High-affinity oxytetracycline-combined nucleic acid molecule and oxytetracycline detection method thereof
By using a method that uses nucleic acid molecules labeled with fluorescent dyes of specific sequences, the problems of complex and costly equipment in existing oxytetracycline detection have been solved, achieving highly sensitive oxytetracycline detection that is suitable for environmental and food testing.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-06
- Publication Date
- 2026-04-07
AI Technical Summary
Existing methods for detecting oxytetracycline require large equipment, are cumbersome to operate, and are costly. Furthermore, antibody preparation is expensive and prone to inactivation, making it difficult to achieve rapid and sensitive detection.
Using nucleic acid molecules with specific sequences as nucleic acid aptamers, fluorescent dyes are labeled and oxytetracycline is detected by changes in fluorescence signal. The method involves incubation with buffer solution and measurement of fluorescence signal, with a detection limit reaching the nM level.
It achieves highly sensitive detection of oxytetracycline with a detection limit at the nM level, making it suitable for research in the fields of environment, food, and bacterial resistance.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of antibiotic detection technology, specifically relating to nucleic acid molecules for detecting oxytetracycline and methods for detecting oxytetracycline. Background Technology
[0002] Oxytetracycline (OTC) belongs to the tetracycline family and is a broad-spectrum antibacterial drug that effectively inhibits both Gram-positive and Gram-negative bacteria. Due to its low cost and broad-spectrum antibacterial properties, oxytetracycline is widely used to treat bacterial infections, tumors, control outbreaks of animal diseases, and promote animal growth. However, due to its stability and persistence, the extensive use of oxytetracycline has led to its accumulation and pollution in water bodies and soil. Residual oxytetracycline in the environment can enter the human body through various routes, including drinking water and food, causing harm such as liver damage, bone diseases, gastrointestinal disorders, and kidney dysfunction. Furthermore, the abundance of oxytetracycline and other antibiotics in the environment can lead to bacterial resistance, resulting in the emergence of drug-resistant bacteria, disrupting the ecological balance, reducing drug efficacy, affecting disease treatment, and ultimately threatening the healthcare system and disease prevention and control. Therefore, developing analytical methods for detecting oxytetracycline is crucial and necessary to address the environmental pollution and health hazards posed by oxytetracycline. Conventional analytical techniques for detecting oxytetracycline mainly include chromatography and mass spectrometry, but these often require large analytical instruments, resulting in high costs and cumbersome, time-consuming procedures, making them unsuitable for rapid on-site analysis. Immunoassay techniques based on antibodies offer advantages such as simple operation and rapid analysis, but antibody preparation requires animals, leading to high costs, and antibodies are prone to inactivation.
[0003] Nucleic acid aptamers are oligonucleotides (DNA or RNA) with affinity recognition capabilities. As a novel type of recognition molecule, they offer many advantages, such as ease of preparation, high purity, good stability, and ease of incorporating labeling groups. Nucleic acid aptamers show promise in the field of detection and analysis, providing a new method for rapid and sensitive detection of oxytetracycline and demonstrating advantages in this area.
[0004] In existing technologies, Liu et al. published a research paper entitled "Label-free and Dye-free Fluorescent Sensing of Tetracyclines Using a Capture-Selected DNA Aptamer" in Analytical Chemistry (2022, 94, 10175-10182). This paper used the capture-SELEX technique on a fixed DNA library to screen for DNA aptamers with sequences as shown in SEQ ID NO:6 that could bind to oxytetracycline, and determined their dissociation constant for oxytetracycline to be 147 nM using isothermal titration calorimetry (ITC). However, further modifications to existing aptamer sequences are still needed to obtain nucleic acid aptamers with higher affinity for oxytetracycline, thereby developing more sensitive detection methods. Summary of the Invention
[0005] Based on this, the object of the present invention is to provide a nucleic acid molecule with higher affinity than nucleic acid molecules having a sequence as shown in SEQ ID NO:6, and a method for sensitively detecting oxytetracycline.
[0006] To achieve the above objectives, the present invention adopts the following technical solution:
[0007] 1. A nucleic acid molecule having a sequence as shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.
[0008] 2. A nucleic acid molecule labeled with a fluorescent dye, said nucleic acid molecule having a sequence as shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.
[0009] 3. The nucleic acid molecule labeled with a fluorescent dye according to item 2, wherein the fluorescent dye is fluorescein and tetramethylrhodamine.
[0010] 4. A nucleic acid molecule labeled with a fluorescent dye according to item 2 or 3, wherein the labeling site of the fluorescent dye is any two of the 5' end, the 18th base, and the 32nd base of the nucleic acid molecule;
[0011] Preferably, the nucleic acid molecule labeled with the fluorescent dye is labeled with tetramethylrhodamine at the 5' end, and with fluorescein labeled at the 18th or 32nd base.
[0012] The number of bases mentioned therein is counted starting from the 5' end.
[0013] 5. A kit for detecting oxytetracycline, said kit comprising the nucleic acid molecule described in item 1 or the nucleic acid molecule labeled with a fluorescent dye as described in any one of items 2-4.
[0014] 6. A method for detecting oxytetracycline in a sample to be tested, the method comprising reacting a nucleic acid molecule labeled with a fluorescent dye as described in any one of items 3-4 with the sample to be tested, measuring the obtained fluorescence signal, and determining the content of oxytetracycline based on the fluorescence signal.
[0015] 7. According to the method described in item 6, the fluorescence signal is the ratio of the fluorescence intensities of the two fluorescent dyes.
[0016] 8. The method according to item 7, wherein the fluorescent dye is fluorescein and tetramethylrhodamine, the excitation wavelength used in the method is 490 nm, the emission fluorescence wavelength of fluorescein is 525 nm, and the emission fluorescence wavelength of tetramethylrhodamine is 575 nm.
[0017] 9. The method according to any one of items 6-8, wherein the detection temperature is 5-30°C, preferably 15°C.
[0018] Use of the nucleic acid molecule described in item 10, or the nucleic acid molecule labeled with a fluorescent dye as described in any one of items 2-4, or the kit described in item 5, in the detection of oxytetracycline.
[0019] The present invention has the following advantages and effects:
[0020] This invention provides a nucleic acid molecule that can bind to oxytetracycline and has a higher affinity than nucleic acid molecules having the sequence shown in SEQ ID NO:6, and a method for detecting oxytetracycline, wherein the detection limit can reach the nM level and the method has high sensitivity. Attached Figure Description
[0021] Figure 1 is a graph showing the affinity between OTC and the nucleic acid molecules of the present invention, characterized by fluorescence anisotropy analysis obtained in Example 1. The horizontal axis represents the concentration of the nucleic acid molecules in nM, and the vertical axis represents the fluorescence anisotropy value (r value). Figure 1a The sequence of the nucleic acid molecule in the sample is SEQ ID NO: 1; Figure 1b The sequence of the nucleic acid molecule in the sample is SEQ ID NO: 6.
[0022] Figure 2The graph shows the results of fluorescence anisotropy analysis used in Example 2 to characterize the affinity between OTC and the nucleic acid molecule with the sequence SEQ ID NO: 2. The horizontal axis represents the concentration of the nucleic acid molecule in nM, and the vertical axis represents the fluorescence anisotropy value (r value).
[0023] Figure 3 The graph shows the results of fluorescence anisotropy analysis used in Example 3 to characterize the affinity between OTC and the nucleic acid molecule with the sequence SEQ ID NO: 3. The horizontal axis represents the concentration of the nucleic acid molecule in nM, and the vertical axis represents the fluorescence anisotropy value (r value).
[0024] Figure 4 The graph shows the results of fluorescence anisotropy analysis used in Example 4 to characterize the affinity between OTC and the nucleic acid molecule with the sequence SEQ ID NO: 4. The horizontal axis represents the concentration of the nucleic acid molecule in nM, and the vertical axis represents the fluorescence anisotropy value (r value).
[0025] Figure 5 The graph shows the results of fluorescence anisotropy analysis used in Example 5 to characterize the affinity between OTC and the nucleic acid molecule with the sequence SEQ ID NO: 5. The horizontal axis represents the concentration of the nucleic acid molecule in nM, and the vertical axis represents the fluorescence anisotropy value (r value).
[0026] Figure 6 The graph shows the results of detecting oxytetracycline using a nucleic acid molecule labeled with a fluorescent dye (a nucleic acid molecule obtained by labeling the 5' end of the SEQ ID NO: 2 sequence with tetramethylrhodamine and the 18th base with fluorescein) obtained in Example 6. The horizontal axis represents the concentration of oxytetracycline in nM, and the vertical axis represents the ratio of fluorescence intensity signals (Fr value).
[0027] Figure 7 The graph shows the results of detecting oxytetracycline using a nucleic acid molecule labeled with a fluorescent dye (a nucleic acid molecule obtained by labeling the 5' end of the SEQ ID NO: 2 sequence with tetramethylrhodamine and the 32nd base with fluorescein) obtained in Example 6. The horizontal axis represents the concentration of oxytetracycline in nM, and the vertical axis represents the ratio of fluorescence intensity signals (Fr value). Detailed Implementation
[0028] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.
[0029] This invention uses nucleic acid molecules with sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 as nucleic acid aptamers for the detection of oxytetracycline. These nucleic acid molecules can be synthesized using chemical methods, such as nucleic acid or oligonucleotide synthesis methods commonly used in the field of nucleic acid chemistry.
[0030] This invention provides a method for determining the affinity of nucleic acid molecules, comprising reacting oxytetracycline with nucleic acid molecules of different concentrations and measuring the fluorescence signal of the OTC. In practice, the nucleic acid molecules can be mixed with oxytetracycline and incubated, and the fluorescence anisotropy value can be measured. Oxytetracycline (OTC) emits fluorescence at an excitation wavelength of 370 nm, with the maximum fluorescence emission wavelength located around 530 nm. OTC molecules that do not bind to nucleic acid aptamers have a small molecular volume and a high rotational speed in solution, exhibiting a low fluorescence anisotropy value (r). However, after OTC binds to the DNA aptamer sequence, the OTC aptamer complex increases in volume, the rotational speed of the fluorescent molecule in solution slows down, and the fluorescence anisotropy value increases. Based on the change in fluorescence anisotropy value, the affinity between the nucleic acid aptamer and OTC can be determined.
[0031] When the nucleic acid molecules of the present invention (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5) are used as nucleic acid aptamers for the detection of OTC, fluorescent dyes can be labeled on specific sites of the nucleic acid aptamers. After the nucleic acid aptamers labeled with fluorescent dyes are incubated with OTC, the fluorescence signal generated by the fluorescent dyes is detected, thereby determining the content of OTC in the sample to be tested.
[0032] The fluorescent dye used in this invention can be any fluorescent dye capable of labeling the nucleic acid molecules of this invention and causing a change in fluorescence signal after labeling, such as tetramethylrhodamine, fluorescein, or Texas Red, with tetramethylrhodamine and / or fluorescein being preferred.
[0033] The present invention does not have any particular limitation on the sites on which the nucleic acid molecules are labeled with fluorescent dyes. It only requires that the nucleic acid molecules labeled with fluorescent dyes can produce obvious changes in fluorescence signals after binding with OTC, which is sufficient for the detection of OTC. Preferably, when OTC is present, the ratio of fluorescence intensities of different fluorescent dyes changes regularly with the concentration of OTC.
[0034] For the purposes of this invention, the preferred sites are the 5' end, the 18th base, or the 32nd base in the nucleic acid molecule sequence of this invention;
[0035] More preferably, the marking site is the 5' end and the 18th base or the 5' end and the 32nd base;
[0036] More preferably, the 5' end is labeled with tetramethylrhodamine, and the 18th or 32nd base is labeled with fluorescein;
[0037] The number of bases mentioned therein is counted starting from the 5' end.
[0038] The fluorescent dye labeling method can be a conventional method in the art, such as chemical coupling.
[0039] The kit for detecting oxytetracycline provided by this invention includes at least one of the nucleic acid molecules described in this invention, i.e., the sequence of the nucleic acid molecule is as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. Preferably, the kit may further include a conventional buffer. More preferably, the buffer may contain different concentrations of NaCl, such as 0 mM to 150 mM NaCl, and different concentrations of MgCl2, such as 1 mM to 20 mM MgCl2. 2。 More preferably, the binding buffer solution is 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2. The binding buffer solution can be prepared by methods known to those skilled in the art. Preferably, the concentration of the nucleic acid molecule used is 25 nM.
[0040] Preferably, the detection temperature is 5-30℃, and more preferably 15℃.
[0041] The incubation can be carried out in a conventional buffer suitable for the reaction, such as a buffer of 25 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 1 mM MgCl2.
[0042] The fluorescence signal determination can be performed under suitable detection conditions according to this method. There are no special limitations on the detection conditions, as long as a suitable fluorescence signal can be detected under those conditions. Preferably, the fluorescence signal detection conditions for the nucleic acid molecule assay obtained by simultaneously labeling FAM and TMR are: excitation wavelength of 490 nm, emission wavelength of FAM of 525 nm, and emission wavelength of TMR of 575 nm.
[0043] The applications of the nucleic acid molecules provided by this invention in the detection of oxytetracycline include, but are not limited to, the detection of oxytetracycline in the environment, such as lake water samples; the detection of oxytetracycline in food and agricultural products, such as milk samples; and the detection of oxytetracycline in other fields such as bacterial resistance research or scientific research and quality control.
[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all experimental materials and reagents used were purchased from conventional reagent companies. All nucleic acid molecular sequences used were synthesized, prepared, and purified by Sangon Biotech (Shanghai) Co., Ltd.
[0045] Example:
[0046] Example 1: Affinity characterization of nucleic acid molecules SEQ ID NO: 1 and SEQ ID NO: 6
[0047] In this embodiment, fluorescence anisotropy analysis was used to investigate the affinity of nucleic acid molecules SEQ ID NO: 1 and SEQ ID NO: 6 for OTC. 100 nM OTC (final concentration of OTC in solution) and different concentrations (0-10000 nM) of nucleic acid molecules SEQ ID NO: 1 and SEQ ID NO: 6 were incubated in binding buffer solution (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2). The fluorescence anisotropy value of OTC was measured using a fluorophotometer at 25°C and an excitation wavelength of 370 nm. The fluorescence emission intensity at 530 nm was recorded, and the corresponding fluorescence anisotropy value was measured. The fluorescence anisotropy value r was plotted against the nucleic acid molecule concentration. Based on the relationship between r and the nucleic acid molecule concentration, the dissociation constant Kd of the corresponding nucleic acid molecule could be calculated.
[0048] The affinity test results between SEQ ID NO: 1 and OTC are as follows: Figure 1a As shown, with increasing concentration of nucleic acid molecule SEQ ID NO: 1, the fluorescence anisotropy value of OTC gradually increases, reaching a plateau. This indicates that nucleic acid molecule SEQ ID NO: 1 can bind to OTC, forming a complex molecule with increased volume, decreasing rotational speed of OTC molecules, and increasing fluorescence anisotropy. Based on the binding curve, the dissociation constant Kd of nucleic acid molecule SEQ ID NO: 1 can be calculated to be 74.8 ± 12.5 nM.
[0049] As a control, the affinity test results of SEQ ID NO: 6 with OTC are as follows: Figure 1b As shown, under the same experimental conditions, the Kd value measured by SEQ ID NO: 6 was 121.3 ± 16.6 nM.
[0050] The results showed that nucleic acid molecule SEQ ID NO: 1 had a higher affinity than nucleic acid molecule SEQ ID NO: 6, and its affinity for OTC was 1.6 times that of nucleic acid molecule SEQ ID NO: 6.
[0051] Example 2: Affinity characterization of nucleic acid molecule SEQ ID NO: 2
[0052] This embodiment employed fluorescence anisotropy analysis to investigate the affinity between nucleic acid molecule SEQ ID NO: 2 and OTC. 100 nM OTC (final OTC concentration in solution) and nucleic acid molecules SEQ ID NO: 2 at different concentrations (0-10000 nM) were incubated in binding buffer solution (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2). The fluorescence anisotropy value of OTC was measured using a fluorophotometer at 25°C and an excitation wavelength of 370 nm. The fluorescence emission intensity at 530 nm was recorded, and the corresponding fluorescence anisotropy value was determined. A graph was plotted between the fluorescence anisotropy value r and the nucleic acid molecule concentration. Based on the relationship between r and the nucleic acid molecule concentration, the corresponding nucleic acid molecule dissociation constant Kd could be calculated.
[0053] Test results as follows Figure 2 As shown, with increasing nucleic acid concentration, the fluorescence anisotropy of OTC gradually increases, reaching a plateau. This indicates that nucleic acid molecule SEQ ID NO: 2 can bind to OTC, forming a complex with increased molecular volume, decreased rotational speed of OTC molecules, and increased fluorescence anisotropy. Based on the binding curve, the dissociation constant Kd of nucleic acid molecule SEQ ID NO: 2 can be calculated to be 36.3 ± 6.9 nM, which is less than the Kd value of nucleic acid molecule SEQ ID NO: 6. This indicates that nucleic acid molecule SEQ ID NO: 2 has a higher affinity, with an affinity for OTC that is 3.3 times that of nucleic acid molecule SEQ ID NO: 6, representing a significant increase in affinity.
[0054] Example 3: Affinity characterization of nucleic acid molecule SEQ ID NO: 3
[0055] This embodiment employed fluorescence anisotropy analysis to investigate the affinity between nucleic acid molecule SEQ ID NO: 3 and OTC. 100 nM OTC (final OTC concentration in solution) and different concentrations (0-10000 nM) of nucleic acid molecule SEQ ID NO: 3 were incubated in binding buffer solution (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2). The fluorescence anisotropy value of OTC was measured using a fluorophotometer at 25°C and an excitation wavelength of 370 nm. The fluorescence emission intensity at 530 nm was recorded, and the corresponding fluorescence anisotropy value was determined. A graph was plotted between the fluorescence anisotropy value r and the nucleic acid molecule concentration. Based on the relationship between r and the nucleic acid molecule concentration, the dissociation constant Kd of the corresponding nucleic acid molecule could be calculated.
[0056] Test results as follows Figure 3As shown, with the increase of the concentration of nucleic acid molecule SEQ ID NO: 3, the fluorescence anisotropy value of OTC gradually increases and reaches a plateau, indicating that nucleic acid molecule SEQ ID NO: 3 can bind to OTC, the volume of the formed complex molecule increases, the rotational speed of the OTC molecule decreases, and the fluorescence anisotropy value increases. Based on the binding curve, the dissociation constant Kd of nucleic acid molecule SEQ ID NO: 3 can be calculated to be 33.5 ± 6.0 nM, which is less than the Kd value of nucleic acid molecule SEQ ID NO: 6, indicating that nucleic acid molecule SEQ ID NO: 3 has a higher affinity, and its affinity for OTC is 3.6 times that of nucleic acid molecule SEQ ID NO: 6, showing a significant increase in affinity.
[0057] Example 4: Affinity characterization of nucleic acid molecule SEQ ID NO: 4
[0058] This embodiment employed fluorescence anisotropy analysis to investigate the affinity of nucleic acid molecule SEQ ID NO: 4. 100 nM OTC (final OTC concentration in solution) and different concentrations (0-10000 nM) of nucleic acid molecule SEQ ID NO: 4 were incubated in binding buffer solution (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2). The fluorescence anisotropy value of OTC was measured using a fluorophotometer at 25°C and an excitation wavelength of 370 nm. The fluorescence emission intensity at 530 nm was recorded, and the corresponding fluorescence anisotropy value was determined. A graph was plotted between the fluorescence anisotropy value r and the nucleic acid molecule concentration. Based on the relationship between r and the nucleic acid molecule concentration, the corresponding nucleic acid molecule dissociation constant Kd could be calculated.
[0059] Test results as follows Figure 4 As shown, with increasing concentration of nucleic acid molecule SEQ ID NO: 4, the fluorescence anisotropy value of OTC gradually increases and reaches a plateau, indicating that nucleic acid molecule SEQ ID NO: 4 can bind to OTC, forming a complex molecule with increased volume, decreasing rotational speed of OTC molecules, and increasing fluorescence anisotropy value. Based on the binding curve, the dissociation constant Kd of nucleic acid molecule SEQ ID NO: 4 can be calculated to be 65.8 ± 10.7 nM, which is less than the Kd value of nucleic acid molecule SEQ ID NO: 6, indicating that nucleic acid molecule SEQ ID NO: 4 has higher affinity.
[0060] Example 5: Affinity characterization of nucleic acid molecule SEQ ID NO: 5
[0061] We investigated the affinity of nucleic acid molecule SEQ ID NO: 5 using fluorescence anisotropy analysis. SEQ ID NO: 5 nucleic acid molecules at different concentrations (0-10000 nM) and 100 nMOTC (final OTC concentration in solution) were incubated in binding buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2). The fluorescence anisotropy of OTC was measured using a fluorophotometer at 25°C and an excitation wavelength of 370 nm. The fluorescence emission intensity at 530 nm was recorded, and the corresponding fluorescence anisotropy value was determined. A graph of the fluorescence anisotropy value r versus nucleic acid molecule concentration was plotted, and the corresponding nucleic acid molecule dissociation constant Kd was calculated based on the relationship between r and nucleic acid molecule concentration.
[0062] Test results as follows Figure 5 As shown, with increasing concentration of nucleic acid molecule SEQ ID NO: 5, the fluorescence anisotropy value of OTC gradually increases and reaches a plateau, indicating that nucleic acid molecule SEQ ID NO: 5 can bind to OTC, forming a complex molecule with increased volume, decreasing rotational speed of OTC molecules, and increasing fluorescence anisotropy value. Based on the binding curve, the dissociation constant Kd of nucleic acid molecule SEQ ID NO: 5 can be calculated to be 45.9 ± 5.2 nM, which is less than the Kd value of nucleic acid molecule SEQ ID NO: 6, indicating that nucleic acid molecule SEQ ID NO: 5 has higher affinity.
[0063] Example 6: Detection of oxytetracycline using the nucleic acid molecules of the present invention
[0064] A nucleic acid molecule with the sequence SEQ ID NO: 2 was labeled with tetramethylrhodamine at its 5' end and with fluorescein at its 18th base. The resulting fluorescently labeled nucleic acid molecule (final concentration of 25 nM) was incubated with different concentrations (0-1250 nM) of OTC in binding buffer (50 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 50 mM NaCl). The fluorescence intensity of fluorescein (FAM) and tetramethylrhodamine was then measured at 15 °C. The excitation wavelength was 490 nm, the emission wavelength of FAM was 525 nm, and the emission wavelength of TMR was 575 nm. The fluorescence intensity ratio Fr of TMR and FAM was used as the detection signal.
[0065] Test results as follows Figure 6 As shown, the Fr value gradually increases with increasing oxytetracycline concentration. The detection limit of this method is 2 nM oxytetracycline.
[0066] Example 7: Detection of oxytetracycline using the nucleic acid molecules of the present invention
[0067] A nucleic acid molecule with the sequence SEQ ID NO: 2 was labeled with tetramethylrhodamine at its 5' end and with fluorescein at its 32nd base. The nucleic acid molecule labeled with the resulting fluorescent dye was then used to detect oxytetracycline using the same method as in Example 6. The detection results are as follows: Figure 7 As shown, the fluorescence intensity ratio (Fr) of TMR and FAM gradually decreases with increasing oxytetracycline concentration. The detection limit of this method is 4 nM oxytetracycline.
[0068] nucleotide sequence
[0069] SEQ ID NO: 1: 5'-ACG ACA TTC TGT TGA TCT CTC CCT TTT GGG TTG GTG TCGT-3'
[0070] SEQ ID NO: 2: 5'-ACG ACA TTC dUGT TGA TCT CTC CCT TTT GGG TTG GTG TCGT-3'
[0071] SEQ ID NO: 3: 5'-ACG ACG TTC CGT TGA TCT CTC CCT TTT GGG TTG GCG TCGT-3'
[0072] SEQ ID NO: 4: 5'-ACG ACC TTC CGT TGA TCT CTC CCT TTT GGG TTG GGG TCGT-3'
[0073] SEQ ID NO: 5: 5'-ACG ACA CTC CGT TGA TCT CTC CCT TTT GGG TTG GTG TCGT-3'
[0074] SEQ ID NO: 6: 5'-ACG ACA TTC CGT TGA TCT CTC CCT TTT GGG TTG GTG TCGT-3'
Claims
1. A nucleic acid molecule, characterized in that, The nucleic acid molecule has a sequence as shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO:
5.
2. A nucleic acid molecule labeled with a fluorescent dye, characterized in that, The nucleic acid molecule has a sequence as shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO:
5.
3. The nucleic acid molecule labeled with a fluorescent dye according to claim 2, characterized in that, The fluorescent dyes are fluorescein and tetramethylrhodamine.
4. The nucleic acid molecule labeled with a fluorescent dye according to claim 2 or 3, characterized in that, The labeling sites of the fluorescent dye are any two of the 5' end, the 18th base, and the 32nd base of the nucleic acid molecule; Preferably, the nucleic acid molecule labeled with the fluorescent dye is labeled with tetramethylrhodamine at the 5' end, and with fluorescein labeled at the 18th or 32nd base. The number of bases mentioned therein is counted starting from the 5' end.
5. A kit for detecting oxytetracycline, characterized in that, The kit comprises the nucleic acid molecule of claim 1 or the nucleic acid molecule labeled with a fluorescent dye as described in any one of claims 2-4.
6. A method for detecting oxytetracycline in a sample, characterized in that, The method includes reacting the nucleic acid molecule labeled with a fluorescent dye according to any one of claims 3-4 with the sample to be tested, measuring the obtained fluorescence signal, and determining the content of oxytetracycline based on the fluorescence signal.
7. The method according to claim 6, characterized in that, The fluorescence signal is the ratio of the fluorescence intensities of the two fluorescent dyes.
8. The method according to claim 7, characterized in that, The fluorescent dyes are fluorescein and tetramethylrhodamine. The excitation wavelength used in the method is 490 nm, the emission wavelength of fluorescein is 525 nm, and the emission wavelength of tetramethylrhodamine is 575 nm.
9. The method according to any one of claims 6-8, characterized in that, The detection temperature is 5-30℃, preferably 15℃.
10. Use of the nucleic acid molecule of claim 1, or the nucleic acid molecule labeled with a fluorescent dye according to any one of claims 2-4, or the kit of claim 5 in the detection of oxytetracycline.