CN113957067B - Tetracycline drug detection method based on TetR protein steric hindrance and gene shearing technology - Google Patents

Abstract

The application discloses a tetracycline drug detection method based on TetR protein steric hindrance and gene shearing technology, a double-stranded DNA molecule for detecting the tetracycline drug, a composition, a kit and application thereof. The double-stranded DNA molecule comprises a tetracycline manipulation gene tetO and a restriction endonuclease recognition site, wherein one end of the double-stranded DNA molecule is connected with a fluorescent group, and the other end of the double-stranded DNA molecule is connected with biotin. The double-stranded DNA molecule can realize the detection of tetracycline drugs.

Description

Detection of tetracycline drugs based on TetR protein steric hindrance and gene shearing technology method

Technical field

The invention relates to the field of biotechnology, and in particular to a detection method for tetracycline drugs based on TetR protein steric hindrance and gene shearing technology.

Background technique

Tetracyclines are broad-spectrum antibiotics that are widely used in animal husbandry and breeding. They can effectively improve feed utilization, promote growth, and prevent and treat diseases. However, extensive use, abuse, and even illegal use of drugs can lead to their residues in animal-derived foods and the environment, endangering human health. Efficient, rapid, sensitive, and multi-target detection is an important means to ensure food safety.

The currently reported detection methods for tetracycline drug residues mainly include microbiological methods, spectroscopic methods, chromatographic methods, liquid mass spectrometry, capillary electrophoresis and immunological methods. Among them, high-performance liquid chromatography and liquid mass spectrometry have higher sensitivity and specificity, but the operation is more complicated, requires large precision instruments, well-trained operators, long detection time, and high detection cost. Enzyme-linked immunosorbent assay (ELISA) combines the antigen-antibody binding reaction and the amplification effect of enzyme labeling. It has the characteristics of simple operation, high sensitivity, strong specificity, and is suitable for rapid screening of large batches of samples. However, due to the large differences in the structures of tetracycline drugs, it is difficult to prepare antibodies that can recognize multiple tetracycline drugs, and the broad spectrum or sensitivity does not meet the requirements. Therefore, it is necessary to prepare a new type of biorecognition material that can identify all tetracycline drugs with high sensitivity.

The tetracycline repressor protein TetR can recognize tetracycline drugs with high affinity and uniformity and is an ideal biorecognition material. Analyzing the three-dimensional structure of TetR, it can be found that the TetR homodimer is composed of two identical monomers. The N-terminus of each monomer is divided into two DNA domains and a regulatory core structure, which participates in dimerization and ligand binding. Therefore, the tetracycline repressor protein TetR has DNA-binding properties. When tetracycline complexed with magnesium ions binds to TetR, the tetracycline repressor protein TetR changes its spatial conformation and detaches from DNA (tetO). ELISA methods based on TetR and tetO have been reported. However, the traditional ELISA method requires multi-step incubation and requires a catalytic substrate to generate a detection signal. The operation is complex and is not conducive to popularization and application. Therefore, there is an urgent need to develop a new detection technology.

Contents of the invention

The invention provides a double-stranded DNA molecule, which includes a tetracycline control gene tetO and a restriction endonuclease recognition site. One end of the double-stranded DNA molecule is connected to a fluorescent group, and the other end is connected to biotin ( Biotin).

This double-stranded DNA molecule was named tetO-X.

Optionally, according to the above-mentioned double-stranded DNA molecule, one end of one strand of the double-stranded DNA molecule is connected to a fluorescent group, and one end of the other strand is connected to biotin. For example, the 3' end of one strand of the double-stranded DNA molecule is connected to a fluorescent group, and the 3' end of the other strand is connected to biotin. The biotin and the fluorescent group are located at the end of the double-stranded DNA molecule. both ends.

The restriction endonuclease can be all restriction endonucleases such as QuickCut ^TM EcoR I; the fluorescent group can be all fluorescent groups such as TAMRA.

Optionally, according to the above-mentioned double-stranded DNA molecule, the sequence of the tetracycline control gene tetO is shown in positions 22-40 of SEQ ID No. 1.

Optionally, according to the above-mentioned double-stranded DNA molecule, the sequence of one strand in the double-stranded DNA molecule is as shown in SEQ ID No. 1, and the sequence of the other strand in the double-stranded DNA molecule is as shown in SEQ ID No. 2. Show.

The single strand shown in the sequence SEQ ID No. 1 is named tetOF, and the single strand shown in the sequence SEQ ID No. 2 is named tetOR, then the double-stranded DNA molecule can be tetOF and 3 connected to TAMRA at 3' 'Double-stranded DNA molecule obtained by complementary hybridization of tetOR linked to biotin.

The present invention also provides a composition for detecting tetracycline and/or its derivatives, which composition includes the above-mentioned double-stranded DNA molecule and at least one of the following substances: tetracycline repressor protein (TetR), the restriction nucleic acid Endonuclease and streptavidin-coated solid phase carrier, the double-stranded DNA molecules are fixed on the streptavidin-coated solid phase carrier. The amino acid sequence of the tetracycline repressor protein may be shown in SEQ ID No. 3.

There are many substances that can be used as the solid phase carrier, such as polystyrene, cellulose, polyacrylamide, polyethylene, polypropylene, cross-linked dextran, glass, silicone rubber, agarose gel, etc. The solid phase carrier can be in the form of test tubes, micro reaction plate concave holes, beads, small discs, etc.

The principle of the above composition for detecting tetracycline and/or its derivatives is: the tetracycline repressor protein TetR can specifically bind to the tetracycline operator gene tetO; the restriction endonuclease can specifically cut the target gene sequence. tetO-X was immobilized onto a solid-phase support via a streptavidin-biotin system. When TetR and the restriction endonuclease are close to each other, only one protein binds to tetO-X due to steric hindrance, so TetR and the restriction endonuclease competitively bind to tetO-X. When tetracycline drugs are present in the sample, the binding of the drug to TetR causes the protein conformation to change, causing TetR to break away from tetO-X, and the steric hindrance is relieved. At this time, the restriction endonuclease can combine with tetO-X to cause gene shearing. This results in a decrease in the fluorescence signal at one end of tetO-X. The intensity of the fluorescence signal can be used to characterize the content of tetracycline drugs and/or tetracycline drugs in the sample.

The present invention also provides a kit for detecting tetracycline and/or its derivatives, which kit includes the above composition and tetracycline or its derivatives as a standard.

The above-mentioned double-stranded DNA molecules or the above-mentioned compositions or the above-mentioned kit applications also fall within the protection scope of the present invention. The application may specifically be an application in at least one of the following:

1) Application in detection or auxiliary detection of tetracycline or its derivatives;

2) Application in the preparation of products for detection or auxiliary detection of tetracycline or its derivatives;

3) Application in detecting or assisting in detecting the content of tetracycline or its derivatives;

4) Application in the preparation of products for detecting or assisting in detecting the content of tetracycline or its derivatives.

Optionally, according to the above application, the detection or auxiliary detection of tetracycline or its derivatives and/or content includes mixing the above-mentioned double-stranded DNA molecules, the tetracycline repressor protein, the restriction endonuclease and the sample to be tested , perform fluorescence detection after washing. For example, it can include diluting tetO-X at a concentration of 10 μM with water 1:2500, then adding it to a streptavidin-coated ELISA plate, incubating for 1 hour, and washing with PBST three times. At the same time, TetR and QuickCut ^TM EcoR I diluted with 10xbuffer were added. The dilution of TetR was 1:2700 and the amount of QuickCu ^TM EcoR I was 0.8 μL. At the same time, add the sample to be tested. After treatment at room temperature for 1 hour, wash three times with PBST and perform fluorescence detection.

Optionally, according to the above application, the product is a kit.

As mentioned above, the derivative of tetracycline may be a tetracycline drug, such as doxycycline, chlortetracycline, minocycline, demeclocycline, meclocycline, oxytetracycline, and rolicycline.

The embodiments of the present invention have proved through experiments that the fluorescence value of tetO-X is stable and successfully fixed on a streptavidin-coated microplate, and then the fluorescence value and restriction of restriction endonuclease when acting alone on tetO-X are detected. The fluorescence value when restriction endonuclease and TetR acted together on tetO-X was found. It was found that TetR prevented the restriction endonuclease from binding to tetO-X, keeping the fluorescence value of tetO-X stable.

The embodiment of the present invention detects the minimum concentration of tetracycline drugs that prevents the combination of TetR and tetO-X. Specifically, the tetracycline standard is diluted twice, and the intensity of the tetO-X fluorescence signal in the presence of different concentrations of tetracycline drugs is detected, and the detection result is obtained. At the minimum concentration, draw a standard curve to determine the range.

The double-stranded DNA molecules provided by the invention can be used to detect tetracycline drugs. The embodiment of the present invention establishes a rapid detection method for tetracycline drugs. The half inhibitory concentration (IC50) value of this method for tetracycline is 0.09ng/mL, the linear range is 0.04-0.23ng/mL, and the method is effective for other tetracycline drugs. The cross-reaction rate ranges from 29% to 987%, enabling multi-residue detection of tetracycline drugs.

The embodiment of the present invention utilizes the binding properties of the tetracycline repressor protein TetR and the tetracycline control gene tetO, the specific cutting target sequence of the restriction endonuclease, and the steric hindrance between proteins to detect the content of tetracycline drugs through the intensity of the fluorescence signal. The operating steps and reaction time of conventional ELSIA are reduced in order to obtain a detection method that is easy to operate and highly sensitive.

Description of the drawings

Figure 1 is the tetracycline standard curve of Example 2.

Detailed ways

The present invention will be described in further detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in literature in the field or in accordance with product instructions. Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

Origin 95 statistical software was used to process the data, and the experimental results were expressed as average values.

QuickCut ^TM EcoRI (item number 1611) used in the following examples was purchased from Takara Biotechnology (Beijing) Co., Ltd. (takara China), including 10xbuffer.

TetR was synthesized artificially, its amino acid sequence is shown in SEQ ID No. 3, and its concentration is 2.5 mg/kg.

Example 1. Competitive binding of TetR and restriction endonuclease to tetO-X

1. Preparation of double-stranded DNA molecules tetO-X for detection of tetracycline and its derivatives

Design and synthesize a DNA fragment (tetO-X) containing the tetracycline control gene tetO and a restriction endonuclease recognition site. The 3' end of one strand is coupled to biotin, and the 3' end of the other strand is coupled to fluorescein. The two strands are complementary hybridized in vitro to form a stretch of double-stranded DNA for later use.

The sequences of the two chains of tetO-X are as follows:

tetOF: 5’-GTTATTTTACCACGGAATTCCTCCCTATCAGTGATAGAGAAA-3’-TAMRA (SEQ ID No. 1)

tetOR: 5’-TTTCTCTATCACTGATAGGGAGGAATTCCGTGGTAAAATAACTTTTTTTTTTTTT-3’-biotin (SEQ ID No. 2).

Among them, the sequence of the tetracycline control gene tetO is shown in positions 22-40 of SEQ ID No. 1, and the sequence of the restriction endonuclease EcoRI recognition site is 5'- GAATTC- 3'.

After mixing the above two chains at a ratio of 1:1, heat at 95°C for 5 minutes and then cool to room temperature. The specific operations are shown in Table 1. Add water to 22.6 μl of single-stranded tetOF to 226 μl and name it tube 1. Add 18.9 μl of single-stranded tetOR to water to 189 μl and name it tube 2. Mix 189 μl of each of tube 1 and tube 2 reagents to obtain double-stranded hybridization. Pre-solution; heat the pre-double-stranded hybridization solution in a constant-temperature metal bath at 95°C for 5 minutes to obtain a post-double-stranded hybridization solution; gradually cool the post-double-stranded hybridization solution to room temperature to obtain the double-stranded DNA molecule tetO for detecting tetracycline and its derivatives. -X (concentration of 10 μM).

Take 2 μl of the pre-double-stranded hybridization solution and the double-stranded DNA molecule tetO-X, add 10 μl of water respectively, mix well, and perform fluorescence detection. The blank control is single-chain tetOR.

Table 1 tetO-X double-stranded hybridization method

The fluorescence detection results are shown in Table 2. The fluorescence value before hybridization is the fluorescence detection result of the solution before double-stranded hybridization. The fluorescence value after hybridization is the fluorescence detection result of the double-stranded DNA molecule tetO-X. 1 and 2 represent respectively. Two parallel experiments, A, B, C and D respectively represent the fluorescence values of the pre-double-stranded hybridization solution or tetO-X at different dilution ratios. This test result proves that heating at 95°C for 5 minutes has no effect on the fluorescence value of fluorescein connected to DNA fragments.

Table 2 Effect of 95℃5min on tetO-X fluorescence value

2. TetR and restriction endonuclease competitively bind to tetO-X

A. When TetR does not exist, there is no steric hindrance between proteins.

(1) The experiment is divided into 8 experimental groups A1-H1. Among them, A1 and B1 are the double-stranded DNA molecules tetO-X prepared above and diluted to 20 times with water. C1 and D1 are the double-stranded DNA molecules tetO-X prepared above. X is diluted to 100 times by adding water, E1 and F1 are diluting the double-stranded DNA molecules tetO-X prepared previously by adding water to 500 times respectively, G1 and H1 are diluting the double-stranded DNA molecules tetO-X prepared previously by adding water to 2500 times respectively. times, add the diluted tetO-X to the streptavidin-coated enzyme plate in an amount of 100 μl/well. After incubation at room temperature for 1 hour, wash three times with PBST and perform fluorescence detection.

(2) Add 1 μl/well of restriction enzyme and 100 μl/well of buffer to the experimental group treated in step (1). The restriction endonuclease is QuickCut ^TM EcoR I, and the buffer is 10x buffer diluted with QuickCut ^TM EcoR I. times obtained. After treatment for 5 minutes at 37°C, wash three times with PBST and perform fluorescence detection.

The blank control is single-chain tetOR.

See Table 3 for specific processing methods and fluorescence detection results. Experiments have shown that restriction endonuclease can bind to tetO-X and specifically recognize the enzyme cutting site, thereby cutting tetO-X and reducing the fluorescence value.

Table 3 Changes in fluorescence value when steric hindrance does not exist

B. When TetR is added first, TetR binds to the specific site on tetO-X first and forms steric hindrance with the restriction endonuclease.

(1) The experiment is divided into 8 experimental groups A1-H1. Among them, A1 and B1 are the double-stranded DNA molecules tetO-X prepared above and diluted to 20 times with water. C1 and D1 are the double-stranded DNA molecules tetO prepared above. -X is diluted to 100 times by adding water, E1 and F1 are diluting the double-stranded DNA molecules tetO-X prepared previously by adding water to 500 times respectively, G1 and H1 are diluting the double-stranded DNA molecules tetO-X prepared previously by adding water to 2500 times, add the diluted tetO-X to the streptavidin-coated enzyme plate in an amount of 100 μl/well. After incubation at room temperature for 1 hour, wash three times with PBST and perform fluorescence detection.

(2) Add tetR diluted 1000 times with water to the experimental groups A1, C1, E1, and G1 treated in step (1), and add 10mM, PBS (solvent) pH 7.4. The amount of reagent added is 100 μl/well. After treatment at room temperature for 1 hour, wash three times with PBST and perform fluorescence detection.

(3) Add 1 μl/well of QuickCut ^TM EcoR I and 100 μl/well of buffer to the experimental groups A1, C1, E1, and G1 treated in step (2) respectively. The buffer is obtained by diluting 10 times of 10xbuffer. Add them to step (2) respectively. ) After treatment, add 100 μl/well of 10mM PBS (solvent) with a pH of 7.4 to the experimental groups B1, D1, F1, and H1. After treatment for 5 minutes at 37°C, wash three times with PBST and perform fluorescence detection.

See Table 4 for specific processing methods and fluorescence detection results. Experiments have shown that when TetR is added first, TetR binds to the specific site on tetO-X first and forms steric hindrance with the restriction endonuclease. When the restriction endonuclease is added, the restriction endonuclease is prevented from binding to tetO-X, and the specific enzyme cutting site cannot be cut, and the fluorescence value remains unchanged.

Table 4 Changes in fluorescence value when steric hindrance exists

In the table, + means added, - means not added.

Example 2. Detection of tetracycline and its derivatives using tetO-X

1. Determine the optimal working concentrations of TetR and EcoRI

After diluting tetO-X prepared in Example 1 with water 1:2500, add it to a streptavidin-coated black ELISA plate, incubate for 1 hour, and wash three times with PBST. At the same time, 100 μl/well of TetR solution and QuickCut ^TM EcoRI solution serially diluted in 10xbuffer were added respectively. From left to right on the ELISA plate, the dilutions of TetR were: 1:100, 1:300, 1:900, 1:2700, 1: 8100, 1:24300. From top to bottom of the ELISA plate, the amounts of QuickCut ^TM EcoR I added are 0.2 μL, 0.4 μL, 0.6 μL, 0.8 μL, 1.0 μL, 1.2 μL, 1.4 μL, and 1.6 μL. At the same time, add 10ng/mL tetracycline. After treatment at room temperature for 1 hour, wash three times with PBST and perform fluorescence detection.

The checkerboard method determined that the optimal working concentration of TetR and QuickCut ^TM EcoR I was TetR 1:2700-fold dilution, and the addition amount of QuickCut ^TM EcoR I was 0.8 μL/well.

2. Draw tetracycline standard curve

The tetO-X prepared in Example 1 was diluted with water 1:2500, then added to a streptavidin-coated black ELISA plate, incubated for 1 hour, and washed three times with PBST. At the same time, 100 μl/well of TetR solution diluted with 10xbuffer and QuickCut ^TM EcoRI solution were added. The dilution of TetR was 1:2700, and the amount of EcoRI added was 0.8 μL. At the same time, 100 μl/well of tetracycline with concentrations of 9.72ng/mL, 3.24ng/mL, 1.08ng/mL, 0.36ng/mL, 0.12ng/mL, 0.04ng/mL, and 0.013ng/mL was added respectively. After treatment at room temperature for 1 hour, wash three times with PBST and perform fluorescence detection.

The standard curve of tetracycline drawn based on the experimental results is shown in Figure 1. The IC ₅₀ value is 0.09ng/mL and the linear range is 0.04-0.23ng/mL.

3. Detection of tetracyclines

The tetO-X prepared in Example 1 was diluted with water 1:2500, then added to a streptavidin-coated black ELISA plate, incubated for 1 hour, and washed three times with PBST. At the same time, TetR and QuickCut ^TM EcoR I diluted in a 10xbuffer series were added. The dilution of TetR was 1:2700, and the amount of QuickCut ^TM EcoR I added was 0.8 μL. At the same time, tetracycline drugs (tetracycline, doxycycline, chlortetracycline, minocycline, demeclocycline, meclocycline, oxytetracycline, and rolicycline) were added respectively. After treatment at room temperature for 1 hour, wash three times with PBST and perform fluorescence detection.

The IC ₅₀ values and cross-reactivity rates of tetracyclines obtained based on the experimental results are shown in Table 5. The calculation formula for the cross-reaction rate is tetracycline drug cross-reaction rate = tetracycline drug IC ₅₀ / tetracycline IC ₅₀ × 100%.

Table 5 Cross-reaction rate table

Drug name IC ₅₀ value (ng/mL) Cross-reaction rate (%) Doxycycline 0.01 987 Chlortetracycline 0.04 214 minocycline 0.06 159 tetracycline 0.09 100 Demeclocycline 0.29 31 meclocycline 0.11 83 Oxytetracycline 0.20 45 Rolicycline 0.31 29

The present invention has been described in detail above. For those skilled in the art, the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without unnecessary experiments. Although specific embodiments of the present invention have been shown, it should be understood that further modifications can be made to the invention. In short, based on the principles of the present invention, this application is intended to include any changes, uses, or improvements to the present invention, including changes that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some essential features may be applied within the scope of the appended claims below.

sequence list

<110> China Agricultural University

<120> Tetracycline drug detection method based on TetR protein steric hindrance and gene shearing technology

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Met Met Ser Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu

1 5 10 15

Leu Leu Asn Glu Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala

20 25 30

Gln Lys Leu Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn

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Lys Arg Ala Leu Leu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His

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His Thr His Phe Cys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu

65 70 75 80

Arg Asn Asn Ala Lys Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp

85 90 95

Gly Ala Lys Val His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu

100 105 110

Thr Leu Glu Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu

115 120 125

Glu Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly

130 135 140

Cys Val Leu Glu Asp Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu

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Thr Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu

165 170 175

Leu Phe Asp His Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu

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Leu Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Ser

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Claims (6)

1. Double-stranded DNA molecule, characterized in that: the double-stranded DNA molecule includes a tetracycline control gene tetO and a restriction endonuclease recognition site, and the 3' end of one strand of the double-stranded DNA molecule is connected to a fluorescent group group, the 3' end of the other chain is connected to biotin, and the biotin and the fluorescent group are located at both ends of the double-stranded DNA molecule; The sequence of one strand of the double-stranded DNA molecule is shown in SEQ ID No. 1, and the sequence of the other strand of the double-stranded DNA molecule is shown in SEQ ID No. 2. 2. A composition for detecting tetracycline drugs, characterized in that: the composition includes the double-stranded DNA molecule of claim 1 and the restriction endonuclease, tetracycline repressor protein, and streptavidin A streptavidin-coated solid phase carrier is used, and the double-stranded DNA molecule is fixed on the streptavidin-coated solid phase carrier. 3. A kit for detecting tetracycline drugs, characterized in that the kit includes the composition of claim 2 and a tetracycline drug as a standard. 4. Application, characterized in that: the application is the application of the double-stranded DNA molecule of claim 1 or the composition of claim 2 or the kit of claim 3 in at least one of the following: 1) Application in the detection or auxiliary detection of tetracyclines for non-disease diagnosis and treatment purposes; 2) Application in the preparation of products for detection or auxiliary detection of tetracycline drugs; 3) Application in detecting or assisting in detecting the content of tetracycline drugs for non-disease diagnosis and treatment purposes; 4) Application in the preparation of products for testing or assisting in detecting the content of tetracycline drugs. 5. The application according to claim 4, characterized in that: in 1) or 3), the application includes mixing the double-stranded DNA molecule and the restriction endonuclease in claim 1 , tetracycline repressor protein, and the sample to be tested, perform fluorescence detection after washing. 6. The application according to claim 5, characterized in that: the product is a test kit.