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Kit for identifying Brucella S2 vaccine strain and wild strain

A technology for Brucella and Brucella suis, which is applied to the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc. strains and wild-type virus-infected strains, etc., to achieve the effect of high accuracy

Inactive Publication Date: 2015-10-28
DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are also key technical bottlenecks that cannot distinguish between vaccine strains and wild virus-infected strains
Although competitive ELISA (cELISA) and fluorescence polarization test (FPT) have certain applications in the serological identification of Brucella attenuated vaccine and wild strain infection due to their high-throughput and operational advantages, but for serum with unknown background For samples, a single test still cannot accurately distinguish between vaccine immunity and wild virus infection

Method used

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  • Kit for identifying Brucella S2 vaccine strain and wild strain
  • Kit for identifying Brucella S2 vaccine strain and wild strain
  • Kit for identifying Brucella S2 vaccine strain and wild strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment one, the using method of the PCR kit of differentiating Brucella S2 vaccine strain and wild strain

[0047] (1) Extract the genomic DNA of the sample to be tested: use the rapid DNA extraction kit to extract the genomic DNA from the blood, milk sample, tissue sample, aerosol and other samples to be tested.

[0048] (2) Using the extracted sample genomic DNA as a template, use the S2-F and S2-R primer pairs for PCR amplification, and use the positive quality control standard and the negative quality control standard as positive and negative controls respectively, and 50 μL Add the components of the reaction system to the PCR reaction tube, respectively 10×PCR Buffer: 5.0 μL, dNTP Mixture (2.5mM): 8.0 μL, 10 μM forward primer: 2.0 μL, 10 μM reverse primer: 2.0 μL , sample DNA to be tested: 5.0 μL, DNA polymerase: 0.5 μL, and finally add 27.5 μL double distilled water (ddH 2 O) Make up to 50 μL. The amplification effect of PCR directly affects the specificity ...

Embodiment 2

[0052] Embodiment two, the assembly of PCR kit

[0053] (1) Prepare PCR reaction solution:

[0054] ① Design 1 pair of primers for identifying the S2 vaccine strain of Brucella suis: SEQ ID No.1 and SEQ ID No.2, named as S2-F and S2-R, the sequences are as follows:

[0055] S2-F: 5'-CCTGCTGAGCGATGACCACCA-3'

[0056] S2-R: 5'-CCGCCAGAACATGCGATTTGA-3'

[0057] ② Preparation of PCR reaction solution: The PCR reaction solution was composed of primers of Brucella suis S2 vaccine strain and dNTPs, Mg 2+ , double distilled water (ddH 2 O) PCR buffer composition, the total volume of PCR reaction solution is 44.5 μ L, the amount of each primer is 2 μ L (primer concentration is 10 μ moL / L), contains dNTPs, Mg 2+ , double distilled water (ddH 2 O) The total amount of PCR buffer is 40.5 μL, in which double distilled water (ddH 2 O) is 27.5 μL.

[0058] (2) Preparation of positive quality control standard:

[0059] ①Template DNA extraction: Bacterial DNA extraction kits were used t...

Embodiment 3

[0065] Embodiment three, Brucella S2 vaccine strain identification kit specificity test

[0066] Adopt PCR kit of the present invention to respectively to Brucella suis S2, Brucella bovis A19, Brucella melis M5-90, Brucella ovis 63 / 290 strain, Brucella canis Bacteria RM6 / 66 strain, Escherichia coli O157:H7, Yersinia enterocolitica O:9, Salmonella, Staphylococcus aureus, Mycobacterium bovis genomic DNA were amplified, and positive and negative controls were set up to verify the PCR reaction. Specificity, PCR reaction conditions: 94°C for 5min, 94°C for 30sec, 60°C for 30sec, 72°C for 30sec, 35 cycles, 72°C for 10min. After the reaction, the PCR product was purified and sequenced.

[0067] The specificity results of Brucella suis S2 vaccine strain identification are as follows: figure 1 As shown, in the results of agarose gel electrophoresis: + is a positive quality control standard, 1-5: Brucella suis S2, Brucella bovis A19, Brucella melis M5-90 respectively , Brucella ovis ...

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Abstract

The invention relates to a kit for identifying a Brucella S2 vaccine strain and a wild strain. The kit comprises a specific primer pair SEQ ID No.1 and SEQ ID No.2 for identifying the Brucella vaccine strain S2. Sequencing analysis is further performed on a PCR amplification product after the kit is used for distributing Brucella and other conventional bacterial strains through a PCR amplification-electrophoresis detection area, and the Brucella S2 vaccine strain in a clinic sample can be rapidly and effectively identified and diagnosed according to the sequencing result.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kit for distinguishing Brucella S2 vaccine strains from wild strains. Background technique [0002] Brucellosis (Brucellosis) is caused by bacteria of the genus Brucella, which is widely distributed around the world and is a zoonotic infectious disease. It not only affects the healthy development of animal husbandry, but also endangers human public health. According to the national statutory infectious disease epidemic data released by the National Health and Family Planning Commission, the number of people infected with Brucella in my country in 2014 was 57,222, and the incidence rate increased by 31.48% compared with 2013. The source of human infection with brucellosis mainly comes from infected animals and contaminated animal products. Therefore, the control and elimination of animal brucellosis is the fundamental guarantee for preventing human infection with brucell...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 何洪彬赵贵民王洪梅贾春涛
Owner DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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