阅读说明：本技术 一种核酸二合一免疫金标速测卡、制备方法及其检测方法 (Nucleic acid two-in-one immune gold-labeled rapid detection card, preparation method and detection method thereof ) 是由 武爱波 吴蔚 徐伟 杨文杰 徐炜 陶渊超 于 2019-10-22 设计创作，主要内容包括：本发明是一种免疫金标速测卡,特别涉及一种核酸二合一免疫金标速测卡、制备方法及其检测方法。包括外壳,所述的外壳中设有检测卡,还包括检测区和控制区,所述的外壳的上部设有观察窗,所述的检测区和控制区分别位于观察窗中,所述的外壳中设有加样孔。可针对PCR扩增产物进行快速、现场和灵敏的检测。(The invention relates to an immune gold-labeled rapid test card, in particular to a nucleic acid two-in-one immune gold-labeled rapid test card, a preparation method and a detection method thereof. The device comprises an outer shell, the shell in be equipped with the detection card, still include detection zone and control area, the upper portion of shell be equipped with the observation window, detection zone and control area be arranged in the observation window respectively, the shell in be equipped with the application of sample hole. Can be used for carrying out rapid, field and sensitive detection on PCR amplification products.)

1. The utility model provides an immune gold mark short-term test card of two unifications of nucleic acid, includes shell (1), shell (1) in be equipped with detection card (2), its characterized in that: still include detection zone and control area, the upper portion of shell (1) be equipped with observation window (4), detection zone and control area be located observation window (4) respectively, shell (1) in be equipped with application of sample hole (3).

2. The nucleic acid two-in-one immune gold-labeled rapid test card according to claim 1, wherein: the detection card (2) comprises a bottom plate (5), and a sample pad (6), a marking pad (7), a nitrocellulose membrane (8) and an absorption pad (9) are sequentially overlapped and stuck on the bottom plate (5);

the labeling pad (7) is coated with a colloidal gold labeled digoxin antibody;

the nitrocellulose membrane (8) comprises a detection line T1(10) coated with an anti-biotin antibody, a detection line T2(11) coated with an anti-FITC antibody, and a quality control line C (12) coated with a secondary antibody.

3. The nucleic acid two-in-one immune gold-labeled rapid test card according to claim 2, wherein:

the coating amount of the digoxin-resistant antibody on the labeling pad (7) is 5 ng-50 ng; the amount of the anti-biotin antibody on the nitrocellulose membrane (8) is 0.2. mu.g to 1.0. mu.g; the amount of anti-FITC antibody on the nitrocellulose membrane (8) is 0.2. mu.g to 1.0. mu.g.

4. The nucleic acid two-in-one immune gold-labeled rapid test card according to claim 3, wherein:

the coating amount of the anti-digoxin antibody on the label pad is 24ng, the amount of the anti-biotin antibody on the nitrocellulose membrane is 0.3. mu.g, and the amount of the anti-FITC antibody on the nitrocellulose membrane is 0.45. mu.g.

5. The nucleic acid two-in-one immune gold-labeled rapid test card according to claim 2, wherein: the size of the sample pad is 3mm multiplied by 15mm, the size of the marking pad is 3mm multiplied by 3mm, the size of the nitrocellulose membrane is 3mm multiplied by 28mm, and the size of the absorption pad is 3mm multiplied by 19 mm.

6. The method for preparing the nucleic acid two-in-one immune gold-labeled rapid test card according to claim 1, which is characterized by comprising the following steps of:

preparation of nitrocellulose membrane containing detection lines T1, T2 and control line C:

1) diluting the biotin antibody to 1mg/mL by using PBS buffer solution with the concentration of 10mM and the pH value of 7.4 to obtain biotin antibody solution serving as the coating antibody of the detection line T1;

diluting the FITC antibody to 1.5mg/mL by using PBS buffer solution with the concentration of 10mM and the pH value of 7.4 to obtain T antigen solution serving as a coating antibody of a detection line T2;

the goat anti-mouse IgG secondary antibody solution is prepared by dissolving goat anti-mouse IgG secondary antibody in PBS buffer solution with the concentration of 10mM and the pH value of 7.4, wherein the concentration of the goat anti-mouse IgG secondary antibody is 1 mg/ml;

2) coating:

selecting a cellulose nitrate membrane of PALL170, and drawing a T1 line on the biotin antibody solution with the concentration of 1.0mg/mL by using a drawing gold spraying machine at 1.0 muL/cm to serve as a detection line T1;

marking a T2 line by 1.0 mu L/cm of FITC antibody solution with the concentration of 1.5mg/mL as a detection line T2;

marking a C line on the goat anti-mouse IgG secondary antibody solution with the concentration of 1mg/mL by 1.0 mu L/cm to be used as a quality control line C;

drying for 24 hours at 37 ℃ for later use; obtaining a nitrocellulose membrane coated with the antigen;

(II) preparing a gold-labeled pad fixed with the specific monoclonal antibody labeled by the colloidal gold:

1) preparing colloidal gold:

a) preparing: cleaning a 500mL beaker, a 20mL small beaker, a rotor, a brown bottle and a glass rod, and then putting the cleaned glass rod into an acid jar to soak for 24 hours;

taking out, washing with tap water for 3-4 times, then washing with ultrapure water for 3-4 times, and drying in a 37 ℃ drying oven for later use;

b) preparation of a gold-sintering solution A: weighing 1g of chloroauric acid powder in a brown bottle by using a plastic weighing spoon, adding 99ml of ultrapure water for full dissolution, and storing in a dark place at the temperature of 4 ℃;

c) preparation of a gold-sintering solution B: 1g of trisodium citrate is weighed out and dissolved in 99ml of ultrapure water and mixed evenly.

d) Preparing colloidal gold: measuring 99ml of ultrapure water in a beaker, adding 1ml of gold-burning solution A, placing the gold-burning solution A on a constant-temperature magnetic stirrer, uniformly stirring, starting to heat until the solution boils, rapidly adding 2ml of newly prepared gold-burning solution B, continuing to stir and heat, gradually changing the solution into bluish black, then making the solution purple black, heating to obtain red, continuing to boil to obtain transparent orange red, continuing to boil for 10min, naturally cooling to room temperature, and adding ultrapure water to fix the volume to 100 ml; pouring into a brown bottle, and storing in a dark place at the temperature of 4 ℃; obtaining a colloidal gold solution;

2) and antibody labeling:

e) labeling of the antibody: taking 1.5ml of the colloidal gold solution prepared in the step (two) d), adjusting the pH value by using 0.1M K2CO3, respectively adding 20ug of digoxin monoclonal antibody, uniformly mixing, and reacting at room temperature for 40 min; adding 10% BSA, stopping, and standing for 30 min;

f) and (3) labeled antibody purification: centrifuging the standing product at low speed, discarding precipitate formed by the coagulated colloidal gold particles, and collecting supernatant; centrifuging at high speed for 30min, carefully removing supernatant, collecting precipitate, redissolving with 0.1M PBS containing 1% BSA by mass and pH7.4, and storing at 4 deg.C; obtaining digoxin antibody labeled colloidal gold solution with the concentration of 0.4 mg/ml;

3) and spraying gold:

diluting 0.4mg/ml of the digoxin antibody labeled colloidal gold solution prepared in the step (two) to 0.04mg/ml, spraying the diluted solution to the pretreated gold-labeled pad test strip at 2.0 muL/cm, and drying the solution for later use to obtain the gold-labeled pad test strip fixed with the specific monoclonal antibody labeled by the colloidal gold;

(III) assembling the nucleic acid two-in-one immune gold-labeled rapid test card:

the plastic plate on the upper layer is provided with two holes, namely a sample adding hole and an observation hole, wherein the size of the sample adding hole is 3mm multiplied by 7mm, and the size of the observation hole is 3mm multiplied by 18 mm;

from adding the sample hole up in proper order for pasting side by side in plastic slab below: a glass fiber membrane is used as a sample pad, the gold-labeled pad test strip which is prepared in the step (two) and is fixed with the specific monoclonal antibody labeled by colloidal gold, the nitrocellulose membrane which is prepared in the step (one) and is coated with the antigen and the absorption pad; then placing the mixture in an environment with the environment of 37 ℃ for vacuum drying for 30min to obtain the nucleic acid two-in-one immune gold-labeled rapid test card.

7. The method for preparing a nucleic acid two-in-one immune gold-labeled rapid test card according to claim 6, wherein the method comprises the following steps:

the proportion of the acid cylinder solution in the step (a) of the second step is as follows: potassium dichromate: concentrated sulfuric acid: 120g of ultrapure water, 200ml of ultrapure water and 1000ml of ultrapure water;

obtaining a colloidal gold solution in the step (two), wherein the particle size of the colloidal gold is 40 nm; the concentration is one ten thousandth;

in the step (two), firstly, centrifuging the standing product at a low speed (1500r/min), wherein the centrifugation low speed is 1500 r/min; then centrifuging for 30 minutes at a high speed, wherein the high-speed centrifugation is 8500 r/min;

and (3) the gold-labeled antibody test strip in the step (III) has the size of 3mm multiplied by 3mm, the nitrocellulose membrane has the size of 3mm multiplied by 28mm, and the absorption pad has the size of 3mm multiplied by 19 mm.

8. The method for detecting the nucleic acid two-in-one immune gold-labeled rapid detection card according to claim 6, which is characterized by comprising the following steps:

two pairs of nucleic acid primer pairs respectively modify digoxin/biotin digoxin/FITC and digoxin/FITC, and PCR reaction is carried out on the extracted detection sample; horizontally placing the quick test card, and adding a reaction product solution into the sample adding hole; the reaction product solution permeates to the test strip of the colloidal gold labeled antibody along the sample pad, if the sample solution contains nucleic acid, the PCR amplification product is combined with the anti-digoxin antibody on the detection card and is combined with the anti-biotin antibody or FITC antibody on the nitrocellulose membrane, after 8-10min, the color change of the detection area T can be observed in the observation hole, namely, the T has a positive strip, one T line indicates that one is positive, and two T lines indicate that two are positive; if the sample solution does not contain the corresponding nucleic acid, no positive band is observed at the detection zone; regardless of whether the sample solution contains the corresponding nucleic acid, when the sample solution reaches the nitrocellulose membrane, the anti-digoxigenin antibody on the detection card can bind to the secondary antibody coated on the control region, thereby developing color.

Technical Field

The invention relates to an immune gold-labeled rapid test card, in particular to a nucleic acid two-in-one immune gold-labeled rapid test card, a preparation method and a detection method thereof.

Background

The PCR technology can amplify the micro target DNA by more than 100 ten thousand times, thereby realizing the detection of the micro target DNA molecules, having the advantages of high sensitivity, strong specificity, high yield, good repeatability, rapidness, simplicity and the like, being widely applied to the fields of microbiology, archaeology, forensic medicine and sports, being popularized to a plurality of common laboratories, greatly simplifying the traditional molecular cloning technology, and further easily analyzing and identifying the target genes.

The PCR amplification product is usually analyzed by agarose gel electrophoresis, a special electrophoresis apparatus and a gel imaging apparatus are required to be equipped, and the PCR primer is modified, so that the amplification product is directly detected without electrophoresis.

Disclosure of Invention

The invention mainly solves the defects in the prior art and provides a nucleic acid two-in-one immune gold-labeled rapid test card capable of directly detecting two PCR amplification products, a preparation method and a detection method thereof.

The technical problem of the invention is mainly solved by the following technical scheme:

the utility model provides an immune gold mark short-term test card of two unifications of nucleic acid, includes the shell, the shell in be equipped with the detection card, still include detection zone and control area, the upper portion of shell be equipped with the observation window, detection zone and control area be located the observation window respectively, the shell in be equipped with the application of sample hole.

Preferably, the detection card comprises a bottom plate, and a sample pad, a marking pad, a nitrocellulose membrane and an absorption pad are sequentially overlapped and stuck on the bottom plate;

the labeling pad is coated with a colloidal gold labeled digoxin antibody;

the nitrocellulose membrane comprises a detection line T1 coated with an anti-biotin antibody, a detection line T2 coated with an anti-FITC antibody, and a quality control line C coated with a secondary antibody.

Preferably, the coating amount of the anti-digoxin antibody on the labeling pad is 5 ng-50 ng; the amount of the anti-biotin antibody on the nitrocellulose membrane is 0.2. mu.g to 1.0. mu.g; the amount of the anti-FITC antibody on the nitrocellulose membrane is 0.2 to 1.0 mu g;

preferably, the coating amount of the anti-digoxin antibody on the label pad is 24ng, the amount of the anti-biotin antibody on the nitrocellulose membrane is 0.3. mu.g, and the amount of the anti-FITC antibody on the nitrocellulose membrane is 0.45. mu.g.

Preferably, the sample pad is 3mm by 15mm in size, the marker pad is 3mm by 3mm in size, the nitrocellulose membrane is 3mm by 28mm in size, and the absorbent pad is 3mm by 19mm in size.

A preparation method of a nucleic acid two-in-one immune gold-labeled rapid test card comprises the following steps:

preparation of nitrocellulose membrane containing detection lines T1, T2 and control line C:

1) diluting the biotin antibody to 1mg/mL by using PBS buffer solution with the concentration of 10mM and the pH value of 7.4 to obtain biotin antibody solution serving as the coating antibody of the detection line T1;

diluting the FITC antibody to 1.5mg/mL by using PBS buffer solution with the concentration of 10mM and the pH value of 7.4 to obtain T antigen solution serving as a coating antibody of a detection line T2;

the goat anti-mouse IgG secondary antibody solution is prepared by dissolving goat anti-mouse IgG secondary antibody in PBS buffer solution with the concentration of 10mM and the pH value of 7.4, wherein the concentration of the goat anti-mouse IgG secondary antibody is 1 mg/ml;

2) coating:

selecting a cellulose nitrate membrane of PALL170, and drawing a T1 line on the biotin antibody solution with the concentration of 1.0mg/mL by using a drawing gold spraying machine at 1.0 muL/cm to serve as a detection line T1;

marking a T2 line by 1.0 mu L/cm of FITC antibody solution with the concentration of 1.5mg/mL as a detection line T2;

marking a C line on the goat anti-mouse IgG secondary antibody solution with the concentration of 1mg/mL by 1.0 mu L/cm to be used as a quality control line C;

drying for 24 hours at 37 ℃ for later use; obtaining a nitrocellulose membrane coated with the antigen;

(II) preparing a gold-labeled pad fixed with the specific monoclonal antibody labeled by the colloidal gold:

1) preparing colloidal gold:

a) preparing: cleaning a 500mL beaker, a 20mL small beaker, a rotor, a brown bottle and a glass rod, and then putting the cleaned glass rod into an acid jar to soak for 24 hours;

taking out, washing with tap water for 3-4 times, then washing with ultrapure water for 3-4 times, and drying in a 37 ℃ drying oven for later use;

b) preparation of a gold-sintering solution A: weighing 1g of chloroauric acid powder in a brown bottle by using a plastic weighing spoon, adding 99ml of ultrapure water for full dissolution, and storing in a dark place at the temperature of 4 ℃;

c) preparation of a gold-sintering solution B: 1g of trisodium citrate is weighed out and dissolved in 99ml of ultrapure water and mixed evenly.

d) Preparing colloidal gold: measuring 99ml of ultrapure water in a beaker, adding 1ml of gold-burning solution A, placing the gold-burning solution A on a constant-temperature magnetic stirrer, uniformly stirring, starting to heat until the solution boils, rapidly adding 2ml of newly prepared gold-burning solution B, continuing to stir and heat, gradually changing the solution into bluish black, then making the solution purple black, heating to obtain red, continuing to boil to obtain transparent orange red, continuing to boil for 10min, naturally cooling to room temperature, and adding ultrapure water to fix the volume to 100 ml; pouring into a brown bottle, and storing in a dark place at the temperature of 4 ℃; obtaining a colloidal gold solution;

2) and antibody labeling:

e) labeling of the antibody: taking 1.5ml of the colloidal gold solution prepared in the step (two) d), adjusting the pH value by using 0.1M K2CO3, respectively adding 20ug of digoxin monoclonal antibody, uniformly mixing, and reacting at room temperature for 40 min; adding 10% BSA, stopping, and standing for 30 min;

f) and (3) labeled antibody purification: centrifuging the standing product at low speed, discarding precipitate formed by the coagulated colloidal gold particles, and collecting supernatant; centrifuging at high speed for 30min, carefully removing supernatant, collecting precipitate, redissolving with 0.1M PBS containing 1% BSA by mass and pH7.4, and storing at 4 deg.C; obtaining digoxin antibody labeled colloidal gold solution with the concentration of 0.4 mg/ml;

3) and spraying gold:

diluting 0.4mg/ml of the digoxin antibody labeled colloidal gold solution prepared in the step (two) to 0.04mg/ml, spraying the diluted solution to the pretreated gold-labeled pad test strip at 2.0 muL/cm, and drying the solution for later use to obtain the gold-labeled pad test strip fixed with the specific monoclonal antibody labeled by the colloidal gold;

(III) assembling the nucleic acid two-in-one immune gold-labeled rapid test card:

the plastic plate on the upper layer is provided with two holes, namely a sample adding hole and an observation hole, wherein the size of the sample adding hole is 3mm multiplied by 7mm, and the size of the observation hole is 3mm multiplied by 18 mm;

from adding the sample hole up in proper order for pasting side by side in plastic slab below: a glass fiber membrane is used as a sample pad, the gold-labeled pad test strip which is prepared in the step (two) and is fixed with the specific monoclonal antibody labeled by colloidal gold, the nitrocellulose membrane which is prepared in the step (one) and is coated with the antigen and the absorption pad; then placing the mixture in an environment with the environment of 37 ℃ for vacuum drying for 30min to obtain the nucleic acid two-in-one immune gold-labeled rapid test card.

Preferably, the ratio of the acid tank solution in the step (two) a) is as follows: potassium dichromate: concentrated sulfuric acid: 120g of ultrapure water, 200ml of ultrapure water and 1000ml of ultrapure water;

obtaining a colloidal gold solution in the step (two), wherein the particle size of the colloidal gold is 40 nm; the concentration is one ten thousandth;

in the step (two), firstly, centrifuging the standing product at a low speed (1500r/min), wherein the centrifugation low speed is 1500 r/min; then centrifuging for 30 minutes at a high speed, wherein the high-speed centrifugation is 8500 r/min;

and (3) the gold-labeled antibody test strip in the step (III) has the size of 3mm multiplied by 3mm, the nitrocellulose membrane has the size of 3mm multiplied by 28mm, and the absorption pad has the size of 3mm multiplied by 19 mm.

A detection method of a nucleic acid two-in-one immune gold-labeled rapid detection card comprises the following steps:

two pairs of nucleic acid primer pairs respectively modify digoxin/biotin digoxin/FITC and digoxin/FITC, and PCR reaction is carried out on the extracted detection sample; horizontally placing the quick test card, and adding a reaction product solution into the sample adding hole; the reaction product solution permeates to the test strip of the colloidal gold labeled antibody along the sample pad, if the sample solution contains nucleic acid, the PCR amplification product is combined with the anti-digoxin antibody on the detection card and is combined with the anti-biotin antibody or FITC antibody on the nitrocellulose membrane, after 8-10min, the color change of the detection area T can be observed in the observation hole, namely, the T has a positive strip, one T line indicates that one is positive, and two T lines indicate that two are positive; if the sample solution does not contain the corresponding nucleic acid, no positive band is observed at the detection zone; regardless of whether the sample solution contains the corresponding nucleic acid, when the sample solution reaches the nitrocellulose membrane, the anti-digoxigenin antibody on the detection card can bind to the secondary antibody coated on the control region, thereby developing color.

The result judgment rule is as follows:

negative result (-): only line C appears;

positive result (+): one or two T lines and C lines appear at the same time;

invalid result: non-outgoing line C line

Compared with the existing gel electrophoresis ultraviolet imaging detection, the nucleic acid two-in-one immune gold-labeled rapid detection card disclosed by the invention has the following specific advantages.

(1) The operation is simple, special instruments and equipment are not needed, and the PCR amplification result can be directly observed by naked eyes.

(2) The detection time is short and is 8-10 min; the result judgment standard is uniform, namely a negative result (-) only has 1C line; positive (+): one or two T and C lines occur simultaneously.

The invention relates to a nucleic acid two-in-one immune gold-labeled rapid detection card, a preparation method and a detection method thereof, which can carry out rapid, on-site and sensitive detection on PCR amplification products.

Drawings

FIG. 1 is a schematic structural view of the present invention;

fig. 2 is a schematic cross-sectional structure of the present invention.

Detailed Description

The technical scheme of the invention is further specifically described by the following embodiments.