Oligonucleotide gene chip and application of oligonucleotide gene chip to detection of various bacteria

A technology of oligonucleotide and nucleotide sequence, which is applied in the field of oligonucleotide gene chip and its application in the detection of various pathogenic bacteria, can solve the problem of long growth cycle of mycobacteria, low detection rate of pathogenic bacteria, It is unfavorable for rapid diagnosis and other problems, and achieves the effect of improving the efficiency of fluorescent labeling, meeting the sensitivity requirements, and good sensitivity and specificity

Inactive Publication Date: 2014-02-19
SOUTH CHINA AGRI UNIV
View PDF5 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the detection of tuberculosis pathogenic bacteria is mainly through the tuberculin test, but due to the influence of physical and biological factors, the detection rate of this type of pathogenic bacteria is not high; bacterial isolation and culture is a classic means of identification, but the growth cycle of mycobacteria is long , generally need to be cultured for 4 to 8 weeks, which is not conducive to rapid diagnosis; the establishment of PCR method can achieve rapid and sensitive detection, but it is easy to be contaminated and lead to false positive results
Mycobacterial infection can also be detected by detecting the level of IFN-γ released by peripheral blood lymphocytes, which has high sensitivity and specificity, but the disadvantage is that the cost is too high, and the detection must be completed within 8 hours after blood collection
For the detection of Brucellosis, test tube agglutination test and tiger red plate agglutination test are mainly used. This method will be affected by physical or biological factors, resulting in false positive or false negative results.
[0005] Therefore, it is urgent to develop a new, fast, accurate, and high-throughput detection technology for the above several pathogenic bacteria. The gene chip method has certain advantages due to its good specificity and high detection throughput. The related technology of pathogenic bacteria needs to be further studied

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
  • Oligonucleotide gene chip and application of oligonucleotide gene chip to detection of various bacteria
  • Oligonucleotide gene chip and application of oligonucleotide gene chip to detection of various bacteria
  • Oligonucleotide gene chip and application of oligonucleotide gene chip to detection of various bacteria

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0083] Preparation of LB solid medium containing Amp: LB solid medium was autoclaved, cooled to 50°C, added Amp with a final concentration of 100 μg / mL, poured into a plate, and stored at 4°C for use.

[0084] 1.5% agarose gel: Weigh 1.5g of agarose into a conical flask, and dilute to 100mL with 1×TAE buffer.

[0085] Other reagents, materials, instruments and methods, unless otherwise specified, are those commonly used in this technical field.

Embodiment 1

[0087] (1) Design and screening of gene chip primers of the present invention

[0088] 1. Primer Design

[0089] The gene sequences of M.tuberculosisH37R, M.bovisAN5, M.avium, M.paratuberculosis and Brucella published by Genebank (http: / / www.ncbi.nlm.nih.gov) were used to select the M.tuberculosisH37Rv The mpt40 gene, the pncA gene of M.bovisAN5, the gyrB gene of M.avian, the IS-900 gene of M.paratuberculosis, and the Alkb gene sequence of Brucella are reference sequences (GeneBank accession number: M.tuberculosis accession number: S69737.1; The retrieval number of M.bovis is: BX248341.1; the retrieval number of M.avium is: NC_008595; the retrieval number of M.paratuberculosis is: S744011; the retrieval number of Brucella is: NC_006933.1), through DNAstar software analysis and comparison, select no variation or variation For the smallest fragment, use PrimerPremier5.0 and Oliga6.0 software to design multiple pairs of primers for comparative analysis. In this example, 2 to 3 p...

Embodiment 2

[0189] Embodiment 2 Application oligonucleotide chip of the present invention detects bovine tuberculosis and brucellosis (Brucellosis)

[0190] Reagents: PCR-related reagents were purchased from Bao Biology (Dalian) Co., Ltd., and SDS and agarose were purchased from Promega Company.

[0191]Instruments: SportArray24 Gene Chip Spotting Apparatus (PerkinElmer, USA), ScanarrayGX Gene Chip Scanner (PerkinElmer, USA), MaiTai Biochip Hybridization Instrument (SciGene), aldehyde-formed gene chip base and gene chip spotting solution were purchased from Beijing Boao Biotechnology Co., Ltd., gel imaging and analysis system (ABI, USA), high-speed refrigerated centrifuge (BeckmanAllegra, USA). PCR instrument (ThermoHybaid Company, UK), micro-adjustable pipette (Eppendorf Company, Germany), autoclave (Sanyo Company), gel electrophoresis instrument (BIO-RAD Company), 1K15 desktop centrifuge (Sigma Company), desktop Constant temperature shaking incubator (Germany GFL company).

[0192] Vi...

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 relates to an oligonucleotide gene chip and its application in detecting various germs. The probes of the oligonucleotide gene chip can be combined with Mycobacterium tuberculosis (M.tuberculosis), Mycobacterium bovis (M.bovis), Mycobacterium avium (M.avium), Mycobacterium paratuberculosis ( M.paratuberculosis) and Brucella (Brucella) PCR amplification products were subjected to hybridization reaction; the probes had the nucleotides shown in SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 37 and SEQ ID NO: 39 respectively sequence. The application of the gene chip in the invention to the detection of the pathogen has good repeatability, and the CV values ​​of the inter-batch and intra-batch variation coefficients are all less than 15%. The detection of pathogenic bacteria samples showed high accuracy, indicating that the method has good specificity and high sensitivity, and realizes high-throughput and parallel detection. The detection results are fast and accurate. From nucleic acid preparation to completion of detection, The whole process only takes 6-8 hours, and has broad prospects in the preparation of reagents and products for pathogen detection, import and export quarantine, and epidemiological analysis.

Description

technical field [0001] The invention belongs to the technical field of gene chips, and in particular relates to an oligonucleotide gene chip and its application in detecting various germs. Background technique [0002] Bovine tuberculosis (Bovis Tuberculosis) is an infectious disease caused by Mycobacterium bovis and Mycobacterium tuberculosis. Safety. [0003] Brucellosis is an infectious disease caused by bacteria such as Brucellosis abortus, which can lead to abortion of cows. At the same time, the bacteria can also infect humans and is difficult to cure. The number of cases of human infection with Brucella has been on the rise this year. , so the detection of this pathogen also has important public health significance. [0004] At present, the detection of tuberculosis pathogenic bacteria is mainly through the tuberculin test, but due to the influence of physical and biological factors, the detection rate of this type of pathogenic bacteria is not high; bacterial isola...

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 Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 李守军贾坤林志雄
Owner SOUTH CHINA AGRI UNIV
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