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Kit for recognizing Brucella wild strain and vaccine strains A19 and S2

A technique for Brucella and Brucella bovis is applied in the field of test kits for identifying wild strains of Brucella and vaccine strains A19 and S2, which can solve the problem of inability to identify vaccine strains and wild virus infection strains, inability to accurately identify vaccine strains and wild virus infection strains. Distinguish between vaccine immunity or wild virus infection

Active Publication Date: 2015-11-04
DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of A19 and S2 vaccine strains has provided an important guarantee for the prevention and control of brucellosis in fauna in my country, but there is also a key technical bottleneck problem that cannot distinguish vaccine strains from 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 recognizing Brucella wild strain and vaccine strains A19 and S2
  • Kit for recognizing Brucella wild strain and vaccine strains A19 and S2
  • Kit for recognizing Brucella wild strain and vaccine strains A19 and S2

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Experimental program
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Effect test

Embodiment 1

[0057] Embodiment one, the usage method of the PCR kit of differentiating brucella wild strain and vaccine strain A19 and S2

[0058] (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.

[0059] (2) Using the extracted sample genomic DNA as a template, use A19-F, A19-R and S2-F, S2-R primer pairs to carry out PCR amplification respectively. As positive and negative controls, add 50 μL of each component of the reaction system to the PCR reaction tube in turn, 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 and sensitivity of the detection results, so it is nec...

Embodiment 2

[0065] Embodiment two, the assembly of PCR kit

[0066] (1) Prepare PCR reaction solution:

[0067] ① Design 2 pairs of primers for identifying the Brucella bovis A19 vaccine strain and the Brucella suis S2 vaccine strain respectively: SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, SEQ ID No. 4. Named A19-F, A19-R and S2-F, S2-R, the sequence is as follows:

[0068] A19-F: 5'-TTCACCGTCATCGGATAATAGGTGCCA-3'

[0069] A19-R: 5'-GGCGGGCTGCATTCTTTTACTCTGC-3'

[0070] S2-F: 5'-CCTGCTGAGCGATGACCACCA-3'

[0071] S2-R: 5'-CCGCCAGAACATGCGATTTGA-3'

[0072] ②Preparation of PCR reaction solution: The PCR reaction solution is composed of primers of Brucella bovis A19 vaccine strain or Brucella suis S2 vaccine strain and containing 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...

Embodiment 3

[0080] Embodiment three, Brucella vaccine strain identification kit specificity test

[0081] Adopt PCR kit of the present invention to respectively to Brucella bovis A19, Brucella suis S2, 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.

[0082] The specificity results of Brucella bovis A19 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 bovis A19, Brucella suis S2, Brucella melis M5-90 , Brucella ovis 63 / 290 strains...

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Abstract

The invention relates to a kit for recognizing a Brucella wild strain and vaccine strains A19 and S2. The kit comprises a special primer pair SEQ ID No.1 and SEQ ID No.2 for recognizing the Brucella vaccine strain A19 and a special primer pair SEQ ID No.3 and SEQ ID No.4 for recognizing the Brucella vaccine strain S2. The kit is used for conducting further sequencing analysis on a PCR amplification product after the Brucella and other conventional bacterial strains are distributed on a PCR amplification-electrophoresis detection area, and the Brucella A19 and S2 vaccine strains in a clinic sample can be rapidly and effectively recognized 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 identifying Brucella wild strains and vaccine strains A19 and S2. 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 bru...

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
Owner DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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