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Method for detecting live attenuated Brucella vaccine strain and wild strain and application of method

A technology of live attenuated vaccines and Brucella, which is applied in the field of detection of live attenuated vaccine strains and wild strains of Brucella, can solve the problems of limited duration of antibodies and achieve high accuracy and universal applicability Broad and specific effects

Pending Publication Date: 2019-12-13
天康生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Routine serological testing techniques are used to detect antibodies produced by antigens, but the duration of antibodies produced by any antigen has certain limitations

Method used

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  • Method for detecting live attenuated Brucella vaccine strain and wild strain and application of method
  • Method for detecting live attenuated Brucella vaccine strain and wild strain and application of method
  • Method for detecting live attenuated Brucella vaccine strain and wild strain and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1 Brucella live attenuated vaccine (A19-ΔVirB12 strain)

[0083] Ten brucellosis antibody-negative calves were selected, 5 of which were inoculated with live attenuated Brucella vaccine (A19-ΔVirB12 strain), and 5 were inoculated with virulent Brucella strain 2308, and kept in isolation. Whole blood was collected on 4, 12, 21, 35, 45, and 60 days after inoculation, and the blood samples were subjected to bacterial nucleic acid extraction and lymphocyte extraction before nucleic acid extraction:

[0084] Bacterial nucleic acid extraction from blood samples can be performed according to Tiangen’s commercial kit.

[0085] The procedure for extracting peripheral blood lymphocytes is as follows:

[0086] 1) Collect 2 mL of anticoagulated blood;

[0087] 2) Inject 2mL of lymphocyte separation solution into a 15mL centrifuge tube (place lymphocyte separation solution at room temperature), use a 5mL syringe to draw 2mL of normal saline, and then absorb 2mL of antic...

Embodiment 2

[0095] Ten brucellosis antibody-negative calves were selected, 5 of which were inoculated with live attenuated Brucella vaccine (S2-ΔVirB12 strain), and 5 were inoculated with virulent Brucella strain 2308, and kept in isolation. Whole blood was collected on 4, 12, 21, 35, 45, and 60 days after inoculation, and the blood samples were subjected to bacterial nucleic acid extraction and lymphocyte extraction before nucleic acid extraction:

[0096] Bacterial nucleic acid extraction from blood samples can be performed according to Tiangen’s commercial kit.

[0097] The procedure for extracting peripheral blood lymphocytes is as follows:

[0098] 1) Collect 2 mL of anticoagulated blood;

[0099] 2) Inject 2mL of lymphocyte separation solution into a 15mL centrifuge tube (place lymphocyte separation solution at room temperature), use a 5mL syringe to draw 2mL of normal saline, and then absorb 2mL of anticoagulant blood. After mixing, pull out the needle of the syringe and centrifug...

Embodiment 3

[0107] Ten brucellosis antibody-negative calves were selected, 5 of which were inoculated with live attenuated Brucella vaccine (M5-ΔBtp1 strain), and 5 were inoculated with virulent Brucella strain 2308, and kept in isolation. Whole blood was collected on 4, 12, 21, 35, 45, and 60 days after inoculation, and the blood samples were subjected to bacterial nucleic acid extraction and lymphocyte extraction before nucleic acid extraction:

[0108] Bacterial nucleic acid extraction from blood samples can be performed according to Tiangen’s commercial kit.

[0109] The procedure for extracting peripheral blood lymphocytes is as follows:

[0110] 1) Collect 2 mL of anticoagulated blood;

[0111] 2) Inject 2mL of lymphocyte separation solution into a 15mL centrifuge tube (place lymphocyte separation solution at room temperature), use a 5mL syringe to draw 2mL of normal saline, and then absorb 2mL of anticoagulant blood. After mixing, pull out the needle of the syringe and centrifuge ...

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Abstract

The invention provides a method for detecting a live attenuated Brucella vaccine strain and a wild strain and an application of the method, and belongs to the field of biotechnologies. The method fordetecting the live attenuated Brucella vaccine strain and the wild strain provided by the invention designs a primer according to a missing gene of the live attenuated Brucella vaccine strain, then adopts the primer to carry out PCR amplification on DNA of a sample to be detected, and judges the sample based on detection results. By the method, the primer is designed according to the missing geneof the live attenuated Brucella vaccine strain, namely the designed primer for amplification is a part or all of the missing gene, so that the primer effectively amplifies a gene of the wild strain without amplifying a gene of the live attenuated vaccine strain, thereby accurately and effectively distinguishing the live attenuated Brucella vaccine strain from the wild strain. The method has broaduniversality, high specificity and high accuracy.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method and application for detecting live attenuated vaccine strains and wild strains of Brucella. Background technique [0002] The currently used brucella vaccines are live attenuated vaccines, which provide a strong protection against brucellosis during the current period of high prevalence of brucellosis. However, the use of live attenuated vaccines also has certain shortcomings, mainly manifested in the inability to distinguish between wild virus infection and vaccine immunity, and there is a risk of infection for personnel. [0003] Gene-marked vaccines are based on the original vaccine strains, which are labeled with nucleic acid using molecular biology techniques, and the labeled antigens are prepared in vitro, and serological and molecular detection techniques are used to distinguish vaccine strains from wild viruses, thereby achieving identification. Conventional serolog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689
Inventor 贺笋何传雨任立松李延涛赵海龙刘梦志吴冬玲
Owner 天康生物制药有限公司
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