CN103048449A - Chromatographic detection kit based on aptamer, as well as preparation method and detection method thereof - Google Patents

Abstract

The invention discloses a nucleic acid aptamer-based chromatography detection kit and its preparation method and detection method. The detection test paper of the kit includes a base plate, a sample pad bonded to the base plate and overlapped sequentially, and a binding pad , a nitrocellulose membrane and a water-absorbing pad; the side of the nitrocellulose membrane close to the binding pad is provided with a detection line, and the side of the nitrocellulose membrane close to the water-absorbing pad is provided with a quality control line; the binding pad is coated with a colloidal gold mark A nucleic acid aptamer; the detection line is coated with a complex of B nucleic acid aptamer and streptavidin; the quality control line is coated with streptavidin; both A nucleic acid aptamer and B nucleic acid aptamer can A biotinylated aptamer that specifically recognizes the same analyte. The kit only needs to directly dilute the sample to be tested and drop it into the sample hole, and the result can be obtained within five minutes. Easy to read, easy to make.

Description

Chromatography detection kit based on nucleic acid aptamer and its preparation method and detection method

technical field

The present invention relates to a medical detection product and a production method thereof, in particular to a nucleic acid aptamer-based chromatography detection kit and a preparation method and detection method thereof.

Background technique

The common immunoassay method is to immobilize a specific labeled antibody on a solid support first, specifically bind to the analyte, and then bind to another antibody, so that the analyte can be qualitatively detected by the naked eye or a specific detection instrument. , so as to realize the specific immunodiagnosis.

The biological macromolecules used in the commonly used immunochromatography method to specifically recognize the detection object are antibodies. Through the specific recognition reaction of antigen and antibody, the immune colloidal particles marked on the antigen or antibody make the detection line and quality control line display a certain color, thereby Realize specific immunodiagnosis. However, this method is not fast and sensitive enough, and antibody detection requires corresponding equipment, which is not easy to operate.

Contents of the invention

The invention aims to overcome the deficiencies of the prior art, a nucleic acid aptamer-based chromatography detection kit and its preparation method and detection method. By providing a detection substance that can be specifically recognized by a nucleic acid aptamer, on the basis of a conventional colloidal gold immunochromatography kit, a colloidal gold immunochromatography detection kit is produced, and the color is developed on the detection line and the quality control line to achieve Implement detection.

In order to realize the above-mentioned purpose of the invention, the technical scheme provided by the invention is:

The invention provides a nucleic acid aptamer-based chromatography detection kit, including a detection test paper; the detection test paper includes a base plate, a sample pad, a binding pad, and a nitrocellulose membrane bonded to the base plate and sequentially overlapped and a water-absorbing pad; the side of the nitrocellulose membrane close to the binding pad is provided with a detection line, and the side of the nitrocellulose membrane close to the water-absorbing pad is provided with a quality control line; the binding pad is coated with nanoparticle-labeled A nucleic acid suitable for Ligand; the detection line is coated with a complex of B nucleic acid aptamer and streptavidin; the quality control line is coated with streptavidin; the A nucleic acid aptamer and B nucleic acid aptamer All are biotin-labeled nucleic acid aptamers that can specifically recognize the same detection object; wherein, the nano-particles are gold nanoparticles, ferric oxide or latex.

Wherein, the 5' end of the DNA sequence of the A nucleic acid aptamer is labeled with a sulfhydryl group, and the 3' end is labeled with biotin; the 3' end of the DNA sequence of the B nucleic acid aptamer is labeled with biotin.

The sequence of the A nucleic acid aptamer and the B nucleic acid aptamer can be designed and adjusted according to the substance to be detected. In one embodiment of the present invention, the DNA sequence of the A nucleic acid aptamer is as SEQ ID NO.1 Shown, the DNA sequence of described B nucleic acid aptamer is as shown in SEQ ID NO.2; In another embodiment of the present invention, the DNA sequence of described A nucleic acid aptamer is as shown in SEQ ID NO.3 , the DNA sequence of the B nucleic acid aptamer is shown in SEQ ID NO.4; in another embodiment of the present invention, the DNA sequence of the A nucleic acid aptamer is shown in SEQ ID NO.5, the The DNA sequence of the B nucleic acid aptamer is shown in SEQ ID NO.4.

The bottom plate is a PVC board; the material of the sample pad is polyacetic acid film; the material of the bonding pad is glass fiber; the water-absorbing pad is water-absorbing filter paper; the test paper is packed by a hard plastic card.

The present invention also provides a method for preparing the above kit, comprising the following steps:

(1) Label A aptamer with nanoparticles;

(2) Soak the sample pad in the sample pad treatment solution containing BSA and Tween PBS for more than 1 hour, and dry it for later use;

(3) Redisperse the nucleic acid aptamer A labeled with nanoparticles in step (1) in PBS buffer dilution containing sucrose, BSA and Tween, spray it on the binding pad, and dry it for later use;

(4) Spray the complex of nucleic acid aptamer B and streptavidin at the position of the detection line on the nitrocellulose membrane, spray some streptavidin at the position of the quality control line, and dry it for later use;

(5) Lap the sample pad, binding pad, nitrocellulose membrane and water-absorbent pad sequentially and glue them on the bottom plate, cut into test strips, and pack the test strips with hard plastic cards.

Among them, the step (1) is to use nanoparticles with a diameter of 10-40nm to label the A nucleic acid aptamer; the B nucleic acid aptamer and streptavidin sprayed on the position of the detection line in the step (4) The complex is that the B nucleic acid aptamer concentration is 0.5-1.5 μ M, preferably 1 μ M solution and concentration is 0.5-1.5 mg/mL, preferably 1 mg/mL streptavidin by volume ratio 1:8-12, It is preferably prepared by mixing 1:10; the streptavidin sprayed on the position of the quality control line is a streptavidin dilution with a concentration of 1.5-2.5 mg/mL, preferably 2 mg/mL.

The present invention also provides a detection method based on nucleic acid aptamers:

The method adopts two nucleic acid aptamers that recognize the same detection object, and on the basis of a conventional chromatography kit, the above-mentioned chromatography detection kit is made, and the nucleic acid aptamer A and the nucleic acid aptamer B are combined with the analyte After specific binding, develop color on the test line and quality control line to achieve antibody-free chromatographic detection: if the quality control line does not develop color, the test results are invalid; if the test line develops color but the quality control line does not develop color, it is also considered is invalid; if both the quality control line and the test line are colored, it means that there is analyte in the test sample.

Below in conjunction with the detailed production process and principle of kit, the present invention will be further described:

The concrete manufacturing process of kit of the present invention is as follows:

(1) Label the aptamer 1 with nanoparticles with a diameter of 10-40nm, dilute the nanoparticles with sterile water, then add the aptamer, centrifuge at 10,000rpm for 15 minutes, remove the supernatant, and redissolve the colloid to 0. In 1 M PBS buffer, this operation was repeated three times, and finally the labeled nucleic acid aptamer 1 was redissolved in 0.01 M pH7.4 PBS buffer containing 20mM Na3PO4 and 1% PEG6000;

(2) Select polyacetate film as the sample pad material, soak it in the sample pad treatment solution containing 3% BSA, 0.05% Tween 20, 0.0l M pH7.4PBS buffer solution for 1 hour, and then dry it at 37°C Dry for 2 hours and store at 4°C for later use;

(3) Choose glass fiber as the binding pad material, and redisperse the colloidal gold-labeled aptamer prepared in step (1) in 0.0l M PBS buffer containing 5% sucrose, 1% BSA, and 0.05% Tween 20 , then spray it on the bonding pad made of glass fiber with a film dispenser, dry it at 37 degrees for 2 hours, and store it at 4 degrees for later use;

(4) Spotting on nitrocellulose membrane: Dissolve the synthesized biotin-labeled B nucleic acid aptamer in sterile water at a concentration of 1 μM, and mix it with streptavidin at a concentration of 1 mg/ml at a ratio of 1:10. Mix the volume ratio, react at room temperature for one hour, then add one volume of 0.01M pH7.4 PBS buffer, then transfer to a centrifugal ultrafiltration tube with a molecular weight cut-off of 30,000, centrifuge at 6,000rpm for 15 minutes, and then add the same volume of 0.01 M pH7.4 PBS buffer solution, 6000rpm centrifugation for 15 minutes, repeated operations like this three times, to obtain the B nucleic acid aptamer streptavidin complex equivalent to the original volume; the streptavidin was dissolved in sterile water, Make streptavidin diluent for coating quality control line with a concentration of 2mg/ml. The film spotting machine spots the B nucleic acid aptamer streptavidin complex and the streptavidin dilution on the detection line on the nitrocellulose membrane (the detection line is located at the center line of the entire test paper) and the quality control The two lines are separated by three millimeters, then put the nitrocellulose membrane in a vacuum dryer, dry for half an hour, then dry at 37°C for 1 hour, seal it well and put it at room temperature for later use;

(5) Paste the treated sample pad, binding pad, nitrocellulose membrane and water-absorbing filter paper on the PVC sheet in sequence, then cut into 4 mm wide test strips and put them into plastic cards, and assemble them into chromatography Method detection kit.

When in use, the sample to be tested is added to the sampling hole, and the sample to be tested is chromatographed along the sample pad, binding pad, and nitrocellulose membrane to the end of the absorbent filter paper under capillary action. The detection substance first specifically binds to the nanoparticle-A nucleic acid aptamer complex on the glass fiber binding pad and reconstitutes it. The B nucleic acid aptamer coated in the line area undergoes an immune reaction, and under the action of a certain concentration of the detection substance, a line of the corresponding color appears; the excess nanoparticle-A nucleic acid aptamer complex continues to move forward, and the A nucleic acid When the biotin labeled on the aptamer reaches the area of the quality control line, it combines with the coated streptavidin, enriches the nanoparticles, and shows the color lines of the nanoparticles. If there is no analyte in the sample, the nanoparticle-A nucleic acid aptamer complex and B nucleic acid aptamer will not act in the detection line region, but continue to forward chromatography through the biostreptavidin reaction. Therefore, the detection line is colorless, while the quality control line shows the color of nanoparticles. The quality control line is used to judge the quality of the kit: when the concentration of the test substance in the sample is too high, or there is high salt, it will cause all the nanoparticle-A nucleic acid aptamers to be enriched in the test line, and there is no excess colloidal gold to continue Forward, the quality control line will not have nanoparticle gold enrichment, and it will appear colorless, indicating that the microenvironment of the test is beyond the scope of application of this test box, and the result is invalid. When the nucleic acid aptamer is detached or degraded from the surface of the nanoparticle, or the quality of the kit changes due to other reasons, there will be no nanoparticles on the detection line and the quality control line, and both the detection line and the quality control line are colorless. Indicates that the test result is invalid. Therefore, in the detection, the detection line and the quality control line show the color of the nanoparticles at the same time, which shows that the detection result is credible.

The quality control line and test line displayed by the immunochromatography method of the present invention are all the colors of nanoparticles. The nucleic acid aptamer can also specifically recognize the detection object, and two nucleic acid aptamers that can specifically recognize the same detection object are respectively coated on the glass fiber binding pad and the nitrocellulose membrane in the colloidal gold immunochromatography. Compared with the prior art, the present invention uses conventional chromatography detection strips to realize antibody-free chromatography: after the labeled A nucleic acid aptamer is combined with the detection substance, the sample will move forward along the nitrocellulose membrane under capillary action. Move, when moving to another nitrocellulose detection line (coated with B nucleic acid aptamer region) that can also specifically recognize the detection substance, the detection substance combined with the A nucleic acid aptamer in the sample is then combined with the B nucleic acid aptamer The nucleic acid aptamer specifically binds, so that the A nucleic acid aptamer labeled with gold nanoparticles, the detection object, and another B nucleic acid aptamer that can also specifically recognize the detection object form a complex, which is a visible nanoparticle Color, thus achieving specific immunochromatographic detection. The present invention does not need to use antibodies when preparing test strips, and does not need other reagents during detection. It only needs to directly dilute the test sample with water and then take 0.12mL and drop it into the sample hole, and the result can be obtained after five minutes. Fast, sensitive and other characteristics, the detection kit does not need to use antibodies, does not use equipment, simple operation, good specificity, easy interpretation of results, and convenient production.

Description of drawings

Fig. 1 is the structural representation of detection test paper in the kit of the present invention;

Figure 2 is a schematic diagram of the principle of the present invention, wherein, there is a detection substance: it means that the concentration of the detection substance is higher than the minimum detection concentration, and the detection line is the color of the nanoparticle (dark color in the figure); no detection substance: it means that the concentration of the detection substance is higher than the minimum detection concentration. The lowest detection concentration, the position of the detection line is colorless (blank in the figure);

Fig. 3 is a judgment diagram of the detection results of the immunochromatography method of the present invention, wherein, T represents the detection line, and C represents the quality control line.

In the figure: 1. bottom plate; 2. sample pad; 3. binding pad; 4. nitrocellulose membrane; 5. detection line; 6. quality control line; 7. absorbent pad.

Detailed ways

The present invention will be further described below in conjunction with specific embodiment:

Example 1

Referring to Fig. 1 to Fig. 3, a kind of colloidal gold immunochromatography detection kit based on nucleic acid aptamer, comprises detection test paper, and described detection test paper comprises base plate 1, the sample pad that is bonded on base plate 1 and lapped successively 2, binding pad 3, nitrocellulose membrane 4 and water-absorbing pad 7; the side of the nitrocellulose membrane 4 close to the binding pad 3 is provided with a detection line 5, and the side of the nitrocellulose membrane 4 close to the water-absorbing pad 7 is provided with Quality control line 6; the binding pad 3 is coated with nanoparticle-labeled A nucleic acid aptamer; the detection line 5 is coated with a complex of B nucleic acid aptamer and streptavidin; the quality control Line 6 is coated with streptavidin; both the A nucleic acid aptamer and the B nucleic acid aptamer are biotin-labeled nucleic acid aptamers that can specifically recognize the same detection object; wherein the nanoparticles are colloidal gold.

Wherein, the 5' end of the DNA sequence of the A nucleic acid aptamer is labeled with a sulfhydryl group, and the 3' end is labeled with biotin; the 3' end of the DNA sequence of the B nucleic acid aptamer is labeled with biotin; the A nucleic acid aptamer The DNA sequence of the ligand is shown in SEQ ID NO.1, and the DNA sequence of the B nucleic acid aptamer is shown in SEQ ID NO.2; the bottom plate 1 is a PVC plate; the material of the sample pad 2 is a polyacetate film; The material of the bonding pad 3 is glass fiber; the water-absorbing pad 7 is water-absorbing filter paper; the detection test paper is packaged by a rigid plastic card.

Example 2

The method for preparing the kit described in Example 1:

(1) Aptamer 1 labeled colloidal gold particles: Centrifuge the synthesized 13nm colloidal gold solution at 10,000rpm for 15 minutes, suck out the supernatant, add the original volume of sterile water, repeat the operation twice, and add 0.5 The ratio of OD nucleic acid aptamer, A nucleic acid aptamer DNA sequence is: 5'-SHC6-ATC AGG GCT AAA GAG TGC AGA GTT ACT TAG TTT TTT TTT T-3'Biotin (SEQ ID NO.1), the The aptamer can specifically recognize lysozyme, and slowly add it to the colloidal gold solution under magnetic stirring. After standing at room temperature for 24 hours, add 0.1 M PB solution to adjust the final concentration of PB to 10mM; add PBS buffer solution to adjust the solution. The NaCl concentration was 0.1M, aged at room temperature for 24 hours; finally, PBS buffer was added to adjust the final concentration of NaCl in the solution to 0.3M, after aging at room temperature for 24 hours, centrifuge at 10,000 rpm, remove the supernatant, and redissolve the colloid to 0.1 M In PBS buffer; this operation was repeated three times, and the labeled nucleic acid aptamer 1 was redissolved in 0.01 M pH7.4 PBS buffer containing 20mM Na3PO4, 1%PEG 6000;

(2) Select polyacetate film as the material of the sample pad, soak the polyacetate film in the sample pad treatment solution containing 3% BSA, 0.05% Tween 20, 0.0l M pH7.4PBS buffer solution for 1 hour, and then 37°C Dry for 2 hours and store at 4°C for later use;

(3) Preparation of binding pads: when used, redisperse the colloidal gold prepared in step (1) in 0.0l M pH7.4 PBS buffer containing 5% sucrose, 5% BSA, 1% PEG20000, 0.05% Tween 20 , then spray it on the bonding pad made of glass fiber with a film dispenser, dry it at 37 degrees for 2 hours, and store it at 4 degrees for later use;

(4) Spotting on nitrocellulose membrane: use synthetic B nucleic acid aptamer labeled with biotin, its DNA sequence is: 5'-GCA GCT AAG CAG GCG GCT CAC AAA ACC ATT CGCATG CGG CTT TTT TTT TT- 3'Biotin (SEQ ID NO.2), dissolved in sterile water at a concentration of 1 μM, mixed with streptavidin at a concentration of 1 mg/ml at a volume ratio of 1:10, reacted at room temperature for one hour, and then added a Double the volume of 0.01M pH7.4 PBS buffer, then transfer to a centrifugal ultrafiltration tube with a molecular weight cut-off of 30,000 Daltons, centrifuge at 6000rpm for 15 minutes, then add the same volume of 0.01MpH7.4 PBS buffer, and centrifuge at 6000rpm For 15 minutes, this operation was repeated three times to obtain the B nucleic acid aptamer streptavidin complex equivalent to the original volume; the streptavidin was dissolved in sterile water to make a coating substance with a concentration of 2 mg/ml Control line with streptavidin diluent. The film spotting machine spots the nucleic acid aptamer 2 streptavidin complex and the streptavidin dilution on the detection line (the detection line is located at the center line of the entire test paper) and the quality control line on the nitrocellulose membrane respectively. The two lines are separated by three millimeters, then put the nitrocellulose membrane in a vacuum dryer, dry for half an hour, and then dry at 37°C for 1 hour, seal it well and put it at room temperature for later use;

(5) Paste the treated sample pad, binding pad, nitrocellulose membrane and water-absorbing filter paper on the PVC sheet in sequence, then cut into 4 mm wide test strips and put them into plastic cards, and assemble them into chromatography method detection kit;

(6) Detection: Take the sample to be tested and dilute it with water, then drop 0.12mL into the sample hole. If the quality control line does not show color, the test results are invalid. If the test line shows red but the quality control line does not show color, it is also considered invalid; Detection concentration (1 μM) of lysozyme.

Example 3

Referring to Fig. 1 to Fig. 3, a kind of colloidal gold immunochromatography detection kit based on nucleic acid aptamer, comprises detection test paper, and described detection test paper comprises base plate 1, the sample pad that is bonded on base plate 1 and lapped successively 2, binding pad 3, nitrocellulose membrane 4 and water-absorbing pad 7; the side of the nitrocellulose membrane 4 close to the binding pad 3 is provided with a detection line 5, and the side of the nitrocellulose membrane 4 close to the water-absorbing pad 7 is provided with Quality control line 6; the binding pad 3 is coated with nanoparticle-labeled A nucleic acid aptamer; the detection line 5 is coated with a complex of B nucleic acid aptamer and streptavidin; the quality control Line 6 is coated with streptavidin; both the A nucleic acid aptamer and the B nucleic acid aptamer are biotin-labeled nucleic acid aptamers that can specifically recognize the same detection object; wherein the nanoparticles are colloidal gold.

Wherein, the 5' end of the DNA sequence of the A nucleic acid aptamer is labeled with a sulfhydryl group, and the 3' end is labeled with biotin; the 3' end of the DNA sequence of the B nucleic acid aptamer is labeled with biotin; the A nucleic acid aptamer The DNA sequence of the ligand is shown in SEQ ID NO.3, and the DNA sequence of the B nucleic acid aptamer is shown in SEQ ID NO.4; the bottom plate 1 is a PVC plate; the material of the sample pad 2 is a polyacetate film; The material of the bonding pad 3 is glass fiber; the water-absorbing pad 7 is water-absorbing filter paper; the detection test paper is packaged by a rigid plastic card.

Example 4

The method for preparing the kit described in Example 3:

(1) Nucleic acid aptamer 1 labeled colloidal gold particles: Centrifuge the synthesized 10nm colloidal gold solution at 10,000rpm for 15 minutes, suck out the supernatant, add the original volume of sterile water, repeat the operation twice, and add 0.5 The ratio of OD nucleic acid aptamer 1, the DNA sequence of the A nucleic acid aptamer that specifically recognizes human thrombin is: 5'SH C6-TTT TTT TTT TGG TTG GTG TGG TTG GTT TTTTTT TT3'Biotin (SEQ ID NO.3), Slowly add to the colloidal gold solution under magnetic stirring, after 24 hours at room temperature, add 100mM PB solution, adjust the final concentration to 10mM; add PBS buffer solution, adjust the NaCl concentration in the solution to 0.1M, and age at room temperature for 24 hours; Finally, add PBS buffer solution, adjust the final concentration of NaCl in the solution to be 0.3M, centrifuge at 10000rpm after aging for 24 hours at room temperature, remove the supernatant, redissolve the colloid in 0.1 M PBS buffer solution, and repeat the operation three times like this, and finally The labeled aptamer 1 was redissolved in 0.01 M pH7.4 PBS buffer containing 20mM Na3PO4 and 1% PEG6000;

(2) Select polyacetate film as the material of the sample pad, soak the polyacetate film in the sample pad treatment solution containing 3% BSA, 0.05% Tween 20, 0.0l M pH7.4PBS buffer solution for 1 hour, and then 37°C Dry for 2 hours and store at 4°C for later use;

(3) Preparation of conjugation pad: When used, redisperse the colloidal gold prepared in step (1) in 0.0l M pH7.2 PBS buffer containing 5% sucrose, 2% BSA, 2% Dextran, 0.05% Tween 20 , then spray it on the glass fiber bonding pad with a film dispenser, dry it at 37°C for 2 hours, and store it at 4°C for later use;

(4) Spotting on nitrocellulose membrane: Use a synthetic nucleic acid aptamer that specifically recognizes human thrombin B labeled with biotin at one end, and its DNA sequence is: 5'-AGT CCG TGG TAG GGC AGG TTGGGG TGA CTT TTT TTT TTT-3'Biotin (SEQ ID NO.4), dissolved in sterile water at a concentration of 1 μM, was mixed with streptavidin at a concentration of 1 mg/ml at a volume ratio of 1:10, and reacted at room temperature for one hour. Add one volume of 0.02M pH7.2 PBS buffer, then transfer to a centrifugal ultrafiltration tube with a molecular weight cut-off of 30,000 Daltons, centrifuge at 6000rpm for 15 minutes, and then add the same volume of 0.02M pH7.2 PBS buffer as above , centrifuge at 6000rpm for 15 minutes, and repeat this operation three times to obtain a nucleic acid aptamer-streptavidin complex equivalent to the original volume; dissolve the streptavidin in sterile water, and coat the quality control line with streptavidin The concentration of avidin diluent is 2mg/ml. The film spotting machine spots the nucleic acid aptamer 2, streptavidin complex and streptavidin dilution on the detection line (the detection line is located at the centerline of the entire test strip) and the quality Control the position of the line, the two lines are separated by three millimeters, then dry the nitrocellulose membrane at 37°C for 2 hours, seal it well and put it at room temperature for later use;

(5) Paste the treated sample pad, binding pad, nitrocellulose membrane and water-absorbing filter paper on the PVC sheet in sequence, then cut into 4 mm wide test strips and put them into plastic cards, and assemble them into chromatography method detection kit;

(6) Detection: Take the sample to be tested and dilute it with water, then drop 0.12mL into the sample hole. If the quality control line does not show color, the test results are invalid. If the test line shows red but the quality control line does not show color, it is also considered invalid; Detection concentration (1 μM) of lysozyme.

Example 5

Referring to Fig. 1 to Fig. 3, a kind of colloidal gold immunochromatography detection kit based on nucleic acid aptamer, comprises detection test paper, and described detection test paper comprises base plate 1, the sample pad that is bonded on base plate 1 and lapped successively 2, binding pad 3, nitrocellulose membrane 4 and water-absorbing pad 7; the side of the nitrocellulose membrane 4 close to the binding pad 3 is provided with a detection line 5, and the side of the nitrocellulose membrane 4 close to the water-absorbing pad 7 is provided with Quality control line 6; the binding pad 3 is coated with nanoparticle-labeled A nucleic acid aptamer; the detection line 5 is coated with a complex of B nucleic acid aptamer and streptavidin; the quality control Line 6 is coated with streptavidin; the A nucleic acid aptamer and the B nucleic acid aptamer are biotin-labeled nucleic acid aptamers that can specifically recognize the same detection object; wherein, the nanoparticles are four Ferric oxide nanoparticles.

Wherein, the 5' end of the DNA sequence of the A nucleic acid aptamer is labeled with a sulfhydryl group; the 3' end of the DNA sequence of the B nucleic acid aptamer is labeled with biotin; the DNA sequence of the A nucleic acid aptamer is as SEQ ID NO .5, the DNA sequence of the B nucleic acid aptamer is as shown in SEQ ID NO.4; the bottom plate 1 is a PVC plate; the material of the sample pad 2 is a polyacetate film; the material of the binding pad 3 is Glass fiber; the absorbent pad 7 is an absorbent filter paper; the detection test paper is packaged by a rigid plastic card.

Example 6

The method for preparing the kit described in Example 5:

(1) Nucleic acid aptamer 1 labeled iron ferric oxide nanoparticles: Centrifuge the brown iron ferric oxide nanoparticle solution with a carboxyl group on the surface of 10 nm in diameter at 14000 rpm for 10 minutes, suck out the supernatant, and add 10 mM EDC In the MES solution, stir at room temperature for 30 minutes, centrifuge to remove the supernatant, add 0.5OD nucleic acid aptamer 1 per milliliter nanoparticle solution, and add 10mM pH7.4 PBS buffer to dissolve the A nucleic acid that specifically recognizes human thrombin The DNA sequence of the aptamer is: 5'-TGG TTGGTG TGG TTG GTT TTT TTT TT-3'NH ₂ (SEQ ID NO.5), slowly added to ferric oxide nanoparticles under magnetic stirring, and left at room temperature for 2 hours Finally, centrifuge to remove the supernatant, then wash twice with 10mM pH7.4 PBS buffer, and finally redissolve the labeled nucleic acid aptamer 1 in 0.01 M pH7.4 PBS containing 20mM Na3PO4, 1% PEG6000 in the buffer;

(2) Select polyacetate film as the material of the sample pad, soak the polyacetate film in the sample pad treatment solution containing 3% BSA, 0.05% Tween 20, 0.0l M pH7.4PBS buffer solution for 1 hour, and then 37°C Dry for 2 hours and store at 4°C for later use;

(3) Preparation of conjugation pads: for use, redisperse the ferric oxide nanoparticles prepared in step (1) in 0.0l M pH7 containing 5% sucrose, 2% BSA, 2% Dextran, 0.05% Tween 20 .2 in PBS buffer solution, then spray it on the glass fiber bonding pad with a film dispenser, dry at 37°C for 2 hours, and store at 4°C for later use;

(4) Spotting on nitrocellulose membrane: Use a synthetic nucleic acid aptamer that specifically recognizes human thrombin B labeled with biotin at one end, and its DNA sequence is: 5'-AGT CCG TGG TAG GGC AGG TTGGGG TGA CTT TTT TTT TTT-3'Biotin (SEQ ID NO.4), dissolved in sterile water at a concentration of 1 μM, was mixed with streptavidin at a concentration of 1 mg/ml at a volume ratio of 1:10, and reacted at room temperature for one hour. Add one volume of 0.02M pH7.2 PBS buffer, then transfer to a centrifugal ultrafiltration tube with a molecular weight cut-off of 30,000 Daltons, centrifuge at 6000rpm for 15 minutes, and then add the same volume of 0.02M pH7.2 PBS buffer as above , centrifuge at 6000rpm for 15 minutes, and repeat this operation three times to obtain a nucleic acid aptamer-streptavidin complex equivalent to the original volume; dissolve the streptavidin in sterile water, and coat the quality control line with streptavidin The concentration of avidin diluent is 2mg/ml. The film spotting machine spots the nucleic acid aptamer 2, streptavidin complex and streptavidin dilution on the detection line (the detection line is located at the centerline of the entire test strip) and the quality Control the position of the line, the two lines are separated by three millimeters, then dry the nitrocellulose membrane at 37°C for 2 hours, seal it well and put it at room temperature for later use;

(5) Paste the treated sample pad, binding pad, nitrocellulose membrane and water-absorbing filter paper on the PVC sheet in sequence, then cut into 4 mm wide test strips and put them into plastic cards, and assemble them into chromatography method detection kit;

(6) Detection: After absorbing the human serum to be tested and diluting it with sterile water, take 0.12mL and drop it into the sample hole. If the quality control line does not develop color, it is invalid. If the test line is brown but the quality control line does not develop color, it is also considered If it is invalid, if both the quality control line and the test line appear brown, it means that there is human thrombin higher than the minimum detection concentration (100nM) in the test sample.

sequence listing

Claims (10)

1. A nucleic acid aptamer-based chromatography detection kit, comprising a detection test paper; the detection test paper comprises a base plate (1), a sample pad (2) bonded to the base plate (1) and overlapped sequentially, A binding pad (3), a nitrocellulose membrane (4) and a water-absorbing pad (7); the side of the nitrocellulose membrane (4) close to the binding pad (3) is provided with a detection line (5), and the nitrocellulose membrane ( 4) A quality control line (6) is provided on the side close to the water-absorbing pad (7); it is characterized in that the binding pad (3) is coated with a nucleic acid aptamer labeled with nanoparticles; the detection line ( 5) A complex of nucleic acid aptamer B and streptavidin is coated on it; the quality control line (6) is coated with streptavidin; the nucleic acid aptamer A and B nucleic acid aptamer are both A biotin-labeled nucleic acid aptamer capable of specifically recognizing the same detection object; wherein, the nanoparticles are gold nanoparticles, ferric oxide or latex. 2. kit as claimed in claim 1, is characterized in that, the DNA sequence 5' end of described A nucleic acid aptamer is labeled by sulfhydryl group, and 3 ' end is labeled by biotin; The DNA of described B nucleic acid aptamer The 3' end of the sequence was labeled with biotin. 3. kit as claimed in claim 1, is characterized in that, the DNA sequence of described A nucleic acid aptamer is as shown in SEQ ID NO.1, and the DNA sequence of described B nucleic acid aptamer is as shown in SEQ ID NO. 2. 4. kit as claimed in claim 1, is characterized in that, the DNA sequence of described A nucleic acid aptamer is as shown in SEQ ID NO.3, and the DNA sequence of described B nucleic acid aptamer is as shown in SEQ ID NO. 4. 5. kit as claimed in claim 1, is characterized in that, the DNA sequence of described A nucleic acid aptamer is as shown in SEQ ID NO.5, and the DNA sequence of described B nucleic acid aptamer is as shown in SEQ ID NO. 4. 6. The kit according to claim 1, characterized in that, the bottom plate (1) is a PVC board; the material of the sample pad (2) is polyacetic acid film; the material of the binding pad (3) is glass fiber ; The water-absorbing pad (7) is water-absorbing filter paper; the detection test paper is packaged by a hard plastic card. 7. The method for preparing the kit according to any one of claims 1 to 6, comprising the steps of: (1) Label A aptamer with nanoparticles; (2) Soak the sample pad in the sample pad treatment solution containing BSA and Tween PBS for more than 1 hour, and dry it for later use; (3) Redisperse the nucleic acid aptamer A labeled with nanoparticle gold in step (1) into PBS buffer dilution containing sucrose, BSA and Tween, spray it on the binding pad, and dry it for later use; (4) Spray the complex of nucleic acid aptamer B and streptavidin at the position of the detection line on the nitrocellulose membrane, spray some streptavidin at the position of the quality control line, and dry it for later use; (5) Lap the sample pad, binding pad, nitrocellulose membrane and water-absorbent pad sequentially and glue them on the bottom plate, cut into test strips, and pack the test strips with hard plastic cards. 8. The method according to claim 7, characterized in that, the step (1) is to use nanoparticles with a diameter of 10-40nm to label the A nucleic acid aptamer; in the step (4), it is sprayed on the detection line The complex of nucleic acid aptamer B and streptavidin at the position is a solution of aptamer B with a concentration of 0.5-1.5 μM and streptavidin with a concentration of 0.5-1.5 mg/mL in a volume ratio of 1 : 8-12 mixed and prepared; the streptavidin sprayed at the position of the quality control line is a streptavidin dilution with a concentration of 1.5-2.5mg/mL. 9. The method according to claim 8, characterized in that, the complex of the B nucleic acid aptamer and streptavidin sprayed on the position of the detection line in step (4) is the B nucleic acid aptamer A solution with a concentration of 1 μM and streptavidin with a concentration of 1 mg/mL was prepared by mixing it at a volume ratio of 1:10; the streptavidin sprayed at the position of the quality control line was 2 mg/mL Streptavidin diluent. 10. A detection method based on a nucleic acid aptamer, characterized in that, the method adopts two nucleic acid aptamers that recognize the same detection object, and on the basis of a conventional chromatography kit, makes any one of claims 1 to 6. The chromatographic detection kit described in item 1, through the specific combination of A nucleic acid aptamer and B nucleic acid aptamer with the analyte, develops color on the detection line and quality control line to achieve antibody-free chromatographic detection: if If the quality control line does not develop color, the test results are invalid; if the detection line develops color but the quality control line does not develop color, it is also considered invalid; thing.

Images