阅读说明：本技术 检测伪结核棒状杆菌抗体的检测卡及其应用和试剂盒 (Detection card for detecting pseudotuberculosis corynebacterium antibodies and application and kit thereof ) 是由 白邦华 许信刚 尹峥 籍修运 于 2021-08-24 设计创作，主要内容包括：本发明适用于生物技术领域,提供了一种检测伪结核棒状杆菌抗体的检测卡及其应用和试剂盒,该检测卡包括：缓冲液垫；以及硝酸纤维素膜,设置在所述缓冲液垫的一侧；所述硝酸纤维素膜与所述缓冲液垫之间设置有胶体金垫；所述硝酸纤维素膜上远离所述胶体金垫的一端依次设置有用抗原蛋白划的测试线以及用第二抗体划的质控线；其中,所述抗原蛋白为伪结核棒状杆菌重组磷脂酶D蛋白；所述胶体金垫上附着有胶体金联接的兔抗羊IgG抗体。本发明实施例将免疫胶体金技术应用到羊伪结核病的快速诊断中,其可以在养殖现场由非专业实验人员操作,并在十多分钟就能得到检测判断结果,可有效检测出羊(包括山羊、绵羊)血清或全血中的伪结核棒状杆抗体。(The invention is suitable for the biotechnology field, provides a detection card for detecting a corynebacterium pseudotuberculosis antibody, and application and a kit thereof, wherein the detection card comprises: a buffer pad; and a nitrocellulose membrane disposed on one side of the buffer pad; a colloidal gold pad is arranged between the nitrocellulose membrane and the buffer solution pad; a test line marked by using antigen protein and a quality control line marked by using a second antibody are sequentially arranged at one end of the nitrocellulose membrane, which is far away from the colloidal gold pad; wherein the antigen protein is a recombinant phospholipase D protein of corynebacterium pseudotuberculosis; the colloidal gold pad is attached with a rabbit anti-sheep IgG antibody connected with colloidal gold. The embodiment of the invention applies the immune colloidal gold technology to the rapid diagnosis of the pseudotuberculosis of sheep, can be operated by non-professional experimenters in a breeding field, can obtain a detection judgment result in more than ten minutes, and can effectively detect the pseudotuberculosis rodlike rod antibody in the serum or the whole blood of sheep (including goats and sheep).)

1. A detection card for detecting a pseudotuberculosis corynebacterium antibody, characterized by comprising:

a buffer pad; and

the nitrocellulose membrane is arranged on one side of the buffer solution pad; a colloidal gold pad is arranged between the nitrocellulose membrane and the buffer solution pad; a test line marked by using antigen protein and a quality control line marked by using a second antibody are sequentially arranged at one end of the nitrocellulose membrane, which is far away from the colloidal gold pad;

wherein the antigen protein is a recombinant phospholipase D protein of corynebacterium pseudotuberculosis; the colloidal gold pad is attached with a rabbit anti-sheep IgG antibody connected with colloidal gold.

2. The detection card for detecting an antibody against Corynebacterium pseudotuberculosis as set forth in claim 1, wherein a buffer well for adding a buffer is provided on said buffer pad.

3. The detection card for detecting an antibody against corynebacterium pseudotuberculosis according to claim 1, wherein a sample hole for adding a sample to be detected is provided on the nitrocellulose membrane, and the sample hole is located between the test line and the colloidal gold pad.

4. The detection card for detecting an antibody against Corynebacterium pseudotuberculosis as set forth in claim 1, wherein evaporation holes for preventing a reaction solution from flowing back are provided on the nitrocellulose membrane.

5. The detection card for detecting an antibody against Corynebacterium pseudotuberculosis as set forth in claim 1, wherein the colloidal gold pad is a glass cellulose film.

6. The detection card for detecting an antibody against Corynebacterium pseudotuberculosis as set forth in any one of claims 1 to 5, further comprising:

and the water absorption filter paper is arranged on one side of the nitrocellulose membrane, which is far away from the buffer pad.

7. Use of a test card according to any one of claims 1 to 6 for the preparation of a kit for the detection of antibodies against corynebacterium pseudotuberculosis.

8. A kit for detecting an antibody against Corynebacterium pseudotuberculosis, comprising the detection card according to any one of claims 1 to 6.

9. The kit for detecting antibodies against Corynebacterium pseudotuberculosis of claim 8, further comprising a buffer.

10. The kit for detecting the antibody against corynebacterium pseudotuberculosis of claim 9, wherein the buffer solution comprises disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, tween-20, sodium azide.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a detection card for detecting a corynebacterium pseudotuberculosis antibody, and application and a kit thereof.

Background

Pseudotuberculosis of sheep (Caseous Lymphadenitis, CLA) is prepared from Corynebacterium pseudotuberculosis (C. pseudotuberculosis)Corynebacterium pseudotuberculosis，C.p) The resulting chronic zoonosis, which is mainly characterized by lymphadenectasis, mainly infects goats and sheep. The sick sheep gradually becomes leaner, the milk yield is reduced, the wool yield is reduced, the reproductive capacity is weakened, and healthy sheep can be infected by nasal secretion, excrement and the like and cause pollution to soil, water, food and pasture. In recent years, the pseudotuberculosis of sheep frequently appears in sheep farms, and the infection tends to spread gradually, so that a convenient and effective diagnosis means is urgently needed to prevent and control the pseudotuberculosis.

At present, the etiological diagnosis method of the sheep pseudotuberculosis comprises isolation culture and biochemical identification of pathogenic bacteria, a synergistic hemolysis test, PCR (polymerase chain reaction) and an enzyme-linked immunoassay test, the methods all need a special laboratory and are equipped with special experiment technicians, and most breeding places cannot carry out the experiments.

Disclosure of Invention

An object of the embodiments of the present invention is to provide a detection card for detecting an antibody against a pseudotuberculosis corynebacterium, and to solve the problems of the background art.

The embodiment of the invention is realized by the detection card for detecting the pseudotuberculosis corynebacterium antibody, which comprises the following steps:

a buffer pad; and

the nitrocellulose membrane is arranged on one side of the buffer solution pad; a colloidal gold pad is arranged between the nitrocellulose membrane and the buffer solution pad; a test line marked by using antigen protein and a quality control line marked by using a second antibody are sequentially arranged at one end of the nitrocellulose membrane, which is far away from the colloidal gold pad;

wherein the antigen protein is a recombinant phospholipase D protein of corynebacterium pseudotuberculosis; the colloidal gold pad is attached with a rabbit anti-sheep IgG antibody connected with colloidal gold.

As a preferable mode of the embodiment of the present invention, the buffer pad is provided with a buffer well for adding a buffer.

As another preferable scheme of the embodiment of the present invention, a sample hole for adding a sample to be detected is provided on the nitrocellulose membrane, and the sample hole is located between the test line and the colloidal gold pad.

As another preferable scheme of the embodiment of the invention, the nitrocellulose membrane is provided with evaporation holes for preventing the reaction liquid from flowing back.

As another preferable scheme of the embodiment of the present invention, the colloidal gold pad is a glass cellulose membrane.

As another preferable scheme of the embodiment of the present invention, the method further includes:

and the water absorption filter paper is arranged on one side of the nitrocellulose membrane, which is far away from the buffer pad.

Another object of the embodiments of the present invention is to provide an application of the above-mentioned detection card in the preparation of a kit for detecting antibodies against Corynebacterium pseudotuberculosis.

Another object of the embodiments of the present invention is to provide a kit for detecting antibodies against Corynebacterium pseudotuberculosis, which includes the above-mentioned detection card.

As another preferable mode of the embodiment of the present invention, a buffer solution is further included.

As another preferable scheme of the embodiment of the invention, the buffer solution comprises disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, Tween-20 and sodium azide.

The detection card for detecting the pseudotuberculosis corynebacterium parvum antibody provided by the embodiment of the invention applies the immune colloidal gold technology to the rapid diagnosis of the pseudotuberculosis of sheep, can be operated by non-professional experimenters in a breeding field, can obtain a detection judgment result in more than ten minutes, and can effectively detect the pseudotuberculosis corynebacterium parvum antibody in serum or whole blood of sheep (including goats and sheep).

Specifically, the embodiment of the invention uses the recombinant phospholipase D (PLD) protein of the corynebacterium pseudotuberculosis as a diagnostic antigen protein, and the antigen protein is fixed on a microporous Nitrocellulose (NC) membrane in a physical adsorption mode. The PLD protein is the main virulence factor of the corynebacterium pseudotuberculosis, contains the major, subgenus and minor species-specific antigenic determinants of the corynebacterium pseudotuberculosis, and can be used as a diagnostic antigen. In addition, in the embodiment of the invention, the rabbit anti-sheep IgG antibody is connected with the colloidal gold microspheres to form immune colloidal gold, and the connector is dried and attached to the colloidal gold pad.

In addition, the embodiment of the invention designs the separated sample hole and the buffer hole, so that the antibody in the sample can preferentially react with the antigen on the nitrocellulose membrane, and the detection sensitivity is improved; in addition, the embodiment of the invention can prevent the reaction liquid from flowing back by arranging the evaporation holes, so that the reaction is more sufficient.

Drawings

FIG. 1 is a schematic structural diagram of a detection card for detecting an antibody against Corynebacterium pseudotuberculosis according to an embodiment of the present invention. In FIG. 1, 1-base plate, 2-buffer pad, 3-colloidal gold pad, 4-nitrocellulose membrane, 5-absorbent filter paper, 6-sample well, 7-test line, 8-quality control line, a-chromatography direction.

FIG. 2 is a schematic diagram of a detection card for detecting an antibody against Corynebacterium pseudotuberculosis according to an embodiment of the present invention; in FIG. 2, B-buffer well, S-sample well, T-test line, C-control line.

FIG. 3 is a diagram showing the judgment of the detection result of a detection card for detecting an antibody against Corynebacterium pseudotuberculosis provided in the embodiment of the present invention; in FIG. 3, T-test line, C-control line.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

As shown in FIG. 1, in one embodiment of the present invention, there is provided a test card for detecting antibodies against Corynebacterium pseudotuberculosis, including:

a buffer pad 2; and

a nitrocellulose membrane 4 disposed on one side of the buffer pad 2; a colloidal gold pad 3 is arranged between the nitrocellulose membrane 4 and the buffer solution pad 2; a test line 7 marked by antigen protein and a quality control line 8 marked by a second antibody are sequentially arranged at one end of the nitrocellulose membrane 4, which is far away from the colloidal gold pad 3;

wherein the antigen protein is a recombinant phospholipase D protein of corynebacterium pseudotuberculosis; the colloidal gold pad 3 is attached with a rabbit anti-sheep IgG antibody connected with colloidal gold.

Specifically, the second antibody has dual properties, and can be used as an antibody to be combined with a corresponding antigen, and also can be used as an antigen to prepare the corresponding antibody; in the present embodiment, the second antibody may be a pseudotuberculosis corynebacterium IgG antibody; the nitrocellulose membrane 4 can be a microporous nitrocellulose membrane, and the recombinant phospholipase D protein of the corynebacterium pseudotuberculosis can be fixed on the microporous nitrocellulose membrane in a physical adsorption mode; the recombinant phospholipase D protein of the corynebacterium pseudotuberculosis is a main virulence factor of the corynebacterium pseudotuberculosis, contains species-specific antigenic determinants of main genera, subgenera and minor species of the corynebacterium pseudotuberculosis, and can be used as a diagnostic antigen.

In practical application, the nucleotide sequence of the recombinant PLD protein of the corynebacterium pseudotuberculosis is as follows: ATGAGGGAGAAATTTGCTTTATTATTATCAATAATTATGGCAATCATGCTTCCGGTAGGGAATGCAGCTGCAGCGCCTGTTGTACATAACCCAGCTTCTACAGCAAATCGGCCAGTCTATGCGATTGCCCACCGCGTTTTAACCACTCAAGGCGTGGATGACGCAGTTGCGATCGGTGCGAATGCGTTGGAAATTGACTTCACTGCGTGGGGTCGTGGCTGGTGGGCAGATCATGATGGTATTCCTACTAGTGCAGGTGCTACTGCAGAGGAAATTTTTAAGCATATAGCTGATAAGCGTAAGCAGGGGGCAAATATTACTTTCACCTGGCTTGACATCAAGAATCCAGACTACTGCAGGGATGCTCGTAGTGTGTGCTCCATAAATGCGTTGCGTGATTTGGCACGTAAATATCTTGAGCCAGCAGGGGTTCGAGTTCTCTATGGGTTCTATAAGACAGTCGGTGGACCTGCCTGGAAGACAATCACCGCTGATCTTCGGGATGGCGAGGCAGTAGCTCTTAGCGGCCCGGCGCAGGACGTATTAAATGATTTTGCAAGGTCTGGAAATAAGATCCTTACTAAACAAAAAATCGCTGACTATGGTTACTACGACATTAACCAAGGGTTTGGTAACTGCTATGGAACCTGGAATAGGACTTGTGATCAACTCCGTAAGTCCAGCGAAGCTCGTGACCAAGGAAAACTCGGTAAAACTTTTGGGTGGACAATCGCTACAGGTCAGGACGCGCGAGTTAATGATCTTTTAGGAAAAGCCAACGTAGATGGGCTGATCTTTGGCTTTAAGATGACTCACTTCTACCGTCATCCAGACACCGAAAATTCTTTCAAAGCCATCAAGAGGTGGGTGGATAAGCACTCCGCTACTCACCATCTGGCTACCGTAGCGGATAACCCGTGGTGA, specifically shown in SEQ ID NO of sequence Listing 1; the recombinant PLD protein of the pseudotuberculous corynebacterium can be prepared by the following preparation method: designing a synthetic primer (an upstream primer: 5'-CGCGGATCCATGAGGGAGAAAGTTGTTTTATTCT-3', shown as a sequence table SEQ ID NO: 2; a downstream primer: 5'-CCGCTCGAGCCACGGGTTATCCGCTACGGTAGCC-3', shown as a sequence table SEQ ID NO: 3), taking a genome of a Shanxi isolated strain of the corynebacterium pseudotuberculosis as a template, carrying out PCR amplification to obtain an amplification product of the PLD protein, connecting the amplification product with an expression vector thereof, carrying out double enzyme digestion simultaneously, and then connecting to construct a recombinant plasmid; and transforming the selected positive recombinant plasmid into escherichia coli BL21(DE3) competent cells to obtain a positive recombinant plasmid monoclonal bacterium, carrying out protein expression on the positive recombinant plasmid monoclonal bacterium under IPTG induction to obtain an induction product, and sequentially carrying out cracking precipitation, purification and six-stage gradient renaturation on the induction product to obtain the recombinant PLD protein.

It should be noted that both the buffer pad 2 and the nitrocellulose membrane 4 can be disposed on the bottom plate 1, and the material of the bottom plate 1 may specifically be polymer material such as polyvinyl chloride, but is not limited thereto.

In the embodiment of the invention, the relevant principles of molecular biology, immunobiology and physical separation technology are combined, and the immune colloidal gold technology is applied to the rapid diagnosis of the pseudotuberculosis of sheep, so that the method can be operated by non-professional experimenters in a breeding field, can obtain a detection judgment result in more than ten minutes, and can effectively detect the pseudotuberculosis rodlike rod antibody in the serum or whole blood of sheep (including goats and sheep).

As shown in fig. 1 and 2, in a preferred embodiment of the present invention, the buffer pad 2 is provided with a buffer well for adding a buffer; a sample hole 6 for adding a sample to be detected is formed in the nitrocellulose membrane 4, and the sample hole 6 is located between the test line 7 and the colloidal gold pad 3; in addition, the nitrocellulose membrane 4 is also provided with evaporation holes for preventing the reaction solution from flowing back.

In the embodiment of the invention, the separated sample hole 6 and the buffer hole are designed, so that the antibody in the sample can preferentially react with the antigen on the nitrocellulose membrane 4, and the detection sensitivity can be improved; in addition, the embodiment of the invention can prevent the reaction liquid from flowing back by arranging the evaporation holes, thereby ensuring that the reaction is more sufficient.

In a preferred embodiment of the present invention, the colloidal gold pad 3 is a glass cellulose membrane, but is not limited thereto, and other cellulose membranes or organic polymer membranes may be used.

In practical application, the rabbit anti-sheep IgG antibody and the colloidal gold microspheres can be linked to form immune colloidal gold, and then the linked substance is dried and attached to the colloidal gold pad 3.

In a preferred embodiment of the present invention, as shown in fig. 1, the test card further comprises:

and the water absorption filter paper 5 is arranged on one side of the nitrocellulose 4 membrane, which is far away from the buffer pad 2.

Through the setting of the water absorption filter paper 5, the reaction liquid is convenient to drain towards the directions of the test line 7 and the quality control line 8.

In another embodiment of the invention, the application of the detection card in preparing a kit for detecting the antibody of the pseudotuberculosis corynebacterium and the kit for detecting the antibody of the pseudotuberculosis are also provided.

Specifically, the kit comprises a detection card, and also can comprise a drying agent, a quantitative sampling tube, a buffer solution, a product instruction and the like. Wherein the buffer solution may comprise disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, tween-20, and sodium azide. In practical applications, the concentration of each component in the buffer solution can be adjusted according to practical situations, for example, the concentration of each component can be adjusted within the range of 0.05-0.5 g/L.

The sample size required by the kit in the detection can be controlled to be about 10 mu L, and the time required by the detection is about 15 minutes; the kit is stored at normal temperature, and the effective period is about 2 years.

In addition, as shown in fig. 1, when the kit provided by the embodiment of the invention is used, an immune colloidal gold lateral flow experiment principle is adopted, a clamping groove is arranged on a base, the detection card is loaded, and an observation window is correspondingly arranged on the detection card; a test line 7 is marked on the nitrocellulose membrane 4 by using the corynebacterium pseudotuberculosis recombinant PLD protein, a quality control line 8 is marked by using a second antibody, and a rabbit anti-sheep IgG antibody connected by colloidal gold is attached to the colloidal gold pad 3. During detection, 10 mu L of a sample to be detected (serum or whole blood sample liquid of a goat or sheep) is sucked by a quantitative sampling tube and dripped into a sample hole 6, the sample is kept still for 5 minutes, and the sample to be detected moves to the other end of a detection card along a nitrocellulose membrane 4 and is combined with a test line 7 fixed with the recombinant PLD protein of the corynebacterium pseudotuberculosis; then 4-5 drops of buffer solution are dripped into the buffer solution hole on the buffer solution pad 2, and the buffer solution is placed at room temperature for 10 minutes, so that the buffer solution moves to the other end of the detection card along the nitrocellulose membrane 4, and the immune colloidal gold on the colloidal gold pad 3 is dissolved and driven to flow forwards (in the direction of a in the figure 1); the immune colloidal gold continuously moves forward to react with the test line 7 and the quality control line 8 one by one for color development, and then whether the sample to be detected contains the pseudo-corynebacterium tuberculosis antibody or not can be detected. Specifically, the method for determining the detection result by the detection card is shown as a, b, c and d in fig. 3: if the quality control line 8 is colored and the test line 7 is not colored, the to-be-detected sample does not contain the pseudo-corynebacterium tuberculosis antibody and is negative; if the quality control line 8 is colored and the test line 7 is colored, the fact that the sample to be detected contains the corynebacterium pseudotuberculosis antibody indicates that the sample to be detected is positive; if the quality control line 8 does not develop color and the test line 7 does not develop color, the detection operation process is incorrect or the detection card fails, and the detection needs to be carried out again.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Shenzhen Shende Biotechnology Limited

Northwest A & F University

<120> detection card for detecting pseudo-corynebacterium tuberculosis antibody, application and kit thereof

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<170> SIPOSequenceListing 1.0

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aatgcagctg cagcgcctgt tgtacataac ccagcttcta cagcaaatcg gccagtctat 120

gcgattgccc accgcgtttt aaccactcaa ggcgtggatg acgcagttgc gatcggtgcg 180

aatgcgttgg aaattgactt cactgcgtgg ggtcgtggct ggtgggcaga tcatgatggt 240

attcctacta gtgcaggtgc tactgcagag gaaattttta agcatatagc tgataagcgt 300

aagcaggggg caaatattac tttcacctgg cttgacatca agaatccaga ctactgcagg 360

gatgctcgta gtgtgtgctc cataaatgcg ttgcgtgatt tggcacgtaa atatcttgag 420

ccagcagggg ttcgagttct ctatgggttc tataagacag tcggtggacc tgcctggaag 480

acaatcaccg ctgatcttcg ggatggcgag gcagtagctc ttagcggccc ggcgcaggac 540

gtattaaatg attttgcaag gtctggaaat aagatcctta ctaaacaaaa aatcgctgac 600

tatggttact acgacattaa ccaagggttt ggtaactgct atggaacctg gaataggact 660

tgtgatcaac tccgtaagtc cagcgaagct cgtgaccaag gaaaactcgg taaaactttt 720

gggtggacaa tcgctacagg tcaggacgcg cgagttaatg atcttttagg aaaagccaac 780

gtagatgggc tgatctttgg ctttaagatg actcacttct accgtcatcc agacaccgaa 840

aattctttca aagccatcaa gaggtgggtg gataagcact ccgctactca ccatctggct 900

accgtagcgg ataacccgtg gtga 924

<210> 2

<211> 34

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