CN108008128B - Kanamycin rapid detection test paper for nucleic acid aptamer recognition and functional magnetic microsphere separation pretreatment, and preparation and application thereof - Google Patents

Abstract

A kanamycin rapid detection test paper for nucleic acid aptamer recognition and functionalized magnetic microsphere separation pretreatment, and preparation and application thereof belong to the field of environmental, food and medical analysis. The qualitative analysis of kanamycin is realized by observing the color development depth of a detection line by utilizing the good separation effect of the magnetic microspheres, the high affinity between the aptamer (Apt) and a target and the indicator mark characteristic of gold nanoparticles (AuNPs), and meanwhile, the quantitative analysis can be performed by utilizing a colloidal gold quantitative analyzer. The cDNA can mediate to form a sandwich structure to capture AuNPs in a detection line, the number of captured AuNPs is linearly related to the cDNA and further the concentration of kanamycin, and therefore kanamycin content can be judged according to the color development depth of the detection line. The test paper method for detecting kanamycin is simple, convenient and quick, the detection is not limited by environmental conditions, the result is visual, the detection process only needs 20 min, and the method is convenient to expand and apply.

Description

Kanamycin rapid detection test paper for nucleic acid aptamer recognition and functional magnetic microsphere separation pretreatment, and preparation and application thereof

Technical Field

The invention relates to a kanamycin rapid detection test paper based on aptamer recognition and functionalized magnetic microsphere separation pretreatment, and preparation and application thereof, and belongs to the field of environmental, food and medical analysis.

Background

Antibiotics (Antibiotics) are secondary metabolites that are metabolized by bacteria, fungi, or animals and plants and have anti-pathogenic properties. Compared with other antibiotics, aminoglycoside antibiotics have the advantages of relatively low cost, good water-soluble effect and wide antibacterial spectrumAnd has good treatment effect on common bacterial diseases, so the traditional Chinese medicine composition is widely applied. Kanamycin, one of the most frequently used aminoglycoside antibiotics, is produced by Streptomyces (Streptomyces)Streptomyces kanamyceticus) Produced by fermentation and widely used in the treatment of infections caused by bacteria. Kanamycin acts by misreading the genetic code and inhibiting translation by combining the 30S subunit of ribosome to disturb the synthesis of bacterial proteins, thereby inhibiting the growth and reproduction of bacteria. Kanamycin is applied to veterinary medicine in the forms of injection and capsules as a second-line antibiotic, and effectively inhibits the growth of gram-negative bacteria and gram-positive bacteria. With the increasing frequency and excessive use of kanamycin, it gradually accumulates in foods of animal origin, and is a hazard to our health, possibly causing ototoxicity, uremia, neurotoxicity and the like. The problem of increasing kanamycin residues worldwide is therefore a serious public health threat.

Because of the existence of a plurality of hazards of kanamycin residues, the development of a practical, simple and rapid detection method is necessary to improve the food quality and safety. Various detection methods for antibiotic residues have been reported, such as microbiological detection, instrumental analysis, biochemical immunoassay, electrochemical detection, and the like. The application of the detection methods has some defects and is not suitable for field detection and scale-up production, so that a convenient, rapid, economic, good-accuracy and high-sensitivity detection method for detecting kanamycin residues in food is established as soon as possible, and the safety of animal-derived food can be effectively improved.

Disclosure of Invention

The invention aims to overcome the defects, provides a kanamycin rapid detection test paper based on nucleic acid aptamer recognition and functionalized magnetic microsphere separation pretreatment, and preparation and application thereof, combines the advantages of a colloidal gold labeled chromatography test paper technology, rapid separation of magnetic microspheres, specificity of nucleic acid aptamers and other technologies, prepares a kanamycin residual detection test paper which is simple, convenient, rapid, low in cost and high in sensitivity, and applies the test paper to sample detection.

The invention provides a kanamycin rapid detection test paper based on aptamer recognition and functionalized magnetic microsphere separation pretreatment, which comprises a sample pad, a gold label pad, a nitrocellulose membrane, a water absorption pad, a PVC (polyvinyl chloride) bottom plate, a detection line and a quality control line; a sample pad, a gold-labeled pad, a nitrocellulose membrane and a water absorption pad are sequentially stuck on the PVC base plate, wherein the sample pad is lapped with the gold-labeled pad, the nitrocellulose membrane is directly arranged on the PVC base plate, one end of the nitrocellulose membrane is lapped with the gold-labeled pad, and the other end of the nitrocellulose membrane is lapped with the water absorption pad; and the nitrocellulose membrane is sequentially provided with a detection line and a quality control line.

The overlapping sections of the sample pad, the gold label pad, the nitrocellulose membrane and the absorbent pad are 2 mm.

Functional gold nanoparticles AuNPs-DNA1 are dripped on the gold label pad, a capture probe 1capDNA1 is modified on the detection line, a capture probe 2 capDNA2 is modified on the quality control line, cDNA can be mediated to form a complementary chain sandwich structure after migrating to the detection line to capture the AuNPs, and redundant AuNPs are captured on the quality control line; the kanamycin concentration is correlated with the number of AuNPs captured on the detection line and the color development depth.

The preparation process of the gold mark pad comprises the following steps: preparing gold nanoparticles, and coupling DNA1 with the gold nanoparticles through covalent action of Au-S bonds to form functionalized gold nanoparticles AuNPs-DNA 1;

wherein the DNA1 sequence is: 5' -ATAGCACAAC CGTC- (CH)₂)₆-HS-3’

Preparing a detection line by modifying capDNA1 with pre-incubated streptavidin and biotin at one end of the nitrocellulose membrane close to the gold-labeled pad, and preparing a quality control line by modifying capDNA2 with streptavidin and biotin at one end close to the water absorption pad;

wherein the sequences of capDNA1 and capDNA2 are:

capDNA1：5’-Biotin-TGACTGTAAG CCG-3’；

capDNA2：5’-GGTTGTGCTA TTATGA-Biotin-3’。

the preparation method of the kanamycin rapid detection test paper based on the aptamer recognition and the functionalized magnetic microsphere separation pretreatment comprises the following steps:

(1) preparing functionalized magnetic microspheres: firstly, designing a section of cDNA partially complementary with an aptamer Apt, carrying out metal bath on the cDNA and the Apt for 3min at 95 ℃, then fully mixing the cDNA and the Apt, and incubating for 1h at 37 ℃ to fully hybridize the Apt-cDNA and the Apt; washing magnetic microspheres MBs for 5 times by using 10mmol/L, pH 7.4.4 PBS buffer solution, mixing the magnetic microspheres with the hybrid DNA, oscillating for 1h at 37 ℃ to form functionalized magnetic microspheres MBs-Apt-cDNA, washing the functionalized magnetic microspheres for 3 times by using washing solution to remove Apt-cDNA which is not bonded to the surfaces of the magnetic microspheres, and storing the pretreated functionalized magnetic microspheres MBs-Apt-cDNA for later use at 4 ℃;

wherein the sequences of Apt and cDNA are respectively:

Apt：5’- Biotin-TGGGGGTTGA GGCTAAGCCG A-3’，

cDNA：5’- TTGTGCTATC GGCTTAC-3’；

(2) preparing functionalized gold nanoparticles: soaking all glass instruments in aqua regia for 30min before preparing gold nanoparticles, cleaning with distilled water, soaking in ultrapure water for 12h, and drying; adding 100mL of chloroauric acid with the mass concentration of 0.01% into a 250mL round-bottom flask, stirring and heating to boil, rapidly adding 3.5mL of trisodium citrate with the mass concentration of 1% under vigorous stirring, continuously heating and stirring for 15min, turning the solution into wine red, stopping heating, continuously stirring for 30min, standing, naturally cooling at room temperature to obtain a gold nanoparticle solution with the particle size of 13nm, and storing at 4 ℃ for later use;

continuously heating the gold nanoparticle solution under the condition of boiling water bath and continuously stirring to evaporate water, stopping boiling water bath until the volume of the solution in the flask reaches 20mL, naturally cooling at room temperature to obtain concentrated gold nanoparticles, and storing at 4 ℃ for later use;

mixing 15 mu L of 1mmol/L tris (2-carboxyethyl) phosphine TCEP with 30 mu L of 100 mu mol/L DNA1, carrying out sulfhydrylation reaction, then adding into 1mL of concentrated gold nanoparticles, and stirring for 1h at room temperature; then, 30 mu L of 15mmol/L deoxyadenosine triphosphate dATP is added into the solution, and stirring is carried out for 35min at room temperature; adding 20 mu after the completion of the shakingPlacing L1 mol/L NaCl solution at room temperature for 30min, and storing at 4 ℃ for 6h to increase the stability of combination; centrifuging at 4 deg.C and 8000r/min for 10min to remove excessive reagent; resuspended in 1mL of 20mmol/L Na containing 0.5% Tween-20, 5% BSA, 2% sucrose, 0.5% TritonX-100₃PO₄Obtaining the functionalized gold nanoparticles AuNPs-DNA1 in a buffer solution, and storing at 4 ℃ in a dark place for later use;

wherein the sequence of the DNA1 is as follows: 5' -ATAGCACAACCGTC- (CH)₂)₆-SH-3', partially complementary to both cDNA and capDNA 2;

(3) pretreating a sample pad and a gold label pad: cutting the gold label pad and the sample pad into proper sizes, completely soaking the gold label pad and the sample pad in 0.1 mol/L Tris-HCl buffer solution containing 1% NaCl, 0.5% Tween-20, 1% BSA, 2% sucrose and 1% TritonX-100 at pH8.2 for 30min, then drying the gold label pad and the sample pad in a constant-temperature drying oven at 37 ℃, and storing the gold label pad and the sample pad under a drying condition for later use;

dripping functional gold nanoparticles AuNPs-DNA1 on the treated gold label pad, drying in a constant-temperature drying oven at 37 ℃, and storing under a drying condition for later use;

(4) pretreating a nitrocellulose membrane: first, biotinylated capDNA1 and capDNA2 were bound to Streptavidin, respectively; uniformly mixing 10 mu L of 1mg/mL streptavidin with 20 mu L of 10 mu mol/L biotinylated capDNA1 and capDNA2 respectively, and then combining for 2h under the condition of 4 ℃ to fully couple the streptavidin with the biotinylated capDNA1 and capDNA 2;

marking a detection line T line at a position 6mm close to one end of the gold standard pad by 1 mu L of combined Streptavidin-biotin-capDNA 1; marking a quality control line C at a position 6mm close to one end of the water absorption pad by 1 mu L of Streptavidin-biotin-capDNA 2; drying the cellulose nitrate membrane with the drawn T, C line at 37 ℃, and storing at 4 ℃ for later use;

(5) assembling the test strip: the test paper strip mainly comprises five parts, namely a PVC bottom plate, a sample pad, a gold label pad, a nitrocellulose membrane and a water absorption pad, wherein the nitrocellulose membrane is firstly adhered to a corresponding position of the PVC bottom plate, the nitrocellulose membrane and the sample pad are lightly pressed to be tightly combined, the treated sample pad and the treated gold label pad are respectively adhered to corresponding positions of the PVC bottom plate, the sample pad and the gold label pad are respectively overlapped by 2mm, the gold label pad and the nitrocellulose membrane are respectively overlapped by 2mm and are adhered to the PVC bottom plate, and the redundant part is cut off; each part is firmly stuck and cut into test strips with the specification of 60 mm multiplied by 4 mm, and the test strips are stored under dry condition for standby.

The application of the kanamycin rapid detection test paper based on the separation pretreatment of the aptamer and the functionalized magnetic microsphere comprises the following steps: washing the functional magnetic microsphere MBs-Apt-cDNA for 3 times by using PBS buffer solution with the concentration of 10mmol/L and the pH value of 7.4, then suspending the functional magnetic microsphere MBs-Apt-cDNA in binding buffer solution, adding a sample to be detected into the binding buffer solution, oscillating the binding buffer solution for 10min to ensure that Apt on the surface of the magnetic microsphere is fully bound with kanamycin contained in the sample, and replacing the cDNA complementary with the Apt into the solution;

the binding buffer solution is specifically Tris-HCl buffer solution containing 1mmol/L EDTA, 50mmol/L NaCl, 50mmol/L of 0.05% Tween-20 and pH7.5;

adsorbing the magnetic microspheres under the action of magnetic force, and taking supernatant containing cDNA as test strip detection liquid; placing the prepared test strip in a test strip card box, and dropwise adding a solution to be detected into a sample adding hole;

when kanamycin does not exist in the detection liquid, the T line does not develop color, and the C line develops color;

when kanamycin exists in the detection liquid, T, C lines are developed; standing for 10min, and after the test strip is completely developed, qualitatively or semi-quantitatively analyzing by observing the color depth of the detection line with naked eyes, or scanning and detecting the test strip in a colloidal gold quantitative analyzer to obtain the peak area values of the detection line and the quality control line, and quantitatively determining the kanamycin concentration in the detection solution according to a standard curve.

The method has the beneficial effects that ① the method utilizes the good separation effect of the magnetic microspheres and the conformation change of the aptamer to realize the change of the optical intensity of the detection line, so as to realize the detection of the kanamycin, the test paper method ② is simple, convenient and quick to detect the kanamycin, the detection is not limited by environmental conditions, the result is visual, the expansion and the application are convenient, the detection process only needs 20 min to obtain the experimental result, the detection efficiency is greatly improved compared with the common method for detecting the kanamycin, the actual application effect is obviously enhanced, ③ the detection method can realize the qualitative analysis of the kanamycin by observing the color intensity, can also carry out quantitative detection by a colloidal gold quantitative instrument, and has important significance for the detection of the kanamycin residue in animal-derived food.

Drawings

FIG. 1 is a schematic diagram of kanamycin detection test paper based on aptamer and functionalized magnetic microsphere separation. A. A magnetic microsphere pretreatment process diagram; B. a test strip composition diagram; C. a negative result chart is detected by test paper; D. a positive result chart is detected by test paper; 1. a sample pad; 2. a gold label pad; 3. a nitrocellulose membrane; 4. a water absorbent pad; 5. a PVC base plate; 6. detecting lines; 7. and (4) quality control line.

FIG. 2 is a test strip for testing kanamycin standard solutions at different concentrations.

FIG. 3 is a standard graph of peak area at the T-line in a standard test solution versus kanamycin concentration.

FIG. 4 is a diagram showing the specificity of the test strip.

FIG. 5 is a graph showing the stability of the test strip assay.

FIG. 6 is a real sample detection image of the test strip. A. Milk; B. honey; C. milk powder.

Detailed Description

Example 1 kanamycin rapid detection test paper based on separation pretreatment of aptamer and magnetic microsphere

The method comprises the steps of pretreatment of magnetic microspheres, preparation and functionalization of gold nanoparticles, pretreatment of a gold label pad and a sample pad, pretreatment of a nitrocellulose membrane, assembly of a test strip, and detection of the color development condition of the test strip; the method comprises the following specific steps:

(1) preparation of functionalized magnetic microspheres

Firstly, designing a section of cDNA partially complementary with an aptamer Apt, and carrying out metal bath on the cDNA and the Apt for 3min at 95 ℃ respectively to open the complementary part of the cDNA to form a single chain, thereby facilitating the complementary hybridization of the cDNA and the Apt in the next step. The cDNA and the Apt are fully mixed and incubated for 1h in a constant temperature incubator at 37 ℃ to fully combine the cDNA and the Apt. Because the surface of the magnetic microsphere is modified with streptavidin and the 5' end of the Apt is modified with biotin, the Apt can be coupled to the surface of the magnetic microsphere through the action of the streptavidin-biotin. And (3) washing the magnetic microspheres with 10mmol/L, pH 7.4.4 PBS buffer solution for 5 times, combining the magnetic microspheres with the Apt-cDNA mixed solution, and shaking for 1h at 37 ℃ to avoid the sedimentation of the magnetic microspheres to form the MBs-Apt-cDNA. And washing the bonded magnetic microspheres for 3 times by using a washing solution to remove Apt-cDNA which is not bonded to the surfaces of the magnetic microspheres. And (3) storing the pretreated functional magnetic microsphere MBs-Apt-cDNA at 4 ℃ for later use.

(2) Preparation and functionalization of gold nanoparticles

Soaking all glass instruments in aqua regia for 30min before preparing gold nanoparticles, cleaning with distilled water, soaking in ultrapure water for 12h, and drying; adding 100mL of chloroauric acid with the mass concentration of 0.01% into a 250mL round-bottom flask, stirring and heating to boil, rapidly adding 3.5mL of trisodium citrate with the mass concentration of 1% under vigorous stirring, continuously heating and stirring for 15min, turning the solution into wine red, stopping heating, continuously stirring for 30min, standing, naturally cooling at room temperature to obtain a gold nanoparticle solution with the particle size of 13nm, and storing at 4 ℃ for later use;

continuously heating the gold nanoparticle solution under the condition of boiling water bath and continuously stirring to evaporate water, stopping boiling water bath until the solution in the flask is 20mL, naturally cooling at room temperature to obtain concentrated gold nanoparticles, and storing at 4 ℃ for later use;

mixing 15 mu L of 1mmol/L tris (2-carboxyethyl) phosphine TCEP with 30 mu L of 100 mu mol/L DNA1, carrying out sulfhydrylation reaction, then adding into 1mL of concentrated gold nanoparticles, and stirring for 1h at room temperature; then, 30 mu L of 15mmol/L deoxyadenosine triphosphate dATP is added into the solution, and stirring is carried out for 35min at room temperature; after the shaking is finished, 20 mu L1 mol/L NaCl solution is added and placed for 30min at room temperature, and the mixture is stored for 6h at 4 ℃ to increase the stability of the combination; centrifuging at 4 deg.C and 8000r/min for 10min to remove excessive reagent; resuspended in 1mL of 20mmol/L Na containing 0.5% Tween-20, 5% BSA, 2% sucrose, 0.5% TritonX-100₃PO₄Storing in buffer solution at 4 deg.C in dark place;

(3) pretreatment of sample pad and gold label pad

Cutting the gold label pad and the sample pad into proper sizes, completely soaking the gold label pad and the sample pad in 0.1 mol/L Tris-HCl buffer solution with the pH of 8.2 and containing 1% NaCl, 0.5% Tween-20, 1% BSA, 2% sucrose and 1% TritonX-100 for 30min, then placing the gold label pad and the sample pad in a 37 ℃ constant temperature drying box for drying, and storing under a drying condition for later use;

dripping functional gold nanoparticles, namely AuNPs-DNA1, on the treated gold label pad, wherein the DNA1 is partially complementary with the cDNA and the capDNA2, drying in a constant-temperature drying oven at 37 ℃, and storing under the drying condition for later use;

(4) nitrocellulose membrane pretreatment

First, streptavidin was bound to biotinylated capDNA1 and capDNA 2. After being uniformly mixed with 20 mu L of 10 mu mol/L biotinylated capDNA1 and capDNA2, 10 mu L of 1mg/mL streptavidin was bound for 2h at 4 ℃, strong binding force between streptavidin and biotin could sufficiently couple the streptavidin to biotinylated capDNA1 and capDNA 2. And marking a detection line (T line) at a position 6mm close to one end of the gold label pad by 1 mu L of combined Streptavidin-biotin-capDNA 1. A quality control line (line C) was scribed at 6mm from 1 mu L of Streptavidin-biotin-capDNA2 near one end of the absorbent pad. And (3) drying the NC film with the marked T, C line at 37 ℃, and storing at 4 ℃ for later use under the drying condition.

(5) Assembly of test strips

The colloidal gold chromatography test strip mainly comprises five parts, namely a PVC (polyvinyl chloride) base plate, a sample pad, a gold label pad, a nitrocellulose membrane and a water absorption pad, wherein the nitrocellulose membrane is firstly adhered to a corresponding position of the PVC base plate and lightly pressed to be tightly combined, the treated sample pad and the treated gold label pad are respectively adhered to corresponding positions of the PVC base plate, the sample pad and the gold label pad are respectively overlapped by 2mm, the gold label pad and the nitrocellulose membrane are respectively overlapped by 2mm, and finally the water absorption pad and the nitrocellulose membrane are overlapped by 2mm and are adhered to the PVC base plate, and the redundant parts are cut off; then all parts are stuck firmly and cut into test strips with the specification of 60 mm multiplied by 4 mm, and the test strips are stored under dry condition for standby.

EXAMPLE 2 test strip for kanamycin Standard solution

(1) Pretreatment of magnetic microspheres

Firstly, designing a section of cDNA partially complementary with an aptamer Apt, and carrying out metal bath on the cDNA and the Apt for 3min at 95 ℃ respectively to open the complementary part of the cDNA to form a single chain, thereby facilitating the complementary hybridization of the cDNA and the Apt in the next step. The cDNA and the Apt are fully mixed and incubated for 1h in a constant temperature incubator at 37 ℃ to fully combine the cDNA and the Apt. Because the surface of the magnetic microsphere is modified with streptavidin and the 5' end of the Apt is modified with biotin, the Apt can be coupled to the surface of the magnetic microsphere through the action of the streptavidin-biotin. And (3) washing the magnetic microspheres with 10mmol/L, pH 7.4.4 PBS buffer solution for 5 times, combining the washed magnetic microspheres with the Apt-cDNA mixed solution, and shaking for 1h at 37 ℃ to avoid the sedimentation of the magnetic microspheres to form the MBs-Apt-cDNA. And washing the bonded magnetic microspheres for 3 times by using a cleaning solution to remove Apt-cDNA which is not bonded to the surfaces of the magnetic microspheres. And storing the pretreated functional magnetic microspheres MBs-Apt-cDNA at 4 ℃ for later use.

(2) The test paper prepared by the method is used for qualitative or semi-quantitative analysis and quantitative detection of kanamycin by a colloidal gold quantitative analyzer. The kanamycin concentration can be determined by determining the cDNA concentration, since the cDNA amount is proportional to the kanamycin concentration, after 0-5 mmol/L kanamycin is treated by the pretreatment step of the magnetic microspheres, and the replaced cDNA is detected by a test strip prepared under the same conditions. As shown in FIG. 2, when kanamycin was not present in the test solution, since cDNA was not replaced, capDNA1 on the test line could not capture gold nanoparticles without cDNA, capDNA2 on the quality control line could capture gold nanoparticles, and therefore only the quality control line developed color on the test strip and the test line did not develop color. When the concentration of kanamycin is gradually increased, the color of the C line is basically unchanged, the color of the T line is gradually increased, and when the concentration of kanamycin reaches 50nmol/L, the detection line can be observed to be slightly colored by naked eyes, so that 50nmol/L is set as the visual detection limit of the detection method. Detecting the peak area of the test strip detection line by using a colloidal gold quantitative instrument, determining the relationship between kanamycin with different concentrations and the peak area of the detection line, as shown in figure 3, the peak area of the detection line gradually increases along with the increase of the kanamycin concentration, when the kanamycin concentration is between 5 and 500 nmol/L, a linear relationship exists between the kanamycin concentration and the peak area of the detection line, and the linear regression equation is as follows: y =678.2684x+263.9928，R²=0.9917, x represents concentration of kanamycin, and y represents peak area of detection line. The theoretical limit of detection calculated from the linear relationship was 4.958nmol/L (S/N = 3). This indicates that the test strip has higher sensitivity.

Example 3 specificity verification of test strip assays

The test paper prepared under the same conditions is used for detecting Streptomycin (STR), Ampicillin (AMP), Neomycin (NEO), Gentamicin (GEN), Kanamycin (KAN) and water with the concentration of 5mmol/L respectively, the detection specificity of the test paper is verified by comparing the color development condition and the peak area size of the detection line after detection, and different antibiotics are used for the peak area graph of the detection line after detection, as shown in figure 4. In FIG. 4, it can be seen that the peak area of the detection line is significantly higher when kanamycin is detected than that of other antibiotics, thus proving that the detection method has higher specificity for kanamycin detection.

Example 4 stability verification of test strip assays

In order to determine the stability of the test strip, the test strip is put at 4 ℃ after being assembled and is stored for different days for detection. In this experiment, the test strips were stored for 0, 3, 7, 15, 30, 60 and 90 days, and then kanamycin (0, 30, 100nmol/L, 10, 100, 500 μmol/L) was detected at different concentrations, and the stability of the detection method is shown in FIG. 5, which is determined by the optical intensity of the detection lines of the test strips at different storage times. In FIG. 5, it can be seen that the peak area of kanamycin detected by the detection method at the same concentration has a slight change within 3 months of storage time, and the peak area of the detection line has a reduction degree of 3.60% -9.84%. The test paper strip has good stability.

EXAMPLE 5 detection of kanamycin residue in actual samples

In order to verify the practicability of the detection method, kanamycin in milk, honey and milk powder is respectively detected. Firstly, kanamycin is added to milk, honey and milk powder samples to reach the concentration of 0, 30 nmol/L, 50nmol/L, 100nmol/L, 200 nmol/L, 500 nmol/L, 1 mu mol/L, 10 mu mol/L and 100 mu mol/L respectively, then the milk, the kanamycin and the milk powder samples are pretreated, 20% trichloroacetic acid solution is dropwise added into the milk, the pH value of the milk is adjusted to 4.6, then the milk is subjected to water bath at 45 ℃ for 10min to precipitate protein, and 10000r/min is centrifuged for 25 min to remove coagulated protein and fat, so that the pretreated milk samples are obtained. Diluting Mel with magnetic microsphere binding solution 10 times. Adding 2 g of milk powder into 5mL of magnetic microsphere binding solution, and mixing uniformly. Centrifuging at 10000r/min for 20 min to remove fat layer, taking supernatant, and diluting the supernatant ten times with magnetic microsphere binding solution to obtain the pretreated milk powder sample. Fully mixing the pretreated milk, honey and milk powder samples with MBs-Apt-cDNA, and detecting the displaced cDNA in the solution by using a test strip after reaction. And detecting and checking the peak condition of the detection line by using a colloidal gold quantitative instrument. In FIG. 6, it can be seen that the test paper strips are used to detect kanamycin in milk, honey and milk powder at the detection limits of 50nmol/L, 50nmol/L and 100nmol/L, respectively. The detection limits of different practical samples are different, the results of detecting the milk and the honey are basically consistent with those of the standard sample, and the detection limit is also within an acceptable range when the milk powder is detected. Therefore, the test strip has good application prospect in actual sample detection.

Example 6 comparison of test strip assays with HPLC assays

High performance liquid chromatography is a common method for antibiotic detection, and the accuracy of a test strip method is verified after HPLC (high performance liquid chromatography) is compared with a test strip detection technology. Kanamycin standard products with different concentrations are detected by test paper strips and HPLC respectively, an HPLC detector used in the invention is an evaporative light detector of Shimadzu, a mobile phase is 0.2% trifluoroacetic acid-methanol (95: 5) solution, the flow rate is 1.0 mL/min, the column temperature is 30 ℃, the sample injection amount is 20 mu L, the drift tube temperature is 80 ℃, and the detection time is 10 min. Comparing the average concentration and the RSD value to determine the accuracy of the detection method. As shown in Table 1, the established test strip detection technology and HPLC are similar to the concentration of the kanamycin standard substance added during detection by analyzing from the angle of average concentration, which indicates that the two detection methods have good accuracy; from the perspective of RSD value, the RSD value detected by the test strip is between 2.15% and 4.96%, and the RSD of HPLC is between 0.25% and 0.59%, which proves that the test strip detection technology is slightly inferior to HPLC, but still has good performance, and meanwhile, the test strip detection method is greatly superior to the HPLC detection method in the aspects of sensitivity and detection limit.

TABLE 1 comparison of test strip detection and HPLC kanamycin detection results

。

Sequence listing

<110> university of south of the Yangtze river

<120> kanamycin rapid detection test paper for nucleic acid aptamer recognition and functionalized magnetic microsphere separation pretreatment, preparation and application thereof

<130>201712031103

<141>2017-12-21

<160>5

<170>SIPOSequenceListing 1.0

<210>1

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<212>DNA

<213> DNA1 sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400>1

atagcacaac cgtc 14

<210>2

<211>13

<212>DNA

<213>capDNA1(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400>2

tgactgtaag ccg 13

<210>3

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<212>DNA

<213>capDNA2(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400>3

ggttgtgcta ttatga 16

<210>4

<211>21

<212>DNA

<213>Apt(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400>4

tgggggttga ggctaagccg a 21

<210>5

<211>17

<212>DNA

<213>cDNA(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400>5

ttgtgctatc ggcttac 17

Claims (5)

1. A kanamycin rapid detection method based on aptamer recognition and functionalized magnetic microsphere separation pretreatment is characterized in that: the test paper used for detection comprises a sample pad (1), a gold label pad (2), a nitrocellulose membrane (3), a water absorption pad (4), a PVC bottom plate (5), a detection line (6) and a quality control line (7); the magnetic microsphere also comprises a functionalized magnetic microsphere MBs-Apt-cDNA;

a sample pad (1), a gold-labeled pad (2), a nitrocellulose membrane (3) and a water absorption pad (4) are sequentially adhered to a PVC base plate (5) of the test paper, wherein the sample pad (1) is lapped with the gold-labeled pad (2), the nitrocellulose membrane (3) is directly arranged on the PVC base plate (5), one end of the nitrocellulose membrane (3) is lapped with the gold-labeled pad (2), and the other end of the nitrocellulose membrane is lapped with the water absorption pad (4); the nitrocellulose membrane (3) is sequentially provided with a detection line (6) and a quality control line (7); overlapping sections of overlapping parts of the sample pad (1), the gold label pad (2), the nitrocellulose membrane (3) and the absorbent pad (4) are 2 mm;

functional gold nanoparticles AuNPs-DNA1 are dripped on the gold-labeled pad (2), a capture probe 1capDNA1 is modified on a detection line (6), a capture probe 2 capDNA2 is modified on a quality control line (7), cDNA can be mediated to form a complementary chain sandwich structure after migrating to the detection line to capture the AuNPs, and redundant AuNPs are captured on the quality control line (7); the kanamycin concentration presents correlation with the number of AuNPs captured on the detection line (6) and the color development depth;

washing the functional magnetic microsphere MBs-Apt-cDNA for 3 times by using PBS buffer solution with the concentration of 10mmol/L and the pH value of 7.4, then suspending the functional magnetic microsphere MBs-Apt-cDNA in binding buffer solution, adding a sample to be detected into the binding buffer solution, oscillating the binding buffer solution for 10min to ensure that the Apt on the surface of the functional magnetic microsphere is fully bound with kanamycin contained in the sample, and replacing the cDNA complementary to the Apt into the solution;

adsorbing the magnetic microspheres under the action of magnetic force, and taking supernatant containing cDNA as test strip detection liquid; placing the prepared test strip in a test strip card box, and dropwise adding a solution to be detected into a sample adding hole;

when kanamycin does not exist in the detection liquid, the T line does not develop color, and the C line develops color;

when kanamycin exists in the detection liquid, T, C lines are developed;

wherein the DNA1 sequence is: 5' -ATAGCACAAC CGTC- (CH)₂)₆-HS-3’；

The sequences of capDNA1 and capDNA2 were:

capDNA1：5’-Biotin-TGACTGTAAG CCG-3’；

capDNA2：5’-GGTTGTGCTA TTATGA-Biotin-3’；

wherein the sequences of Apt and cDNA are respectively:

Apt：5’- Biotin-TGGGGGTTGA GGCTAAGCCG A-3’，

cDNA：5’- TTGTGCTATC GGCTTAC-3’。

2. the kanamycin rapid detection method based on aptamer recognition and magnetic microsphere separation pretreatment of claim 1, characterized in that the gold-labeled pad (2) is prepared by the following steps: preparing gold nanoparticles and coupling DNA1 with the gold nanoparticles through the covalent action of Au-S bonds to form the functionalized gold nanoparticles AuNPs-DNA 1.

3. The kanamycin rapid detection method based on aptamer recognition and magnetic microsphere separation pretreatment of claim 1, which is characterized in that: preparing a detection line (6) by modifying capDNA1 with pre-incubated streptavidin and biotin at one end of the nitrocellulose membrane (3) close to the gold-labeled pad (2), and preparing a quality control line (7) by modifying capDNA2 with streptavidin and biotin at one end close to the water absorption pad (4).

4. The method for rapidly detecting kanamycin based on aptamer recognition and magnetic microsphere separation pretreatment of claim 1, wherein the preparation steps of the detection test paper are as follows:

(1) preparing functionalized magnetic microspheres: firstly, designing a section of cDNA partially complementary with an aptamer Apt, carrying out metal bath on the cDNA and the Apt for 3min at 95 ℃, then fully mixing the cDNA and the Apt, and incubating for 1h at 37 ℃ to fully hybridize the cDNA and the Apt; washing magnetic microspheres MBs for 5 times by using 10mmol/L, pH 7.4.4 PBS buffer solution, mixing the magnetic microspheres with the hybrid Apt-cDNA, oscillating for 1h at 37 ℃ to form functionalized magnetic microspheres MBs-Apt-cDNA, washing the functionalized magnetic microspheres for 3 times by using washing solution to remove Apt-cDNA which is not combined to the surfaces of the magnetic microspheres, and storing the pretreated functionalized magnetic microspheres MBs-Apt-cDNA at 4 ℃ for later use;

(2) preparing functionalized gold nanoparticles: soaking all glass instruments in aqua regia for 30min before preparing gold nanoparticles, cleaning with distilled water, soaking in ultrapure water for 12h, and drying; adding 100mL of chloroauric acid with the mass concentration of 0.01% into a 250mL round-bottom flask, stirring and heating to boil, rapidly adding 3.5mL of trisodium citrate with the mass concentration of 1% under vigorous stirring, continuously heating and stirring for 15min, turning the solution into wine red, stopping heating, continuously stirring for 30min, standing, naturally cooling at room temperature to obtain a gold nanoparticle solution with the particle size of 13nm, and storing at 4 ℃ for later use;

continuously heating the gold nanoparticle solution under the condition of boiling water bath and continuously stirring to evaporate water, stopping boiling water bath until the volume of the solution in the flask reaches 20mL, naturally cooling at room temperature to obtain concentrated gold nanoparticles, and storing at 4 ℃ for later use;

mixing 15 mu L of 1mmol/L tris (2-carboxyethyl) phosphine TCEP with 30 mu L of 100 mu mol/L DNA1, carrying out sulfhydrylation reaction, then adding into 1mL of concentrated gold nanoparticles, and stirring for 1h at room temperature; then 30 mu L of 15mmol/L deoxyadenosine triphosphateAdding dATP into the solution, and stirring at room temperature for 35 min; after the oscillation is finished, 20 mu L of 1mol/L NaCl solution is added and placed for 30min at room temperature, and the mixture is stored for 6h at 4 ℃ to increase the stability of combination; centrifuging at 4 deg.C and 8000r/min for 10min to remove excessive reagent; resuspended in 1mL of 20mmol/L Na containing 0.5% Tween-20, 5% BSA, 2% sucrose, 0.5% TritonX-100₃PO₄Obtaining the functionalized gold nanoparticles AuNPs-DNA1 in a buffer solution, and storing at 4 ℃ in a dark place for later use;

wherein DNA1 is partially complementary to both cDNA and capDNA 2;

(3) pretreating a sample pad and a gold label pad: cutting the gold label pad and the sample pad into proper sizes, completely soaking the gold label pad and the sample pad in 0.1 mol/L Tris-HCl buffer solution containing 1% NaCl, 0.5% Tween-20, 1% BSA, 2% sucrose and 1% TritonX-100 at pH8.2 for 30min, then drying the gold label pad and the sample pad in a constant-temperature drying oven at 37 ℃, and storing the gold label pad and the sample pad under a drying condition for later use;

dripping functional gold nanoparticles AuNPs-DNA1 on the treated gold label pad, drying in a constant-temperature drying oven at 37 ℃, and storing under a drying condition for later use;

(4) pretreating a nitrocellulose membrane: first, biotinylated capDNA1 and capDNA2 were bound to Streptavidin, respectively; uniformly mixing 10 mu L of 1mg/mL streptavidin with 20 mu L of 10 mu mol/L biotinylated capDNA1 and capDNA2 respectively, and then combining for 2h under the condition of 4 ℃ to fully couple the streptavidin with the biotinylated capDNA1 and capDNA 2;

marking a detection line T line at a position 6mm close to one end of the gold standard pad by 1 mu L of combined Streptavidin-biotin-capDNA 1; marking a quality control line C at a position 6mm close to one end of the water absorption pad by 1 mu L of Streptavidin-biotin-capDNA 2; drying the cellulose nitrate membrane with the drawn T, C line at 37 ℃, and storing at 4 ℃ for later use;

(5) assembling the test strip: the test strip mainly comprises five parts, namely a PVC (polyvinyl chloride) base plate (5), a sample pad (1), a gold label pad (2), a nitrocellulose membrane (3) and a water absorption pad (4), wherein the nitrocellulose membrane is firstly adhered to a corresponding position of the PVC base plate, the nitrocellulose membrane and the sample pad are lightly pressed to be tightly combined, the treated sample pad and the treated gold label pad are respectively adhered to corresponding positions of the PVC base plate, the sample pad and the gold label pad are respectively overlapped by 2mm, the gold label pad and the nitrocellulose membrane are respectively overlapped by 2mm, finally the water absorption pad and the nitrocellulose membrane are overlapped by 2mm and adhered to the PVC base plate, and redundant parts are cut off; each part is firmly stuck and cut into test strips with the specification of 60 mm multiplied by 4 mm, and the test strips are stored under dry condition for standby.

5. The use of test paper for rapid kanamycin detection prepared by the method of claim 4, which is characterized in that: the functionalized magnetic microsphere MBs-Apt-cDNA is resuspended in a binding buffer solution, wherein the binding buffer solution is specifically 50mmol/L Tris-HCl buffer solution containing 1mmol/L EDTA, 50mmol/L NaCl, 0.05% Tween-20 and pH7.5;

adsorbing the magnetic microspheres under the action of magnetic force, and taking supernatant containing cDNA as test strip detection liquid; placing the prepared test strip in a test strip card box, and dropwise adding a solution to be detected into a sample adding hole;

when kanamycin does not exist in the detection liquid, the T line does not develop color, and the C line develops color;

when kanamycin exists in the detection liquid, T, C lines are developed; standing for 10min, and after the test strip is completely developed, qualitatively or semi-quantitatively analyzing by observing the color depth of the detection line with naked eyes, or scanning and detecting the test strip in a colloidal gold quantitative analyzer to obtain the peak area values of the detection line and the quality control line, and quantitatively determining the kanamycin concentration in the detection solution according to a standard curve.

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