Primer for identifying brucellosis vaccine strain and wild strain, probe and test kit

A technology for Brucella and Brucella bovis, which is applied in the field of primers, probes and kits for identifying vaccine strains and wild strains of Brucellosis, and can solve problems such as the inability to distinguish vaccine strains from wild strains , to achieve rapid detection, high specificity and good sensitivity

Pending Publication Date: 2020-10-16
VETERINARY INST XINJINAG ACADEMY OF ANIMAL SCI CLINIC MEDICAL SCI RES CENT XINJIANG ACADEMY OF ANIMAL HUSBANDRY SCI
View PDF2 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing real-time fluorescent PCR method for Brucella cannot distinguish vaccine strains from wild strains

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Primer for identifying brucellosis vaccine strain and wild strain, probe and test kit
  • Primer for identifying brucellosis vaccine strain and wild strain, probe and test kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The construction of embodiment 1 detection method

[0038] 1. Design of primers and probes

[0039] According to the NCBI nucleic acid database GenBank Brucella VirB12 gene (GenBank number) sequence, a highly conserved and specific region was selected to design primers and TaqMan probes.

[0040] The primer sequences are as follows:

[0041] VirB12-F: 5'-GTGCGCTGGCTATCTATAA-3'

[0042] VirB12-R: 5'-GCATTTGATGCGTAATCTTT-3'

[0043]Specific TaqMan probe sequences are:

[0044] VirB12-Probe: 5'-FAM-ATTGGCTGATCAATCAAGGCGTACC-BHQ1-3'.

[0045] 2. Construction of detection method

[0046] 2.1 DNA extraction of samples to be tested

[0047] The DNA extraction kit used in the present invention is the bacterial whole genome DNA extraction kit of Tiangen Company.

[0048] 2.2 Primers and probes

[0049] The primers and probes used in the present invention were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0050] 2.3 Fluorescent quantitative PCR amplificatio...

Embodiment 2

[0056] Embodiment 2 sensitivity monitoring

[0057] The standard positive plasmid in embodiment 1 is respectively gradiently diluted to 10 10 -10 0 Copies, each dilution is done 3 parallel repeated detections, the Log value of the concentration of each standard is used as the X-axis, and the cycle number (Ct value) is used as the Y-axis to draw a standard curve ( figure 1 ), correlation coefficient R 2 =0.997, good repeatability.

[0058] The minimum detection limit of the real-time fluorescent PCR method between the Brucella bovis A19-ΔVirB12 molecularly labeled vaccine strain of Brucella bovis and the existing vaccine strains and wild strains of Brucella in the present invention is 100 copies / reaction.

Embodiment 3

[0059] Embodiment 3 Specificity detection

[0060] The invention can mark the genomic DNA of the Brucella A19-ΔVirB12 molecular marker vaccine strain, the genomic DNA of the Brucella bovis A19 vaccine strain, the genomic DNA of the Brucella melis M5 vaccine strain, and the Brucella suis S2 vaccine strain Genomic DNA, genomic DNA of wild strains of Brucella, and genomic DNA of Escherichia coli (E.coil) were detected by real-time fluorescent PCR according to the established reaction system and reaction conditions.

[0061] Such as figure 2 As shown in the results, it can be seen that the genomic DNA of the Brucella A19-ΔVirB12 molecular marker vaccine strain, the genomic DNA of Escherichia coli (E.coil), and the negative control have no amplification curve and no Ct value, which is judged as negative. The remaining samples all showed amplification curves, obtained Ct values, and were judged as positive. The invention can specifically detect the Brucella VirB12 gene, so as to ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention provides a primer for identifying a brucellosis vaccine strain and a brucellosis wild strain, a probe and a test kit, the nucleotide sequences of the primer are shown as SEQ ID NO.1-2, the nucleotide sequence of the probe is shown as SEQ ID NO.3, and the test kit comprises the primer and the probe. Pure brucella nucleic acid DNA can be identified, trace brucella nucleic acid DNA suchas tissue samples and the like can also be identified, and brucella abortus A19-delta VirB12 molecular marker vaccine immune samples are distinguished from brucella existing vaccine immune samples and wild strain infection samples. The method has the advantages of being high in safety, convenient to operate, high in sensitivity, high in specificity and the like, can detect a large number of samples at the same time, and has practical application value on prevention and control and diagnosis of brucellosis.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a primer, a probe and a kit for identifying brucellosis molecular marker vaccine strains and existing brucellosis vaccine strains and wild strains, specifically an identification Real-time fluorescent PCR specific primers, probes and kits for Brucella bovis A19-ΔVirB12 molecularly marked vaccine strains, existing vaccine strains and wild strains of Brucella. Background technique [0002] Brucellosis, referred to as brucellosis, is a zoonotic infectious disease caused by Brucella. The prevention, control, diagnosis and purification of brucellosis is an important basis for national public health security and the healthy development of animal husbandry. [0003] At present, the international and domestic prevention and control of brucellosis in animals mainly adopts brucellosis attenuated live vaccines, and the main vaccines in the world are S19, RB51, and Rev1. At present,...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/06C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6851C12Q2531/113C12Q2561/101C12Q2545/114
Inventor 叶锋马晓菁谷文喜刘丽娅易新萍谢彩云陈荣贵刘帅钟旗古丽努尔·吐尔逊张旭
Owner VETERINARY INST XINJINAG ACADEMY OF ANIMAL SCI CLINIC MEDICAL SCI RES CENT XINJIANG ACADEMY OF ANIMAL HUSBANDRY SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap