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Bovine tuberculosis serological diagnostic marker and clinical application thereof

A technology of tuberculosis and markers, which is applied in the application field of preparing bovine tuberculosis diagnostic kits, can solve the problems of strict production conditions, inability to differentiate and diagnose latently infected cattle and active tuberculosis cattle, false positive results, etc., and achieve improvement The effects of diagnostic efficiency, simplified operation requirements, and strong specificity

Active Publication Date: 2022-01-04
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the tuberculin skin test uses bovine tuberculin (PPD-B) as the stimulus, and a large amount of Mycobacterium bovis needs to be cultivated in the production process, which requires strict production conditions, and bovine tuberculin and poultry tuberculin ( PPD-A), environmental mycobacteria and Bacillus Calmette-Guerin (BCG) have common antigens, which can easily lead to false positive results in clinical applications, and cannot differentiate between latently infected cattle and active tuberculosis cattle.
Serological detection methods have the advantages of convenience, rapidity, and high throughput. However, none of the currently established serological diagnostic methods for bovine tuberculosis can achieve the required sensitivity and specificity, so they have not been recommended by international organizations.

Method used

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  • Bovine tuberculosis serological diagnostic marker and clinical application thereof
  • Bovine tuberculosis serological diagnostic marker and clinical application thereof
  • Bovine tuberculosis serological diagnostic marker and clinical application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1 main detection method

[0026] tuberculin skin test

[0027] The tuberculin skin test was performed according to the "Diagnostic Criteria for Bovine Tuberculosis" (GB / T 18645-2002). Shave the upper 1 / 3 of the neck of the cow, and inject 0.1 mL of purified tuberculin bovis (PPD-B, 250 IU / head) intradermally. The skin thickness at the injection site was measured by the same operator with a vernier caliper before and 72 hours after the injection, and the difference in skin thickness was calculated. When the difference in skin thickness is greater than or equal to 4mm, the cow is positive for tuberculosis; when the difference in skin thickness is less than 2mm, it is judged as negative for tuberculosis; The skin test was performed 60 days after the first test. If the difference in skin thickness in the second test was greater than or equal to 2mm, it was determined to be positive for tuberculosis.

[0028] PCR detection of nasal swab secretion

[0029] Put ...

Embodiment 2

[0031] Example 2 Screening of Molecular Markers

[0032] Sample collection and preparation

[0033] Aseptically collect 5ml of venous blood from cattle, without adding anticoagulant, let stand at 37°C for 1h and then overnight at 4°C, after the serum naturally precipitates, centrifuge at 3000r / min for 10min, separate the serum and transfer it into a 1.5ml centrifuge tube, store at -80 ℃ for later use.

[0034] bovine paratuberculosis detection

[0035] Utilize the method of embodiment 1 to screen out tuberculosis negative cattle (NC) at random, the method for " sample collection and preparation " prepares the serum sample of this batch of cattle, utilizes bovine paratuberculosis antibody ELISA kit to detect the antibody level in the serum then, screens out Tuberculosis negative, paratuberculosis positive cattle (paraTB).

[0036] ELISA Validation Molecular Markers

[0037] Utilize the method for embodiment 1 to randomly screen tuberculosis bovine PCR positive (bTB PCR-P ) 1...

Embodiment 3

[0040] Example 3 Determination of cutoff value for detecting bovine tuberculosis method

[0041] In this study, tuberculin skin test and PCR detection of nasal swab secretion were used to screen tuberculosis bovine PCR-positive (bTB PCR-P ) 17 head, tuberculosis cattle PCR negative (bTB PCR-N ) and 5 tuberculosis-negative cattle (NC), the venous blood of the above-mentioned total of 105 cattle was aseptically collected, and the concentrations of KLK12, MMP-1 and MMP-9 in the serum were detected by ELISA kits, and KLK12 was analyzed according to the working characteristic curve , MMP-1 and MMP-9 to distinguish the cutoff value of tuberculosis positive cattle and negative cattle, and analyze the cutoff value of KLK12 to distinguish active and inactive tuberculosis cattle.

[0042] The results showed that when distinguishing tuberculosis positive cattle from negative cattle, the AUC of KLK12 and MMP-1 was 1, and the selection specificity and detection sensitivity could reach 100...

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Abstract

The invention provides a bovine tuberculosis serological diagnostic marker and clinical application thereof. Serum markers, namely KLK12, MMP-1 and MMP-9, capable of identifying negative and positive bovine tuberculosis are obtained through transcriptome sequencing analysis, mouse animal experiments and clinical tests. The bovine tuberculosis serological diagnostic marker provided by the invention has the advantages that the diagnostic result of the bovine tuberculosis serological diagnostic marker is 100% consistent with that of a tubercle bacillus skin test, and the bovine tuberculosis serological diagnostic marker can identify and diagnose latent infected cattle and active tuberculosis cattle, is not influenced by infection of other mycobacteria, and has relatively strong specificity and relatively high sensitivity. The diagnostic marker provides a new target spot for diagnosis of bovine tuberculosis, can be used for preparing a serological diagnostic kit for bovine tuberculosis, does not need to collect anticoagulant and specific antigen stimulation, simplifies operation requirements, improves diagnosis efficiency, is beneficial to batch and rapid detection and elimination of tuberculosis cattle, has a wide application prospect, and provides a technical support for prevention, control and purification of bovine tuberculosis.

Description

technical field [0001] The invention belongs to the field of prevention and treatment of bovine tuberculosis in the veterinary field. Specifically, the application provides bovine tuberculosis serological diagnostic markers KLK12, MMP-1, MMP-9 and their application in the preparation of bovine tuberculosis diagnostic kits Background technique [0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis complex. Mycobacterium tuberculosis and Mycobacterium bovis (Mycobacterium bovis, referred to as M.bovis) are the main pathogenic bacteria. Bovine tuberculosis caused by Mycobacterium bovis is a Important zoonotic diseases are also major infectious diseases that endanger the development of animal husbandry and seriously threaten public health and food safety. Studies have shown that in addition to infecting cattle, Mycobacterium bovis can infect different species of warm-blooded animals, including more than 50 mammals including primates, marsupial...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/6893G01N2800/065
Inventor 周向梅王元智梁正敏刘一朵葛昕
Owner CHINA AGRI UNIV
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