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Primer and probe for identifying brucella vaccine strain A19 and wild strain, and application thereof

A technology of Brucella and wild strains, applied in the direction of microorganisms, recombinant DNA technology, and methods based on microorganisms, can solve the problems of high cost of detection and typing, low detection cost, etc., and achieve fast detection speed, high sensitivity, The effect of improving correctness

Inactive Publication Date: 2021-06-25
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The currently reported methods for the identification and detection of vaccine strain A19 from wild strains include the MGB probe method for single nucleotide polymorphism (SNP) site detection (Gopaul K K, Sells J, Bricker B J, et al. Rapid and ReliableSingle Nucleotide Polymorphism-Based Differentiation of Brucella Live VaccineStrains from Field Strains[J].Journal of Clinical Microbiology,2010,48(4):1461-4.) and Cycling probe method (Tan Pengfei. Based on PCR method Brucella vaccine strains and The establishment of a differential diagnosis method for wild strains [D]. Yangzhou University, 2012.), but both methods require dual probes, the cost of detection and typing is high, and it is necessary to develop typing with lower detection costs and wider application value Detection method

Method used

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  • Primer and probe for identifying brucella vaccine strain A19 and wild strain, and application thereof
  • Primer and probe for identifying brucella vaccine strain A19 and wild strain, and application thereof
  • Primer and probe for identifying brucella vaccine strain A19 and wild strain, and application thereof

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Experimental program
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Effect test

Embodiment 1

[0031] Design and screening of embodiment 1 primers and probes

[0032] After screening a large number of primers and probes designed, it was found that the primer pair F, R, and probe P fluorescent method had the best effect in distinguishing Brucella A19 (S19) vaccine strains from wild strains. As follows.

[0033] Primer F: 5'-CATCGATGTGCCGTTCATCATG-3' (SEQ ID NO: 1),

[0034] Primer R: 5'-CACATCTTCGCCGACATAGC-3' (SEQ ID NO: 2),

[0035] Probe P: 5'-ATGCGACGACGCTGACGCAAGCC-3' (SEQ ID NO: 3).

[0036]A fluorescent group FAM is labeled at the 5' end of the probe P, and a quencher group BHQ1 is labeled at its 3' end.

[0037] like figure 1 As shown, at the probe P position, there is only one SNP site (C18G) between the Brucella A19 (S19) vaccine strain and the wild strain.

Embodiment 2

[0038] The preparation of embodiment 2 standard samples, fluorescent PCR amplification and melting curve analysis

[0039] 1. Preparation of standard samples

[0040] In order to verify the feasibility and reliability of the method of the present invention, a standard positive sample was prepared to provide a positive control for subsequent method establishment, sensitivity, and specificity tests.

[0041] according to figure 1 Probe sequence comparison analysis, the corresponding Brucella A19 (S19) vaccine strain sequence of probe P is one group, and other wild strains are another group.

[0042] The genome nucleic acid of the Brucella A19 vaccine and wild strain representative strain 544A used in the present invention is donated by China Veterinary Drug Control Institute. Using the nucleic acid of the A19 (S19) vaccine and the genomic nucleic acid of the field strain 544A as templates, the following primer pairs were used:

[0043] CF: 5'-GTTCTGTCGGTTGCGGTTCA-3' (SEQ ID N...

Embodiment 3

[0055] Embodiment 3 specificity experiment

[0056] The genome nucleic acid of Brucella vaccine A19 strain, Brucella bovis 544A strain, Brucella suis 1330S strain, Brucella ovis 16M strain donated by the China Veterinary Drug Administration and other common samples were respectively selected. Pathogens of common bacterial diseases in pigs isolated and preserved in the laboratory: E. coli, Pasteurella, Streptococcus suis, Pseudomonas aeruginosa, Actinobacillus pleuropneumoniae ( Actinobacillus pleuropneumoniae).

[0057] Utilize DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract the above-mentioned bacterial nucleic acid, and use the extracted nucleic acid and water (negative control) as PCR templates respectively, with the PCR amplification reaction in the above-mentioned embodiment 2 and The melting curve analysis method was used for analysis, and compared with the positive standard samples pA19 and pW, the peak shape of the melting curve was ...

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Abstract

The invention discloses a primer and a probe for identifying a brucella vaccine strain A19 and a wild strain, and application thereof, and belongs to the field of gene detection. The primer comprises an upstream primer as shown in SEQ ID NO: 1 and a downstream primer as shown in SEQ ID NO: 2. The nucleotide sequence of the probe is as shown in SEQ ID NO: 3. The primer and the probe are used for fluorescence detection, and rapid, high-throughput, high-sensitivity and specific region distribution of a vaccine strain and a wild strain of the Brucella A19 (S19) can be realized.

Description

technical field [0001] The invention relates to the technical field of gene detection, relates to a primer, probe and application for distinguishing Brucella vaccine strain A19 from wild strains, in particular to primer probes and reagents for quickly distinguishing Brucella vaccine strain A19 from wild strains Boxes and applications and methods. Background technique [0002] Brucellosis (abbreviated as brucellosis) is a zoonotic infectious disease caused by Brucella. According to its pathogenicity and host selectivity, it can be divided into at least 10 species, among which Brucella melitensis, Brucella abortus and Brucella suis ( Brucella suis) is the most infectious. The bacteria can infect humans and cattle, sheep, pigs and other animals. At present, my country mainly adopts prevention-based epidemic prevention measures. The varieties of live brucellosis vaccines approved for production in China mainly include: Brucella bovis strain A19, Brucella pig strain S2, Brucel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/686
Inventor 张春红刘志成沈海燕马祥孙俊颖张建峰廖明
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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