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Double real-time fluorescent quantitative pcr identification method of brucella s2 vaccine strain and the kit of reagents used

A Brucella and non-Brucella technology, applied in the biological field, can solve the problem that immune antibodies cannot be distinguished from natural infection antibodies, and achieve the goals of preventing large-scale outbreaks, shortening detection time, ensuring development and public health safety Effect

Active Publication Date: 2022-04-01
CHINA ANIMAL DISEASE CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The detection of animal brucellosis is mainly based on serological detection methods, but the existing serological detection methods have cross-reactions with some serotypes of Escherichia coli and Yersinia, and the immune antibodies produced by the brucellosis vaccine used in my country and Natural infection antibodies of wild strains cannot be distinguished
Up to now, there is no identification method that can quickly identify Brucella S2 vaccine strains

Method used

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  • Double real-time fluorescent quantitative pcr identification method of brucella s2 vaccine strain and the kit of reagents used
  • Double real-time fluorescent quantitative pcr identification method of brucella s2 vaccine strain and the kit of reagents used
  • Double real-time fluorescent quantitative pcr identification method of brucella s2 vaccine strain and the kit of reagents used

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1, be used for the preparation of the test kit identifying Brucella S2 vaccine strain

[0066] 1. Screening of a complete set of reagents for identifying Brucella S2 vaccine strains

[0067] 1. Through a large number of sequence acquisition, analysis, comparison and preliminary experiments, the inventor of the present invention finally takes the IS711 gene in the Brucella genome as the target gene, screens and synthesizes a combination of primers and probes for detecting Brucella Bru-F1 / Bru-R1 / Bru-Probe1; against deletions specific to the S2 vaccine strain (e.g. figure 1 shown), design and synthesize the primer-probe combinations S2-F1 / S2-R1 / S2-Probe1, S2-F2 / S2-R2 / S2-Probe2 and S2-F3 / for identifying Brucella S2 vaccine strains S2-R3 / S2-Probe3. Perform Bru-F1 / Bru-R1 / Bru-Probe1 with S2-F1 / S2-R1 / S2-Probe1, S2-F2 / S2-R2 / S2-Probe2 and S2-F3 / S2-R3 / S2-Probe3 respectively Combining, the set of reagents 1, set of reagents 2 and set of reagents 3 are obtained in seq...

Embodiment 2

[0077] Embodiment 2, establishment of the method for identifying Brucella S2 vaccine strain by double real-time fluorescent quantitative PCR

[0078] 1. Drawing of standard curve

[0079] 1. Refer to the whole genome sequence of Brucella S2 vaccine strain, Brucella A19 vaccine strain, Brucella melis 16M strain, and Brucella bovis 2308 strain in the NCBI database, provided by Yingweijieji Trading Co., Ltd. Co., Ltd. artificially synthesized plasmid IS711 and plasmid S2.

[0080] Plasmid IS711 contains the nucleotide sequence of Brucella IS711 gene. The nucleotide sequence of the Brucella IS711 gene is shown in SEQ ID NO:10.

[0081] Plasmid S2 contains specific DNA sequences. The nucleotide sequence of the specific DNA sequence is shown in SEQ ID NO:11.

[0082] The Brucella S2 vaccine strain does not contain this specific DNA sequence, but other Brucella bacteria contain this specific DNA sequence.

[0083] 2. Detect the concentration of plasmid IS711 and plasmid S2 with ...

Embodiment 3

[0110] Embodiment 3, specific detection

[0111] 1. According to the method of step 3 in Example 2, specificity detection is carried out. The samples to be tested are Brucella S2 vaccine strain, Brucella A19 vaccine strain, Brucella ovis 16M strain, Brucella bovis 2308 strain, Brucella suis 1330 strain, dog cloth Rutella strain 6 / 66, Staphylococcus aureus, Salmonella typhimurium, Escherichia coli O:157 and Yersinia O:9.

[0112] See some results Figure 4 (A is the Brucella A19 vaccine strain, B is the Brucella S2 vaccine strain). The results showed that Brucella A19 vaccine strain, Brucella melis 16M strain, Brucella bovis 2308 strains, Brucella suis 1330 strains and Brucella canis 6 / 66 strains (all Brucella, which is a non-Brucella S2 vaccine strain), has two "S"-shaped amplification curves, Brucella S2 vaccine strain has one "S"-shaped amplification curve, Staphylococcus aureus, Salmonella typhimurium, E. coli O:157, Yersinia O:9, and the negative control all had no "S"...

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Abstract

The invention discloses a method for identifying Brucella S2 vaccine strain by double real-time fluorescence quantitative PCR and a complete set of reagents used therefor. The complete set of reagents consists of primer Bru‑F1, primer Bru‑R1, probe Bru‑Probe1, primer S2‑F2, primer S2‑R2 and probe S2‑Probe2. The nucleotide sequence is as follows: SEQ ID NO: 1—SEQ ID NO: Shown in 6. Compared with other brucella nucleic acid detection methods, the method established by the present invention is to directly carry out qualitative and quantitative brucella S2 vaccine strains through fluorescence information, and analyze and identify at the same time, which has the advantages of convenience, accuracy, sensitivity, specificity, etc. The advantage is that the detection time is greatly shortened, and it has great application value for the prevention and control of brucellosis. It is helpful to control the epidemic from the source, effectively prevent large-scale outbreaks of brucellosis, and ensure the development of animal husbandry and public health safety. .

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for identifying Brucella S2 vaccine strain by double real-time fluorescent quantitative PCR and a complete set of reagents used therefor. Background technique [0002] Brucellosis (referred to as brucellosis) is an important zoonotic infectious disease caused by Brucella spp. The infection rate of brucellosis in herds and humans is increasing year by year and remains at a relatively high level, which continues to pose a threat to public health security and the development of animal husbandry. [0003] Vaccination is one of the most effective ways to control brucellosis in areas of high prevalence. According to the relevant provisions of the "National Brucellosis Prevention and Control Plan (2016-2020)" issued by the former Ministry of Agriculture and the Health and Family Planning Commission, cattle and sheep farms in Class I areas will be fully immunized, and d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/686C12Q2561/113C12Q2563/107C12Q2545/114C12Q2537/143
Inventor 董浩原霖王传彬顾小雪魏巍韩焘赵柏林毕一鸣徐琦
Owner CHINA ANIMAL DISEASE CONTROL CENT
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