amide-bonded carbon dot-labeled signal probe.

[0015] Furthermore, in the method of using the above-mentioned multi-antibiotic visual detection test strip, the strand displacement probe and the aptamer are pre-annealed and hybridized to form a complex, and the complex is added to the sample extract together with the carbon dot-labeled signal probe, mixed, and dripped into the central sample area of ​​the test strip for detection.

[0016] The multi-antibiotic visual detection test strip based on strand displacement-surface proximity hybridization prepared by the above method is circular, with the spot hole at the center of the circle, and the detection area is distributed in a 360° fan shape. From the spot hole at the center of the circle to the outside, there are detection lines fixed with detection probes and quality control lines fixed with quality control probes.

[0017] Furthermore, the detection probe and the quality control probe respectively use streptavidin as an intermediate to form a streptavidin-biotin-DNA complex and are fixed on the corresponding detection line and quality control line on the NC membrane.

[0018] The above-mentioned multi-antibiotic visual detection test strip can be used in the detection of oxytetracycline and / or chloramphenicol in tap water and agricultural and sideline products for non-disease diagnosis purposes.

[0019] Furthermore, the detection sensitivity of oxytetracycline is ≥24.6 μg / L; the detection sensitivity of chloramphenicol is >10.26 μg / L.

[0020] The present invention has the following beneficial effects:

[0021] 1. This application uses antibiotic nucleic acid aptamers as molecular recognition elements and optical nanomaterial carbon dots as transducers, adopts chain displacement signal conduction technology, and then outputs signals through surface proximity hybridization reactions. The chain displacement probe and the aptamer are pre-annealed and hybridized to form a complex, and the sample extract is added together with the signal probe labeled with optical nanomaterial carbon dots, mixed, and dropped into the central sample area of ​​the test paper. In the presence of the target, the chain displacement probe is competed off and continues to diffuse outward along the test paper with the signal probe along with the extract to reach the detection line. Therefore, the brightness of the detection band in this detection scheme is proportional to the concentration of the target. The visual detection test strip for determining multiple antibiotics established by this method has the advantages of economy, simplicity, convenience, and high specificity.

[0022] 2. According to the principle of surface proximity hybridization, the strand displacement probe and the signal probe form a hybridization complex with the detection probe fixed on the detection band through synergistic hybridization and develop color; in the absence of the target, the strand displacement probe does not dissociate from the aptamer, and no surface proximity hybridization reaction occurs on the detection band, so no color is developed. Therefore, the brightness of the detection band in this detection scheme is proportional to the concentration of the target. This method is a signal-on detection mode. Through specific experiments and LCMS instrumental analysis, this method has the advantages of being economical, fast, specific, highly accurate and convenient, and is expected to be used in the rapid detection market demand of oxytetracycline and chloramphenicol. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 This is a design diagram of a test strip that can detect multiple antibiotics simultaneously.

[0025] Figure 2 This is the transmission electron microscopy image of carbon dot CD-1.

[0026] Figure 3 Figure a shows the excitation wavelength of the carbon dot solution, figure b shows the emission wavelength, and the color photograph of the solution (inset in figure a).

[0027] Figure 4 This is an investigation of the luminescence intensity of carbon dot CD1 solution at different excitation wavelengths.

[0028] Figure 5 This is the detection principle of the visual detection test strip of this application.

[0029] Figure 6 It is a graph of the specificity validation results of the test strips, where OTC-oxytetracycline, CAP-chloramphenicol, oxytetracycline-oxytetracycline, and tetra-tetracycline.

[0030] Figure 7 The test results of tap water spiked with antibiotics are shown in the following figure. The concentrations of tap water spiked with oxytetracycline are: A, 1.1μg / L; B, 70.2μg / L; C, 10.87μg / L; D, 30.48μg / L; E, 5.15μg / L; F, 24.6μg / L. The concentrations of tap water spiked with chloramphenicol are: G, 50.47μg / L; H, 10.2μg / L; 1, 2, 3 represent 3 repeated tests of samples with the same concentration. DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Example

[0032] The preparation method of a multi-antibiotic visual detection test strip based on strand displacement-surface proximity hybridization comprises the following steps:

[0033] 1. Design of test strips

[0034] The design of antibiotic test strips Figure 1 As shown, the spot hole is at the center of the circle, and can be divided into multiple detection areas according to the type of antibiotics to be detected. The advantages of the circular test strip design are: it can ensure that the target in the sample extract reaches the detection band or the quality control band to migrate to the same distance, eliminating the inconsistency of the test results caused by the distance difference; the sample in the central sample hole can be evenly diffused and migrated to the surroundings at the same time, and a small amount of sample can achieve simultaneous detection of multiple targets; the length of the detection strip and quality control strip around the central sample hole is adjustable, which is convenient for adding more target strips of interest at any time as needed in the later stage, without increasing the cost too much.

[0035] 2. Detection steps

[0036] (1) Probe design

[0037] All probes designed by the present invention are shown in the following table:

[0038] Table 1. Aptamer sequences and related probe sequences used in the experiment

[0039]

[0040] Note: Underscore sequences match each other

[0041] (2) Biofunctionalization of carbon dot surfaces

[0042] a. Preparation method of carbon dots CD1

[0043] Citric acid-ethylenediamine carbon dots: 1.26 g of citric acid and 1608 μl of EDA were dissolved in 30 mL of double distilled water, mixed evenly, and transferred to a hydrothermal reactor for reaction at 180°C for 5 h. The obtained product was purified by dialyzing with a dialysis bag with a molecular weight cutoff of 300 Da to obtain carbon dots CD1 with rich surface carboxyl groups, which were stored at 4°C.

[0044] Transmission electron microscopy image of carbon dot CD1 Figure 2As shown in FIG. 1 , it can be seen that the average particle size of the carbon dots CD1 of the present application is 10 nm. Figure 3 From a and b, we can see that the excitation wavelength of the carbon dot CD1 is 280 nm and the emission wavelength is 420 nm. We specifically used different wavelengths of excitation light to irradiate the carbon dot CD1 solution and detect the luminescence intensity and luminescence performance of the solution. Figure 4 As shown: The maximum excitation wavelength of the carbon dot CD1 prepared by the hydrothermal synthesis method in this study does not change with the movement of the excitation wavelength, so it is suitable as a modified carbon dot as a signal probe.

[0045] b. Take 5 mg of surface carboxyl-rich carbon dots CD1 and dissolve it in 7.5 mL of Tris-HCl buffer (0.1M, pH 7.2), add 3 mg of EDC and 4.5 mg of NHS and react for 30 min. Then add 50 µL of signal probe and react for 12 h, then dialysis and purification with dialysis membrane, and the prepared nucleic acid functionalized carbon dots (carbon dot-labeled signal probes) are stored at 4°C for future use.

[0046] (3) Fixing the probe on the test strip

[0047] In order to fix the corresponding DNA (detection probe and quality control probe) on the corresponding detection line and quality control line on the NC membrane, streptavidin is used as an intermediate to react with biotin-labeled DNA (bio-DNA) to form a streptavidin-biotin-DNA complex.

[0048] The specific steps are as follows: spray 40uL of 10uM bio-DNA and 40uL of 1mg / mL streptavidin mixture on the test line and quality control line, incubate at 4℃ for 2h, and then add 50uL PBS (0.6% PEG, 1% sucrose, 0.2% Tween 20, 0.02% MgSO 4 , 0.05%(NH 4 ) 2 SO 4 and 1% OVA, all are mass fractions).

[0049] The detection principle of the visual test paper in this application (such as Figure 5As shown in the figure, the strand displacement probe (black probe in the figure) and the nucleic acid aptamer (green probe) are pre-annealed and hybridized to form a complex, and the sample extract is added together with the signal probe labeled with optical nanomaterials (orange-red probe in the figure), mixed, and dropped into the central sample area of ​​the test strip. Because the binding force between the target and the nucleic acid aptamer is greater than the binding force between the strand displacement probe and the nucleic acid aptamer, in the presence of the target, the strand displacement probe will be competed by the target, and together with the signal probe, it will continue to diffuse outward along the test strip with the extract and migrate to the detection line. At this time, according to the principle of surface proximity hybridization, the strand displacement probe and the signal probe form a hybridization complex with the detection probe (cyan probe in the figure) fixed on the detection strip through synergistic hybridization and develop color. In the absence of the target, the strand displacement probe will not dissociate from the nucleic acid aptamer, and no surface proximity hybridization reaction will occur on the detection strip. Therefore, the signal probe will continue to diffuse outward and migrate to the quality control line, and will be captured by the quality control probe (dark blue probe in the figure) fixed on it and fixed and develop color. Therefore, the brightness of the detection strip in this detection scheme is proportional to the concentration of the target.

[0050] Implementation effect example

[0051] 1. Specificity of the detection method:

[0052] In order to investigate the specificity of the method, we collected 4 water samples containing 100 μg / L (national limit value) of oxytetracycline (OTC), chloramphenicol (CAP), levofloxacin, and tetracycline, and tested them after simple filtration. Take 100 μL of the water sample to be tested, 100 μL of premixed probe solution (oxytetracycline and chloramphenicol matching probe concentrations are: 1 μM carbon dot-labeled signal probe, 1 μM aptamer, 1 μM chain displacement probe), 300 μL of purified water, 500 μL of 2× reaction buffer solution (200 mM NaCl, 40 mM Tris-HCl, 4 mM MgCl 2 , 10 mM KCl, 2 mM CaCl 2 , 0.04 Tween20, pH 7.6. ), mix well and react at room temperature for 5 minutes, add dropwise to the center of the circle in the center of the test strip, observe the color of the test strip after 3 minutes, and the result is as follows Figure 6 As shown, the test paper can well identify oxytetracycline and chloramphenicol, and the test line and control line are clearly visible at this concentration.

[0053] 2. Comparison with LC-MS results

[0054] In order to verify the reliability of the method, we selected tap water spiked with oxytetracycline and chloramphenicol at different concentrations and tested them using the LCMS method and the test paper prepared by the present invention respectively:

[0055] Table 2 Verification analysis of spiked tap water

[0056]

[0057] *Note: The instrument test result is the average of 3 results for each sample; the judgment standard of the test paper test result is: if more than 2 of the 3 test results are positive, it is judged as positive.

[0058] The statistical results are shown in Table 2, and the test paper test results are shown in Table 2. Figure 7 As shown. The concentrations of tap water spiked with oxytetracycline are: A, 1.1 μg / L; B, 70.2 μg / L; C, 10.87 μg / L; D, 30.48 μg / L; E, 5.15 μg / L; F, 24.6 μg / L. The concentrations of tap water spiked with chloramphenicol are: G, 50.47 μg / L; H, 10.2 μg / L. Through comparative experiments, we found that the test paper test results of tap water samples spiked with OTC concentration ≥24.6 μg / L matched the results of LCMS instrumental analysis method; the test paper could not detect the spiked tap water samples with OTC concentration <24.6 μg / L. The test paper test results of tap water samples spiked with CAP concentration ≥50.47 μg / L were consistent with the results of LCMS instrumental analysis method; the test paper could not detect the spiked tap water samples with CAP concentration ≤10.26 μg / L.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention. <110> Institute of Agricultural Quality Standards and Testing Technology, Henan Academy of Agricultural Sciences <120> Multi-antibiotic visual detection test strip based on strand displacement-surface proximity hybridization and preparation method and application thereof use <130> Probe-associated sequence <150> 202210528209X <151> 2022-05-16 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 33 <212> DNA <213> Unknown <220> <223> Artificial sequence <400> 1 gatcgggtgt ggttggcgta aagggagcat cgg 33 <210> 2 <211> 15 <212> DNA <213> Unknown <220> <223> Artificial sequence <400> 2 ccgatgctcc cttta 15 <210> 3 <211> 25 <212> DNA <213> Unknown <220> <223> Artificial sequence <400> 3 tttttttttt agcatcggcc gttag 25 <210> 4 <211> twenty four <212> DNA <213> Unknown <220> <223> Artificial sequence <400> 4 tttttttttt atgctcttaa aggg 24 <210> 5 <211> twenty one <212> DNA <213> Unknown <220> <223> Artificial sequence <400> 5 tttttttttt gatgctccct t 21 <210> 6 <211> 40 <212> DNA <213> Unknown <220> <223> Artificial sequence <400> 6 acttcagtga gttgtcccac ggtcggcgag tcggtggtag 40 <210> 7 <211> twenty one <212> DNA <213> Unknown <220> <223> Artificial sequence <400> 7 ctaccaccga ctcgccgacc g 21 <210> 8 <211> 28 <212> DNA <213> Unknown <220> <223> Artificial sequence <400> 8 tttttttttt gtcggtggta gtagccgt 28 <210> 9 <211> 27 <212> DNA <213> Unknown <220> <223> Artificial sequence <400> 9 tttttttttt atgctctcgg tcggcga 27 <210> 10 <211> 25 <212> DNA <213> Unknown <220> <223> Artificial sequence <400> 10 tttttttttt ccaccgactc gccga 25

Claims

1. A method for preparing a multi-antibiotic visual detection test strip based on strand displacement-surface proximity hybridization, characterized in that: Here are the steps: (1) Design aptamers, strand displacement probes, signal probes, detection probes, and quality control probes for oxytetracycline and chloramphenicol, respectively; (2) preparing carbon dots CD1 and reacting with the signal probe to obtain the carbon dots labeled signal probe; (3) Fixing the detection probe and the quality control probe on the detection line and the quality control line on the NC membrane respectively; In the step (1), the sequence of the oxytetracycline aptamer is 5'-bio-(CH2)6-GATCGGGTGTGGTTGGCGTAAAGGGAGCATCGG-3', the sequence of the strand displacement probe is 5'-CCGATGCTCCCTTTA-3', and the sequence of the signal probe is 5'-NH2-(CH2) 12 -TTTTTTTTTTAGCATCGGCCGTTAG-3', the sequence of the detection probe is 5'-bio-(CH2)6-TTTTTTTTTTATGCTCTTAAAGGG-3', and the sequence of the quality control probe is 5'-bio-(CH2)6-TTTTTTTTTTGATGCTCCCTT-3'; In the step (1), the aptamer sequence of chloramphenicol is 5'-bio-(CH2)6-ACTTCAGTGAGTTGTCCCACGGTCGGCGAGTCGGTGGTAG-3', the sequence of the strand displacement probe is 5'-CTACCACCGACTCGCCGACCG-3', and the sequence of the signal probe is 5'-NH2-(CH2) 12 -TTTTTTTTTTGTCGGTGGTAGTAGCCGT-3', the sequence of the detection probe is 5'-bio-(CH2)6-TTTTTTTTTTATGCTCTCGGTCGGCGA-3', and the sequence of the quality control probe is 5'-bio-(CH2)6-TTTTTTTTTTCCACCGACTCGCCGA-3'.

2. The preparation method according to claim 1, characterized in that: The preparation method of the carbon dot CD1 in step (2) is as follows: 1.26 g of citric acid and 1608 μl of EDA are dissolved in 30 mL of double distilled water, mixed evenly and transferred to a hydrothermal reactor, reacted at 180° C. for 5 h, and the obtained product was purified by dialysis to obtain the carbon dot CD1.

3. The preparation method according to claim 1, characterized in that: The preparation method of the carbon dot-labeled signal probe in step (2) is as follows: 5 mg of carbon dot CD1 is dissolved in 7.5 mL of 0.1M, pH 7.2 Tris-HCl buffer, 3 mg of EDC and 4.5 mg of NHS are added to react for 30 min, and then 50 μL of the signal probe is added to react for 12 h, followed by purification by dialyzing with a dialysis membrane to obtain the carbon dot-labeled signal probe.

4. The preparation method according to claim 1, characterized in that: The fixing mechanism of the carbon dot-labeled signal probe in step (2) is to use EDC and NHS as cross-linking agents to fix the amino-modified signal probe to the carbon dots containing carboxyl groups on the surface, thereby forming an amide-bonded carbon dot-labeled signal probe.

5. A multi-antibiotic visual detection test strip based on strand displacement-surface proximity hybridization prepared by the method according to any one of claims 1 to 4, wherein the multi-antibiotic visual detection test strip is circular, the sample well is at the center of the circle, and the detection area is distributed in a 360° sector shape, characterized in that: From the center of the sample spot hole outward, there are the detection line with the detection probe fixed thereon and the quality control line with the quality control probe fixed thereon.

6. The method for using the multi-antibiotic visual detection test strip according to claim 5, characterized in that: The strand displacement probe and the aptamer are pre-annealed and hybridized to form a complex, and then added to the sample extract together with the carbon dot-labeled signal probe, mixed evenly, and dropped into the central sample area of ​​the test paper for detection.

7. The multi-antibiotic visual detection test strip according to claim 6 is used in the detection of oxytetracycline and / or chloramphenicol in tap water and agricultural and sideline products for non-disease diagnosis purposes.

8. The use according to claim 7, characterized in that: The detection sensitivity of oxytetracycline is ≥24.6 μg / L; the detection sensitivity of chloramphenicol is >10.26 μg / L.

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