CN101294958A - Bovine Tuberculosis Dot-ELISA Rapid Detection Kit - Google Patents

Abstract

The invention provides a rapid and sensitive diagnosis method for bovine tuberculosis. In this method, Escherichia coli is used to co-express the early secretory proteins EAST6 and CFP10 of Mycobacterium bovis in vitro through gene recombination technology, and the expressed products are purified and quantified and then adsorbed on nitrocellulose membrane as the detection antigen, thus establishing a Bovine tuberculosis Dot-ELISA diagnostic kit with good sensitivity and specificity.

Description

Bovine Tuberculosis Dot-ELISA Rapid Detection Kit

technical field

The invention relates to a biological product for diagnosing bovine tuberculosis, which belongs to the field of veterinary biological products.

technical background

Consumptive infectious diseases of cattle. This disease can take place throughout the year, is the world epidemic, and in the severe epidemic area, the infection rate can reach 60%. More than ten countries in the world, including the United States, Canada, Australia, etc., have reported tuberculosis-free cattle, but in recent years, bovine tuberculosis is mainly caused by Mycobacterium bovis, a chronic disease caused by wild The occurrence and prevalence of animal and human tuberculosis have put cattle in countries with good tuberculosis control under constant quarantine and high vigilance. Bovine tuberculosis not only leads to the decline of the productivity of the cattle industry itself, but more importantly, it poses a serious threat to human health. Humans are more sensitive to bovine tuberculosis, and many cases have a history of contact with infected animals. Because the relationship between cattle and humans (milk, beef and its products, etc.) is closer than other animals, about 10% of human tuberculosis is caused by Mycobacterium bovis; studies have shown that in areas with tuberculosis cows, human Tuberculosis infection rates can be as high as 75%.

In order to fundamentally control the occurrence and prevalence of bovine tuberculosis, it is first necessary to strengthen the research on the diagnosis of the disease. In fact, domestic and foreign scholars have never stopped improving the diagnostic methods of bovine tuberculosis. At present, there are mainly three methods for bovine tuberculosis detection: tuberculin intradermal test (tuberculin test, TST), bovine serum ELISA detection method and bovine IFN-γELISA detection method. TST is a classic diagnostic method for bovine tuberculosis, and it is widely used in the world, but this method has exposed many shortcomings, mainly in: ① high proportion of false positives and false negatives; ② inability to distinguish immunized animals from infected animals; It takes more than 30 days to repeat the test to detect suspicious samples; ④Two tests must be carried out on the living body, which is cumbersome to operate and is not conducive to large-scale epidemiological investigations. In view of the defects of TST, there is a great need for a highly specific and sensitive serological diagnostic method to make up for its shortcomings. The bovine serum ELISA detection method is favored because of its rapidity, simplicity and low cost. Initially, people used PPD as the diagnostic antigen for establishing ELISA method, but there was also strong non-specificity; with the deepening of the molecular biology research of M. It was expressed and used as a diagnostic antigen, and an indirect ELISA diagnostic method was established. Compared with PPD-ELISA, the specificity of this method was improved, but the sensitivity was reduced. The current commercial bovine tuberculin IFN-γ ELISA kit is only the monoclonal antibody sandwich ELISA kit produced by Bovigam, Australia. This kit is not only expensive, but also not suitable for my country's national conditions. Due to the high degree of homology between bovine tuberculosis and avian tuberculosis (paratuberculosis), stimulation of peripheral blood cells with bovine PPD (PPDB) can also produce higher levels of IFN-γ. In order to avoid this problem, Bovigam’s kit cleverly uses poultry PPD (PPDA) as a control, and when the result is judged, use PPDB and PPDA to stimulate the difference in the ELISA reading of the test sample (OD _PPDB -OD _PPDA > 0.1) as a basis for judgment. This method is more suitable for countries without poultry tuberculosis (paratuberculosis) or poultry tuberculosis (paratuberculosis) infection is relatively mild, but because poultry tuberculosis (paratuberculosis) infection is also quite serious in my country, it is even worse than bovine tuberculosis to some extent In severe cases, use of this kit tends to result in a high rate of false negative results.

Although there have been reports on the expression of CFP10 and EAST6 in vitro, no one has yet used the expression products as a coating antigen to adsorb onto NC membranes and detect bovine tuberculosis by Dot-ELISA.

The object of the present invention is to provide a fast, highly specific and highly sensitive bovine tuberculosis detection kit aiming at the deficiencies of the above several detection methods.

Contents of the invention

The technology involved in the present invention includes: extracting Mycobacterium bovis DNA as a template for PCR reaction to amplify esxB and esxA gene fragments, using gene splicing technology to finally amplify the target gene (esxB-esxB gene), and cloning it into the plasmid , and then transformed into Escherichia coli, the obtained recombinant bacteria containing positive expression plasmids were induced to express according to conventional methods, the expressed proteins were purified and quantified, and then coated with nitrocellulose membrane (NC membrane), and analyzed by Dot-ELISA method to detect.

1 Construction of co-expression plasmids of Mycobacterium bovis EAST6 and CFP10

According to conventional methods, the DNA of Mycobacterium bovis (CVCC68001) strain was extracted as the template of PCR reaction to amplify the esxB and esxA gene fragments, and the target gene (esxB-esxB gene) was finally amplified by gene splicing technology, and cloned into pET- 32a plasmid, and then transformed into Escherichia coli BL21 (DE3), the results of double enzyme digestion, PCR identification and sequencing all proved that the obtained plasmid was positive, and the recombinant bacteria was named Escherichia coli (Escherichia coli) pET-CFP10 -ESAT6 strain bacteria (on May 7, 2008, the Japanese strain has been sent to the General Microbiology Center of the China Microbiological Culture Collection Management Committee for preservation, and the number is: CGMCC No.2843).

2 Purification and quantification of CFP10-EAST6 protein

Centrifuge the expression-induced bacterial cells at 12000r/min for 10min, resuspend the precipitate in PBS at a concentration ratio of 10 times, lyse the bacterial cells by ultrasonic waves, take the supernatant, and pass through a NI-NTA column (Beijing Weishi Bohui Chromatography Technology Co., Ltd. ) Purify the recombinant protein by using the imidazole concentration gradient elution method to purify the expressed protein, and determine the concentration of the recombinant protein according to the Bradford method.

Determination of 3Dot-ELISA reaction program

Soak the NC membrane in the purified recombinant protein diluted in a certain proportion for 10 minutes to become the detection antigen. After diluting the serum to be tested 10 times, spot it on the membrane with 1μl sample volume, act at 37°C for 0.5h, and wash 3 times with TBST. Add secondary antibody and incubate for 0.5h, wash 3 times with TBST, and develop color with chromogenic solution.

Specific Embodiments of the Invention

The protein involved in the invention is a secreted protein in the early stage of bovine tuberculosis infection, and is the main dominant antigen mediating cellular immunity. The technology involved in the present invention includes: extracting Mycobacterium bovis (CVCC68001) bacterial strain DNA to amplify esxB and esxA gene fragments as the template of PCR reaction, finally amplifying the target gene (esxB-esxB gene) with gene splicing technology, and combining them Cloned into the pET-32a plasmid, and transformed into Escherichia coli BL21 (DE3), the recombinant bacteria Escherichia coli pET-CFP10-ESAT6 ( CGMCC No.2843), and then the recombinant strain CGMCC No.2843 was induced to express according to conventional methods ("Molecular Cloning Experiment Guide", the second edition, J. Sambrook et al., Jin Dongyan et al. translation, 1996, P822-847), The obtained expressed protein was purified and quantified, used to coat NC membrane, and detected by Dot-ELISA method.

1 Amplification of the target gene

The present invention adopts the technique of gene splicing by over lap extension, that is, the overlapping PCR method realizes the splicing of the target gene through two PCRs. A total of 4 primers were designed, P1 is the upstream primer of esxB gene (encoding CFP10 protein); the first half of P2 (underlined part) is the linker sequence (Linker, encoding 15 hydrophobic amino acids), the second half is the downstream primer of esxB gene; the first half of P3 The segment (underlined part) is the base sequence (Linker) complementary to the underlined part of primer P2, and the second half is the upstream primer of the esxA gene (encoding ESAT-6 protein); P4 is the downstream primer of the esxA gene.

First, the esxB gene was amplified with primers P1 and P2, and the esxA gene was amplified with primers P3 and P4. Then, the two gene products were mixed in equal volumes as templates, and the esxB-esxA spliced gene was amplified with primers P1 and P4.

The specific operation method is as follows:

1.1 Primer design

Design primers according to Mycobacterium bovis AF2122/97 (accession number NC002945) in GenBank:

P1: 5'- GCGAATTC ATGGCAGAGATGAAG-3'

P2:

5'- GCTTCCACCTCCTCCGCTTCCACCACCTCCGCTTCCACCGCCACC GAAGCCCATTTGCG-3'

P3: 5'- GGTGGCGGTGGAAGCGGAGGTGGTGGAAGCGGAGGAGGTGGAAGC ATGACAG

AGCAGCAG-3'

P4: 5'-CC CTCGAG TTACTATGCGAACATCCCAGTG-3'

The underlined parts of P1 and P4 are EcoRI and HindIII restriction sites respectively, and the underlined parts of P2 and P3 are complementary 45-base linkers.

1.2 Amplification of the target fragment

The PCR amplification conditions were: 94°C/10min, 94°C/30s, 55°C/30s, 72°C/2min (30 cycles), 72°C/10min. First, P1, P2, P3, and P4 were used as primers respectively, and the DNA of Mycobacterium bovis (CVCC68001) strain was extracted as a template for PCR reaction to amplify the esxB and esxA gene fragments, and the PCR products were recovered. The product was used as a template, and the esxB-esxA fusion gene was amplified by using P1 and P4 as primers. PCR product purification kit to recover the target fragment.

2 Construction of recombinant expression plasmid pET-32a(+)-cfp10-esat6

The recovered esxB-esxA PCR product and pET-32a(+) were double-digested with EcoRI and HindIII respectively, and the two digested products were recovered and ligated at a molar ratio of 100:1, and transformed into Escherichia coli BL21(DE3 ), extract the plasmid, preliminarily screen the positive clones by double enzyme digestion, and verify the clones by sequence determination.

3 Induced expression of recombinant protein and SDS-PAGE analysis

The recombinant bacteria containing the positive plasmid—Escherichia coli pET-CFP10-ESAT6 strain (the Japanese strain on May 7, 2008) has been sent to the General Microbiology Center of China Microbiological Culture Collection Management Committee for preservation, and the number is: CGMCC No. 2843), cultured in LB liquid medium with shaking at 37°C overnight, inoculated LB medium at a ratio of 1:100, cultured with shaking at 37°C for 3 hours, added IPTG to a final concentration of 1 mmol/L, and continued to cultivate at 30°C for 6 hours. Collect 1mL of uninduced and 6h-induced bacterial liquid, centrifuge to get the precipitate, resuspend with 200μl of PBS with pH 7.4, add 50μL of 5×loading Buffer, mix well and boil for 5min, take 10μl of supernatant and standard molecular weight protein Maker SDS-PAGE electrophoresis followed by Coomassie brilliant blue staining.

4 Purification of recombinant protein

Centrifuge the induced bacteria at 5000r/min at 4°C for 15 minutes, discard the supernatant, resuspend the precipitate in PBS with a pH of 7.4 at a concentration ratio of 10 times, lyse the bacteria with ultrasonic (power 200W, work for 10s, intermittent for 10s) for 20 minutes, and again for 4 Centrifuge at 5000r/min for 20min, collect the supernatant, filter through a 0.45μm filter membrane, pass through a NI-NTA column (Beijing Weishi Bohui Chromatography Technology Co., Ltd., the specific operation method is carried out according to the product manual), and wash through the concentration gradient of imidazole. The recombinant protein was depurified, and the concentration of the recombinant protein was determined according to the Bradford method.

Establishment of 5Dot-ELISA diagnostic method

Dilute the purified expressed protein (coated antigen) to a certain ratio. Soak the sheared NC membrane in protein diluent, let it react at room temperature for 10 minutes, take it out and dry it at 37°C for 5 minutes, wash it with TBST with pH 7.4 three times, each time for 5 minutes (the same below). Block with 5% skimmed milk for 30 minutes, then three times with TBST, and dry in an incubator for 5 minutes (antigen detection). After the serum to be tested was diluted 1:10, 1 μl was taken and spotted on the membrane, incubated at 37°C for 30 minutes, and washed 3 times with TBST. The membrane was incubated in 1:1000 diluted secondary antibody (goat anti-bovine IgG-HRP) at 37°C for 30 min, washed 3 times with TBST, and then added with chromogenic solution to develop color.

After the color development stops, if there are spots visible to the naked eye, it is judged as positive; otherwise, it is negative.

6 Preparation and use of the kit

6.1 Preparation of positive serum and negative serum

6.1.1 Preparation of positive serum Collect bovine PPD intradermal test (TST) and bovine tuberculin IFN-γELISA to detect positive bovine serum. After the serum is diluted 1:5, use Dot among the present invention. -ELISA detection test titer, the lowest dilution of 20 times the concentration as a positive serum sample, and add 1/10000 sodium azide as a preservative.

6.1.2 Preparation of negative serum The bovine serum that was negative in the bovine PPD intradermal test (TST) and bovine tuberculin IFN-γELISA was collected as a negative serum sample, and 1/10000 sodium azide was added as a preservative.

6.1.3 HRP-anti-bovine-IgG antibody was purchased from Santa Cruz Company.

6.2 Solution preparation

6.2.110×washing liquid: Ttis base (Sigma) 60.6g, NaCl 87.8g, Tween-2010mL, add deionized water to 1L.

6.2.2 Substrate solution A: Na ₂ HPO ₄ ·12H ₂ O 3.68g, citric acid 0.933g, deionized water 200mL, mix well and store at 4°C for later use.

6.2.3 Substrate solution B: Dissolve 0.08g of o-phenylenediamine in 10mL of substrate solution A, aliquot 0.5mL/cartridge, and store at -20°C in the dark.

6.2.4 Chromogenic solution: take 0.5mL of substrate solution B, thaw quickly in the dark, add 9.5mL of substrate solution A, add 16μL of 30% H ₂ O ₂ when used, shake well, and use immediately.

6.3 Kit composition and packaging

The Dot-ELISA detection kit involved in the present invention consists of coated antigen, nitrocellulose membrane (NC membrane), positive control serum, negative control serum, 10 × washing liquid, HRP-anti-bovine-IgG antibody (Santa Cruz company ), substrate solution A, substrate solution B, and chromogenic solution.

This kit is used for bovine tuberculosis Dot-ELISA test for quarantine and diagnosis of bovine tuberculosis.

6.4 Preparation before use

6.4.1 Before using this kit, please carefully open the coated antigen, fully dissolve it with 1mL deionized water, and avoid blowing it hard.

6.4.2 Dilute the 10× washing solution with deionized water to the working concentration (1× washing solution).

6.5 Detection steps

6.5.1 Take an appropriate amount of dissolved coated antigen, dilute it at 1:200, put it into a suitable container, soak the NC membrane in it for 10 minutes, take it out, and dry it at 37°C (or room temperature).

6.5.2 After diluting the negative and positive sera and the sera to be tested 1:10 with 1× washing solution, take 1 μl and spot on the NC membrane. Place at room temperature for 5 minutes.

6.5.3 Wash 3 times with 15mL 1×washing solution, 5min each time.

6.5.4 Soak the NC membrane in 1:1000 diluted goat anti-bovine IgG-HRP antibody, incubate at room temperature for 30 minutes, and shake several times in the middle.

6.5.5 Wash 3 times with 15mL 1× washing solution, 5min each time.

6.5.6 Mix 9.5mL of substrate solution A and 0.5mL of substrate solution B, add 16.5μl of H ₂ O ₂ and mix well, then put the NC membrane into it immediately, and develop color at room temperature for 1-2min in the dark.

6.5.7 After the color development is completed, rinse with deionized H ₂ O to stop the color development.

6.6 Judgment of test results

After the color development stops, if there are spots visible to the naked eye, it is judged as positive; otherwise, it is negative.

6.7 Precautions

6.7.1 Do not directly touch the NC membrane with your hands during the test. It is recommended to wear gloves to operate and take the membrane with tweezers;

6.7.2 It is recommended to dry the membrane on a clean filter paper;

6.7.3 When developing color, you can refer to negative and positive controls to determine the stop time.

Description of drawings

Accompanying drawing 1 is the SDS-PAGE result of the induced expression product and the purified product of the recombinant bacteria.

1. Medium protein marker;

2. Escherichia coli BL21 (DE3) lysate supernatant containing pET-32a plasmid, without IPTG induction;

3. Escherichia coli BL21 (DE3) lysate supernatant containing pET-32a plasmid, induced by IPTG;

4. Escherichia coli BL21 (DE3) cell lysate supernatant containing pET-CFP10-ESAT6 recombinant plasmid, not induced by IPTG;

5. Blank PBS control;

6. Escherichia coli BL21 (DE3) cell lysate supernatant containing pET-CFP10-ESAT6 recombinant plasmid, induced by IPTG;

7. The recombinant expression product purified by Ni-NTA column.

Accompanying drawing 2 Dot-ELISA detection result to some samples.

Fig. 2a 1 is the bovine tuberculosis positive serum control; 2 to 24 are the test results of TST tuberculosis positive bovine serum samples, and 25 is the tuberculosis negative serum control;

Figure 2b 1 is the bovine tuberculosis positive serum control; 2 to 16 are the test results of TST tuberculosis positive bovine serum samples, and 17 is the tuberculosis negative serum control.

Advantages of the invention

1 The diagnostic antigen used in the present invention is the main dominant antigen that mediates cellular immunity in the early stage of bovine tuberculosis infection, and the antigen can persist in the body after tuberculosis infection; but poultry tuberculosis, paratuberculosis, and BCG, etc. can easily interfere with the diagnosis of bovine tuberculosis Bacteria do not express this protein. Therefore, the kit can not only diagnose bovine tuberculosis infection in time, but also has great significance in differential diagnosis.

2 The method of gene splicing by over lap extension is used to connect the two genes, and a hydrophobic amino acid linker is introduced in the middle of the two genes, which can ensure the maximum possible spatial structure between the two antigens during the connection, so as to maintain the natural activity of the protein.

3 Bovine tuberculosis Dot-ELISA diagnostic method, compared with the conventional bovine tuberculosis diagnostic method, has the advantages of simple and fast operation, simple reagents and experimental conditions, and has good specificity and sensitivity, providing a solid foundation for the diagnosis of bovine tuberculosis. good way.

Positive effects of the present invention: the bovine tuberculosis can be diagnosed quickly, sensitively and specifically by using the kit involved in the present invention, and one detection can be completed within 2 hours.

<110> China Veterinary Drug Control Institute

<120> Bovine Tuberculosis Dot-ELISA Rapid Detection Kit

<140>2008101064024

<141>2008-05-13

<160>4

<210>1

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> Design primer P1 according to Mycobacterium bovis AF2122/97 (accession number NC 002945) in GenBank

<400>1

gcgaattcat ggcagagatg aag 23

<210>2

<211>59

<212>DNA

<213> Artificial sequence

<220>

<223> Design primer P2 according to Mycobacterium bovis AF2122/97 (accession number NC 002945) in GenBank

<400>2

gcttccacct cctccgcttc caccacctcc gcttccaccg ccaccgaagc ccatttgcg 59

<210>3

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<212>DNA

<213> Artificial sequence

<220>

<223> Design primer P3 according to Mycobacterium bovis AF2122/97 (accession number NC 002945) in GenBank

<400>3

ggtggcggtg gaagcggagg tggtggaagc ggaggaggtg gaagcatgac agagcagcag 60

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<213> Artificial sequence

<220>

<223> Design primer P4 according to Mycobacterium bovis AF2122/97 (accession number NC 002945) in GenBank

<400>4

ccctcgagtt actatgcgaa catcccagtg 30

Claims (4)

1. dot-ELISA fast detecting reagent kit for bovine tuberculosis, it is characterized in that envelope antigen, nitrocellulose filter, positive control serum, negative control sera, HRP-anti-bovine-IgG antibody, 10 * cleansing solution, substrate solution A, the substrate solution B of this kit by reorganization bacterium CGMCC No.2843 preparation, colour developing liquid is formed.

2. a kind of dot-ELISA fast detecting reagent kit for bovine tuberculosis as claimed in claim 1, the bacterium that it is characterized in that recombinating is to go out esxB and esxA genetic fragment by extracting the template amplification of CVCC68001 strain mycobacterium bovis DNA as the PCR reaction, finally amplify genes of interest esxB-esxB gene, its clone is entered in the pET-32a plasmid with the gene splicing technology, change colon bacillus again over to, obtain to contain the colon bacillus pET-CFP10-ESAT6 reorganization bacterium that the positive expression plasmid is numbered CGMCC No.2843.

3. a kind of dot-ELISA fast detecting reagent kit for bovine tuberculosis as claimed in claim 1, it is characterized in that its envelope antigen be CGMCC No.2843 reorganization bacterium is cultivated in the LB nutrient culture media after the product of abduction delivering purified, quantitatively form.

4. a kind of dot-ELISA fast detecting reagent kit for bovine tuberculosis as claimed in claim 1 is characterized in that being used for detecting as detecting antigen by envelope antigen being adsorbed onto on the nitrocellulose filter.

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