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Detection kit for distinguishing cow pathogenic mycobacteria infection from non-pathogenic mycobacteria infection and method thereof

A technology of mycobacterium bovis and reagents, applied in the field of immunoassay, can solve the problems of false positive, poor detection specificity, and low sensitivity of serological detection methods, and achieve the effect of strong specificity and sensitivity

Active Publication Date: 2012-09-26
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complex antigenic components contained in PPD, some of the antigens are found in bovine pathogenic mycobacteria such as Mycobacterium tuberculosis and Mycobacterium bovis, and bovine non-pathogenic mycobacteria such as Mycobacterium avium and non-pathogenic environmental mycobacteria. Bacilli are widely present in the bacteria, resulting in poor detection specificity. At the same time, studies have shown that PPD cannot effectively distinguish between Mycobacterium bovis infection and BCG immunity, and false positives often occur in actual use.
[0006] Therefore, there is an urgent need to develop a detection reagent and method that can improve the specificity and sensitivity of detection at the same time, which can not only overcome the low sensitivity of serological detection methods, but also have a higher detection rate than PPD-stimulated IFN-γ release test. specificity

Method used

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  • Detection kit for distinguishing cow pathogenic mycobacteria infection from non-pathogenic mycobacteria infection and method thereof
  • Detection kit for distinguishing cow pathogenic mycobacteria infection from non-pathogenic mycobacteria infection and method thereof
  • Detection kit for distinguishing cow pathogenic mycobacteria infection from non-pathogenic mycobacteria infection and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Construction of recombinant plasmid pET-E6-M63-H70

[0028] 1.1 Extraction of Mycobacterium bovis genomic DNA

[0029] Refer to the method described in the instructions of the Bacterial Genomic DNA Small Amount Rapid Extraction Kit (purchased from Beijing Biotech Gene Technology Co., Ltd.).

[0030] 1.2 Design of primers

[0031] Specific primers were designed according to the ESAT-6, MPB63 and HSP70 gene sequences of Mycobacterium bovis genomic DNA (Accession No. BX248333) in GenBank. The primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd., and the sequences are shown in Table 1 (the underline is the enzyme cut point, the shaded place is Linker).

[0032] Table 1PCR primer name, sequence and size of amplified product

[0033]

[0034] 1.3 PCR amplification of target gene and product recovery

[0035] Using Mycobacterium bovis genomic DNA as a template, LA Taq DNA polymerase was used to amplify ESAT-6, MPB63 and HSP70 genes respectively....

Embodiment 2

[0048] Example 2 Expression and Purification of Mycobacterium bovis Recombinant Fusion Protein rE6-M63-H70

[0049] 2.1 Induced expression, purification, SDS-PAGE and Western-blot analysis of recombinant fusion protein

[0050] Transform the recombinant plasmid pET-E6-M63-H70 prepared in Example 1 into BL21(DE3) competent cells, pick a single colony and inoculate it into 200 mL of LB medium containing a final concentration of 25 μg / ml ampicillin, and shake at 37°C Bed culture until OD600=0.7, add IPTG with a final concentration of 1mM, induce culture at 30°C, 160rpm shaker for 10h, centrifuge at 9000rpm for 10min to collect the bacteria, resuspend the bacteria in 60mL PBS (pH 7.4), and sonicate the bacteria in an ice bath After crushing, the mixture was centrifuged at 12000rpm and 4°C for 30min, then the supernatant was taken, and the His Link TM Protein Purification Resin (purchased from Beijing Zeping Technology Co., Ltd.) operation manual to purify the recombinant fusion ...

Embodiment 3

[0052] The establishment of embodiment 3 Mycobacterium bovis detection method

[0053] 3.1 Collection and culture of bovine whole blood to be tested

[0054] ①Collect 5ml of bovine heparin anticoagulated whole blood under aseptic conditions, transport it to the laboratory at room temperature (22±5°C) and culture it within 8 hours after blood collection. ② Add anticoagulant blood to 24-well tissue culture plate, 1.5ml / well, 2 wells for each cow to be tested, then aseptically add 50μl Tris-Cl (100mM, pH 8.0, as negative control stimulus), 50μl containing rE6-M63-H70 solution (20μg) into the corresponding well, mix well, 37 ℃ CO 2 Incubate in the incubator for 24h. ③ After incubation, carefully draw 400 μl of the upper layer of plasma and transfer it to a 1.5ml centrifuge tube (plasma can be stored at 2-8°C for 7 days, and at -20°C for several months), and draw 50 μl of plasma from each tube as the sample to be tested ELISA detection of bovine IFN-γ release level.

[0055] 3....

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Abstract

The invention belongs to the field of immunodetection and relates to a detection kit for distinguishing cow pathogenic mycobacteria infection from non-pathogenic mycobacteria infection and a method thereof. The detection reagent comprises combined fusion protein rE6-M63-H70 used as a specific stimulation origin, the combined fusion protein can effectively stimulate sensitized peripheral blood lymphocyte cultured in vitro to release Gamma-interferon (IFN-Gamma) at a high level. The cow IFN-Gamma release test established by using the detection reagent rE6-M63-H70 combined fusion protein as the stimulation origin overcomes the insufficiencies of serology detection method and the IFN-Gamma release test with PPD as the stimulation origin, thus enjoying very high sensitivity and specificity anddistinguishing cow pathogenic mycobacteria ( such as mycobacterium bovis) infection from non-pathogenic mycobacteria (such as mycobacterium avium or non-pathogenic mycobacteria) infection and even distinguishing the cow pathogenic mycobacteria infection from BGG immunity; therefore, the detection kit and the method of the invention can be effectively used to detect the clinical cow pathogenic mycobacteria infection.

Description

technical field [0001] The invention belongs to the technical field of immunoassay. The present invention relates to kits and methods for differentiating between pathogenic and non-pathogenic mycobacterial infections in cattle. Background technique [0002] Bovine tuberculosis is a zoonotic chronic wasting infectious disease caused by Mycobacterium bovis and Mycobacterium tuberculosis. Cross-infection between humans and animals causes the disease to spread widely, so it has a very important public health meaning. The World Health Organization (WHO) pointed out: "In countries where bovine tuberculosis exists, human beings are always threatened. Unless bovine tuberculosis is eliminated, the control of human tuberculosis will not be successful." At present, some relatively developed countries and regions, such as the United States, Bovine tuberculosis has been basically eradicated in Australia and Northern Europe. However, bovine tuberculosis is still one of the most common ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/543C07K19/00
Inventor 朱鸿飞侯绍华郝辉贾红张泉毛开荣
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI