阅读说明：本技术 一种检测副溶血性弧菌td毒素的胶体金塑封检测卡及其制备方法和应用 (Colloidal gold plastic-sealed detection card for detecting vibrio parahaemolyticus TD toxin and preparation method and application thereof ) 是由 冀德君 于 2019-11-29 设计创作，主要内容包括：本发明涉及一种检测副溶血性弧菌TD毒素的胶体金塑封检测卡及其制备方法和应用,所述的检测卡由按顺序依次固定的样品垫、胶体金垫、硝酸纤维素膜以及吸水垫组成,其中胶体金垫内有胶体金标记抗副溶血性弧菌TD毒素抗原的单表位抗体；硝酸纤维素膜上设有检测线和质控线；对上述检测核心进行冷裱塑封,并在加样处留出加样孔。通过本发明的实施,开发出低成本易制作的副溶血性弧菌TD毒素快速免疫检测卡的生产制备技术及其产品,推进本领域的技术进步。(The invention relates to a colloidal gold plastic-sealed detection card for detecting vibrio parahaemolyticus TD toxin and a preparation method and application thereof, wherein the detection card consists of a sample pad, a colloidal gold pad, a nitrocellulose membrane and a water absorption pad which are sequentially fixed, wherein the colloidal gold pad is internally provided with a colloidal gold-labeled single epitope antibody for resisting vibrio parahaemolyticus TD toxin antigen; the nitrocellulose membrane is provided with a detection line and a quality control line; and carrying out cold mounting and plastic packaging on the detection core, and reserving a sample adding hole at a sample adding position. Through the implementation of the invention, the production and preparation technology of the vibrio parahaemolyticus TD toxin rapid immunoassay card and the product thereof which are low in cost and easy to manufacture are developed, and the technical progress in the field is promoted.)

1. A colloidal gold plastic-sealed detection card for detecting vibrio parahaemolyticus TD toxin comprises a test strip, and is characterized in that the test strip comprises a back plate, a sample pad, a colloidal gold pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad, the colloidal gold pad, the nitrocellulose membrane and the water absorption pad are sequentially fixed on the back plate, and the colloidal gold pad contains a colloidal gold-labeled single epitope antibody against vibrio parahaemolyticus TD toxin antigen; the nitrocellulose membrane is provided with a detection line and a quality control line; the detection card is provided with a sample hole, the sample hole is positioned right above the sample pad, and the test strip is also provided with a cover film.

2. The colloidal gold plastic-sealed detection card for detecting the TD toxin of Vibrio parahaemolyticus according to claim 1, wherein the detection line and the quality control line are arranged in parallel; in the colloidal gold pad, the particle size of the colloidal gold is 10 nm; the pore diameter of the nitrocellulose membrane is 6 um; the dosage of the single epitope antibody of the anti-vibrio parahaemolyticus TD toxin antigen is 20ug, and the dosage of the detection line envelope epitope antibody is 25 ug; 1-2mm overlapping areas are arranged among the sample pad, the colloidal gold pad, the nitrocellulose membrane and the water absorption pad; the sample pad and the colloidal gold pad are both made of glass fiber, and the water absorption pad is made of cellulose fiber; the back plate is a PVC plate; the test strip is 60mm multiplied by 3.7mm in size; the sample hole is a rectangular hole, and the cross section area of the sample hole is linearly and sequentially decreased from top to bottom.

3. A preparation method of a colloidal gold plastic-sealed detection card for detecting vibrio parahaemolyticus TD toxin is characterized by comprising the following steps:

cracking and desalting the vibrio parahaemolyticus culture solution to obtain vibrio parahaemolyticus lysate;

preparing purified vibrio parahaemolyticus TD toxin antigen from the prepared vibrio parahaemolyticus lysate by a weak anion exchange chromatography method;

preparing a single epitope antibody of the anti-vibrio parahaemolyticus TD toxin antigen;

preparing a colloidal gold-labeled single epitope antibody of the anti-vibrio parahemolyticus TD toxin antigen;

and assembling the detection card.

4. The method for preparing the colloidal gold plastic-encapsulated detection card for detecting the TD toxin of Vibrio parahaemolyticus according to claim 3, wherein the antigen of the TD toxin of Vibrio parahaemolyticus is prepared from a small peptide synthesized according to the sequence wtrnvkrkppykdygqsgvsvftttsgtkwl.

5. The method for preparing the colloidal gold plastic-sealed detection card for detecting the TD toxin of Vibrio parahaemolyticus according to claim 4, wherein the method for preparing the single epitope antibody of the TD toxin antigen comprises: the muscle injection type non-emulsified water-soluble adjuvant and the TD antigen small peptide obtained by synthesis are uniformly mixed in equal volume, the rabbit is immunized, whole blood is taken, blood serum is taken after blood coagulation, and affinity purification is further carried out by adopting an affinity chromatography column.

6. The method for preparing the colloidal gold plastic-sealed detection card for detecting the TD toxin of Vibrio parahaemolyticus according to claim 3, wherein the step of assembling the detection card comprises: sticking a nitrocellulose membrane to a back plate, respectively taking the coated goat anti-rabbit secondary antibody and anti-vibrio parahaemolyticus TD toxin single epitope antibodies as a C line and a T line on the nitrocellulose membrane by using a membrane scratching instrument, spraying the diluted gold-labeled antibodies on a colloidal gold pad, drying, sticking the gold-labeled antibodies on the back plate, sequentially sticking a sample pad and absorbent paper, cutting the assembled test paper strip by using a slitter, and further preparing the product by adopting a compression molding process.

7. The method for preparing the colloidal gold plastic-sealed detection card for detecting the TD toxin of Vibrio parahaemolyticus according to claim 3, wherein the method for preparing the colloidal gold-labeled antibody comprises:

dialyzing to remove redundant salt ions; adding a potassium carbonate solution to adjust the pH value of the colloidal gold solution, dropwise adding the antibody solution into the colloidal gold solution, uniformly mixing, and standing; adding BSA solution and PEG20000 solution to block residual epitopes on the surface of the gold particles to aggregate the particles, and standing; centrifuging, taking supernatant, transferring into a new centrifuge tube, and removing unconnected gold particles in the labeling process; centrifuging the sucked supernatant, discarding the supernatant, and removing the antibody which is not successfully labeled and the colloidal gold particles to obtain the colloidal gold labeled antibody.

8. The method for preparing the colloidal gold plastic-sealed detection card for detecting the TD toxin of Vibrio parahaemolyticus according to claim 7, wherein the quality identification method for the colloidal gold-labeled antibody comprises: after the goat anti-rabbit secondary antibody is diluted by 100 times by PB and spotted on an NC membrane in a manner of 0.6 mu l/strip, after the coating is completed, the diluted gold-labeled antibody solution is dripped on the position of the secondary antibody, and directly reacts with the secondary antibody, and the result is observed.

9. The method for preparing the colloidal gold plastic-sealed detection card for detecting the TD toxin of Vibrio parahaemolyticus according to claim 7, wherein the concentration of the BSA solution and the concentration of the PEG20000 solution are both 20%.

10. The colloidal gold plastic-sealed detection card for detecting the TD toxin of Vibrio parahaemolyticus of claims 1-2 and the preparation method of the colloidal gold plastic-sealed detection card for detecting the TD toxin of Vibrio parahaemolyticus of claims 3-9 are applied to the detection of the TD toxin of Vibrio parahaemolyticus.

Technical Field

The invention relates to a colloidal gold detection card for detecting vibrio parahaemolyticus TD toxin, and a preparation method and application thereof.

Background

According to WHO estimation, the rate of missing reports of food-borne diseases in developed countries is more than 90%, and the rate of missing reports in developing countries is more than 95%. Based on the inference, the food poisoning data currently mastered by China is only one corner of the iceberg which is the food-borne disease actually occurring in China. Such a high rate of false negative is an important factor in the detection of pathogenic microorganisms and the limitation of the tracing means, in addition to the problem of management. Currently, bacterial food poisoning mainly includes infection type poisoning and toxin type poisoning. The vibrio parahaemolyticus TD toxin is a relatively common food-borne pathogen in the near term, has a remarkable rising trend in occurrence scale and population exposure scale, is high in microbial food poisoning at the first place, probably because the proportion of sea products in food of people is increased, and the sea products generally carry the vibrio parahaemolyticus, and the vibrio parahaemolyticus in the sea products is proliferated and generates toxin due to incomplete refrigeration conditions, so that food poisoning symptoms such as vomiting and diarrhea are caused after eating by mistake.

At present, food safety research teams, equipment and expenses in China are quite lack, and compared with foreign countries, the food safety research teams still have large gaps, which mainly show that the food safety research teams have few detection items and unstable quality and cannot meet the requirements of food detection work. The critical reality places higher demands on microbial detection-more accurate and faster detection and monitoring of pathogens.

The traditional pathogen detection and identification means of food-borne diseases in China still remain in the traditional pathogen culture, the biggest weakness of the detection method is slow, and the detection method is difficult to adapt to diagnosis and treatment, and the method mainly comprises two methods: firstly, the PCR molecular identification technology detects pathogenic bacteria, although the sensitivity is high and the detection is rapid and can be directly carried out, the requirements on laboratories and reagents are higher, otherwise, the result is very unreliable; and secondly, although the enzyme-linked immunosorbent assay (ELISA) detection is a mature immunoassay technology, the method is complex in operation, long in detection time, and required to be carried out in a laboratory, and the requirement of on-site rapid detection cannot be met. Thus, the backward nature of current pathogenic microorganism detection methods has limited the ability to trace and identify pathogenic bacteria causing food-borne diseases to a considerable extent. When food poisoning happens, most people may get ill in a short time due to short poisoning latency and rapid coming, the onset curve tends to rise suddenly, and spreading and situation expansion of food poisoning can be controlled only by grasping the food poisoning happening situation in the shortest time and determining the poisoning reason, so that the health of people is guaranteed. Therefore, the development of a fast and sensitive food safety detection technology platform applied to the field is a trend of food safety detection in the future.

Disclosure of Invention

In order to overcome the defects, the invention provides a colloidal gold detection card for detecting vibrio parahemolyticus TD toxin and a preparation method and application thereof.

In order to achieve the purpose of the invention, the invention adopts the technical scheme that:

a colloidal gold plastic-sealed detection card for detecting vibrio parahaemolyticus TD toxin comprises a test strip, wherein the test strip comprises a back plate, a sample pad, a colloidal gold pad, a nitrocellulose membrane and a water absorption pad, the sample pad, the colloidal gold pad, the nitrocellulose membrane and the water absorption pad are sequentially fixed on the back plate, and the colloidal gold pad contains a colloidal gold-labeled single epitope antibody against vibrio parahaemolyticus TD toxin antigen; the nitrocellulose membrane is provided with a detection line and a quality control line; the detection card is provided with a sample hole, the sample hole is positioned right above the sample pad, and the test strip is also provided with a cover film.

Further, the detection line and the quality control line are arranged in parallel; in the colloidal gold pad, the particle size of the colloidal gold is 10 nm; the pore diameter of the nitrocellulose membrane is 6 um; the dosage of the single epitope antibody of the anti-vibrio parahaemolyticus TD toxin antigen is 20ug, and the dosage of the detection line envelope epitope antibody is 25 ug; 1-2mm overlapping areas are arranged among the sample pad, the colloidal gold pad, the nitrocellulose membrane and the water absorption pad; the sample pad and the colloidal gold pad are both made of glass fiber, and the water absorption pad is made of cellulose fiber; the back plate is a PVC plate; the test strip size is 60mm 3.7 mm.

The invention also provides a preparation method of the colloidal gold plastic-sealed detection card for detecting the vibrio parahaemolyticus TD toxin, which comprises the following steps:

cracking and desalting the vibrio parahaemolyticus culture solution to obtain vibrio parahaemolyticus lysate;

preparing purified vibrio parahaemolyticus TD toxin antigen from the prepared vibrio parahaemolyticus lysate by a weak anion exchange chromatography method;

preparing a single epitope antibody of the anti-vibrio parahaemolyticus TD toxin antigen;

preparing a colloidal gold-labeled single epitope antibody of the anti-vibrio parahemolyticus TD toxin antigen;

and assembling the detection card.

Further, the vibrio parahaemolyticus TD toxin antigen is prepared according to a small peptide synthesized by a sequence wtnnrnvkkpykdyqsvfttsgtkwl.

Further, the preparation method of the single epitope antibody of the TD toxin antigen comprises the following steps: the muscle injection type non-emulsified water-soluble adjuvant and the TD antigen small peptide obtained by synthesis are uniformly mixed in equal volume, the rabbit is immunized, whole blood is taken, blood serum is taken after blood coagulation, and affinity purification is further carried out by adopting an affinity chromatography column.

Further, the step of assembling the test card includes: sticking a nitrocellulose membrane to a back plate, respectively taking the coated goat anti-rabbit secondary antibody and anti-vibrio parahaemolyticus TD toxin single epitope antibodies as a C line and a T line on the nitrocellulose membrane by using a membrane scratching instrument, spraying the diluted gold-labeled antibodies on a colloidal gold pad, drying, sticking the gold-labeled antibodies on the back plate, sequentially sticking a sample pad and absorbent paper, cutting the assembled test paper strip by using a slitter, and further preparing the product by adopting a compression molding process.

Further, the preparation method of the colloidal gold labeled antibody comprises the following steps:

dialyzing to remove redundant salt ions; adding a potassium carbonate solution to adjust the pH value of the colloidal gold solution, dropwise adding the antibody solution into the colloidal gold solution, uniformly mixing, and standing; adding BSA solution and PEG20000 solution to block residual epitopes on the surface of the gold particles to aggregate the particles, and standing; centrifuging, taking supernatant, transferring into a new centrifuge tube, and removing unconnected gold particles in the labeling process; centrifuging the sucked supernatant, discarding the supernatant, and removing the antibody which is not successfully labeled and the colloidal gold particles to obtain the colloidal gold labeled antibody.

Further, the quality identification method of the colloidal gold labeled antibody comprises the following steps: after the goat anti-rabbit secondary antibody is diluted by 100 times by PB and spotted on an NC membrane in a manner of 0.6 mu l/strip, after the coating is completed, the diluted gold-labeled antibody solution is dripped on the position of the secondary antibody, and directly reacts with the secondary antibody, and the result is observed.

Further, the concentration of the BSA solution and the PEG20000 solution are both 20%.

The invention also provides application of the detection card in detection of the vibrio parahaemolyticus TD toxin.

The invention also provides application of the preparation method in vibrio parahaemolyticus TD toxin detection.

Compared with the prior art, the invention has the beneficial effects that:

the immune colloidal gold chromatography technology is a fast and sensitive food safety detection method. The method is a colloidal gold rapid immunoassay technology which is used for qualitatively, semi-quantitatively or quantitatively detecting a substance to be detected in a sample by using a large-aperture microporous filter membrane as a carrier and colloidal gold as a solid-phase marker according to an immunoreaction principle. The principle is as follows: if the sample contains the antigen, the antigen is firstly combined with the antibody marked by the colloidal gold particles under the sample adding pad to form an antigen-antibody-colloidal gold complex, and the antigen-antibody-colloidal gold complex moves to the detection line by virtue of capillary action. And another single epitope antibody is fixed on a detection line of the nitrocellulose membrane of the detection plate. When the sample processing solution is chromatographed to the detection line, an antibody-antigen-antibody colloidal gold sandwich complex may be further formed and accumulate on the detection line to show a visible red line. The absence of the red line indicates that no antigen exists in the sample, and the method is convenient and quick.

Through the implementation of the invention, a production and preparation technology of the plastic-packaged vibrio parahemolyticus TD toxin rapid immunoassay card is developed, and the technical progress in the field is promoted. By popularizing the product, the cognition of people on the persistence and distribution of food-borne pathogens in daily life is improved, and a convenient monitoring means is provided for preventing the occurrence of mass food-borne public health events and promoting the stable development of public health services. After the invention is implemented, a new product series is provided, and the sales is expected to be enlarged by more than 300 ten thousand yuan.

Drawings

FIG. 1 is a schematic view of a test strip of a test card;

FIG. 2 is a schematic diagram of the detection result of the detection card;

FIG. 3 shows a pre-manufactured plastic package test card; and (3) plastic packaging is carried out by adopting a multi-linked test strip, and detection can be started after tearing.

FIG. 4 is a strong recognition epitope (aa 63-78) of the Vibrio parahaemolyticus TD toxin;

Detailed Description

The following description and specific examples are presented to further illustrate the present invention for the purpose of illustrating its technical solutions and objectives.