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Oligonucleotide for detection and identification of mycobacteria

A technology of oligonucleotides and mycobacteria, which is applied in the field of oligonucleotides for detection and identification of mycobacteria, and can solve problems such as no distinction

Inactive Publication Date: 2004-11-03
SJHIGHTECH +2
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  • Summary
  • 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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  • Oligonucleotide for detection and identification of mycobacteria
  • Oligonucleotide for detection and identification of mycobacteria
  • Oligonucleotide for detection and identification of mycobacteria

Examples

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example 1

[0056] Example 1. Mycobacterium culture and genomic DNA isolation

[0057] The standard strains of mycobacteria are derived from KCTC (Korea Model Collection) and ATCC (American Model Collection), and clinical strains are from the Korean National Tuberculosis Association, National Masan Tuberculosis Hospital, and clinical microbiology experts from Pusan ​​National University Hospital Manage the storage and cultivation of strains. The mycobacteria and pathogens used in the examples are listed in Table 8.

[0058] The extraction process of mycobacterial DNA is as follows: culture mycobacteria with Ogawa medium. Then place the cultured bacteria in a platinum ring in a microcentrifuge tube, mix with 200 μl of InstaGene substrate (Bio-Rad Co.), and incubate at 56°C for 30 minutes. After another 10 minutes of vortex mixing, the mixture was placed at 100°C for 8 minutes. After another 10 minutes of vortex mixing, the mixture was centrifuged at 12,000 rPM for 3 minutes. Take the supernata...

example 2

[0059] Example 2. Amplify mycobacterial ITS to make primers

[0060] The mycobacterial ITS and the primers used to amplify the ITS by PCR are listed in Figure 1. The primers for amplifying the ITS of mycobacteria were constructed based on several fragments of the conserved regions of 16SrRNA and 23SrRNA of mycobacteria. The DNA sequences of 16SrRNA and 23SrRNA of Mycobacterium from the gene bank were analyzed by multiple sequence comparison and immature cell inspection. Then, primers were designed by part of the DNA of 16SrRNA and 23SrRNA, so that about 500bp of ITS region can be selectively placed. The DNA sequences of these primers are listed in SEQ ID NOs: 242 (IT SF) and 243 (ITSR). All the primers used in this example were prepared by Derkin-Elmer DNA Synthesizer (BioBasic, Canada) with a concentration of 50 nmol.

example 3

[0061] Example 3. PCR and product identification

[0062] In PCR, 2 μl of the DNA solution in Example 1 was used. 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 , 1pmol primer, 1μTag DNA polymerase (Bio Basic Inc.). After denaturation at 94°C for 5 minutes, the solution was denatured at 94°C for 1 minute, annealed at 60°C for 1 minute and 72°C for an extension of 1 minute and circulated 30 times. A further extension of 72°C for 10 minutes ensures that the extension is over. After the reaction, the PCR product was identified by 1.5% agarose gel electrophoresis. As predicted from GenBank data, the ITS amplified by the primers prepared from the conserved regions of 16S rRNA and 23S rRNA was about 500 bP.

[0063] Figure 2 is a photograph of the electrophoresis result after PCR. PCR uses several mycobacterial strains and a pair of ITS amplification primers, including a part of mycobacterial 16S r...

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Abstract

This invention relates to oligonucleotides sequence of probes or primers for detection or identication of Mycobacterium. In the claimed invention, oligonucleotide sequences of ITS (Internal Transcribing Spacer Region) from M. fortuitium, M. chelonae, M. abscessus, M. vaccae, M. flavescence, M. Asiaticum, M. porcinum, M. acapulcensis and M. diernhoferi have been identified. Using these ITS sequences, PCR primers or hybridization probes for detection or identication of Mycobacterium have been developed and presented as seq ID : 10 to seq ID : 241.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 00808201.4. The patent application No. 00808201.4 was filed on May 16, 2000. The title of the invention is oligonucleotides for detection and identification of mycobacteria. Technical field [0002] The present invention relates to oligonucleotides for detecting and identifying mycobacteria. More specifically, the present invention is used to identify the nucleotide sequence of ITS (Internal Transcribed Spacer Region) of non-tuberculous mycobacteria. These non-tuberculous mycobacteria include: 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 by these mycobacteria. Background technique [0003] Although the number of tuberculosis patients has steadily decreased in re...

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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