Oligouncleotide for detection and identification of mycobacteria

A technology of oligonucleotide and mycobacteria, applied in the field of nucleotide sequence of ITS, can solve the problem of no distinction

Inactive Publication Date: 2002-06-12
SJHIGHTECH +2
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, Mycobacterium chelonis is divided into Mycobacterium chelonis and Mycobacterium abscessus, and there is no method to distinguish them

Method used

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  • Oligouncleotide for detection and identification of mycobacteria
  • Oligouncleotide for detection and identification of mycobacteria
  • Oligouncleotide for detection and identification of mycobacteria

Examples

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example 1

[0054] Below will introduce some better examples of the present invention and its reference accompanying drawing through embodiment. Example 1. Mycobacterial Culture and Genomic DNA Isolation

[0055] Mycobacterium standard strains come from KCTC (Korea Type Culture Collection) and ATCC (American Type Culture Collection), and clinical strains come from the Korean National Tuberculosis Association, National Masan Tuberculosis Hospital and Pusan ​​National University Hospital clinical microbiology experts Manage the storage and cultivation of strains. The mycobacteria and pathogens used in the examples are listed in Table 8.

example 2

[0056] The extraction process of mycobacterial DNA was as follows: Mycobacteria were cultured in Ogawa medium. Then, the strain cultured with a platinum ring was placed in a microcentrifuge tube, mixed with 200 μl of InstaGene matrix (Bio-Rad Co.), and incubated at 56° C. for 30 minutes. After an additional 10 minutes of vortex mixing, the mixture was placed at 100°C for 8 minutes. After an additional 10 minutes of vortex mixing, the mixture was centrifuged at 12,000 rpm for 3 minutes. Take the supernatant and put it in another test tube, and store it at -20°C. In the following PCR, 2 μl of the DNA solution of each strain will be used. Example 2. Amplification of Mycobacterial ITS Primers for Manufacturing

example 3

[0057] Mycobacterial ITS and the primers used to amplify ITS by PCR are listed in figure 1 middle. Primers for amplifying mycobacterial ITS were constructed based on several fragments of mycobacterial 16SrRNA and 23SrRNA conserved regions. The DNA sequences of 16SrRNA and 23SrRNA of mycobacteria in the gene bank were analyzed by multiple sequence alignment and immature cell inspection. Then, primers were designed through a part of DNA of 16SrRNA and 23SrRNA, thereby selectively amplifying the ITS region of about 500 bp. The DNA sequences of these primers are listed in SEQ ID NOs: 242 (IT SF) and 243 (ITSR). All primers used in this example were prepared by Derkin-Elmer DNA synthesizer (BioBasic, Canada) at a concentration of 50 nmol. Example 3. PCR and product identification

[0058] 2 µl of the DNA solution in Example 1 was used in PCR. The reaction solution includes: 500mM KCl, 100mM Tris HCl (pH9.0), 1% Triton X-100, 0.2mM dNTP (dATP, dGTP, dTTP and dCTP), 1.5mM MgCl 2...

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Abstract

Provided is oligonucleotide sequences of probes or primers for the detection or identification of Mycobacterium. The oligonucleotide sequences of ITS (Internal Transcribing Spacer Region) from M. fortuitium, M. chelonae, M. abscessus, M. vaccae, M. flavescens, M. Asiaticum, M. porcinum, M. acapulcensis and M. diernhoferi have been identified. Using these ITS sequences represented, PCR primers or hybridization probes for the detection or identification of Mycobacterium have been developed and presented. The oligonucleotide probe is hybridized with only specific nucleotide sequence presenting in ITS of mycobacteria. Therefore, it is attached on the solid support and detects specifically and sensitively mycobacteria strains. Through PCR magnifying, the primers with similar oligonucleotide sequences as above can also detect specifically and sensitively mycobacteria strains. By adopting this oligonucleotide sequences of probes or primers based on the ITS (Internal Transcribing Spacer Region), the accurate and effective detection or identification of Mycobacterium, identification of tubercle bacillus and NTM can be realized.

Description

technical field [0001] The present invention relates to oligonucleotides for the detection and identification of mycobacteria. More specifically, the present invention is used to identify the nucleotide sequence of the ITS (Internal Transcribed Spacer Region, internal transcribed spacer region) of non-tuberculous mycobacteria, these non-tuberculous mycobacteria include: fortuitum mycobacterium (Mycobacterium fortuitum), Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium vaccae, Mycobacterium flavescens, Mycobacterium asiaticum, Mycobacterium suis ( Mycobacterium porcinum), Mycobacterium acapulcensis and Mycobacterium diernhoferi. oligonucleotide primers or probes through these mycobacteria. Background technique [0002] Although the number of tuberculosis patients has been steadily decreasing in recent years, about 8,000,000 people still suffer from tuberculosis and 3,000,000 patients die each year. In underdeveloped countries, the number of chronic patients w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C07K14/35C12N15/11
CPCC07K14/35C12N15/11
Inventor 金哲民朴希卿张贤贞
Owner SJHIGHTECH
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