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Composition for detection of m. tuberculosis complex or mycobacteria genus and simultaneous detection method for m. tuberculosis complex and mycobacteria genus with multiplex real time PCR using the same

A technology for Mycobacterium tuberculosis and Mycobacterium, which is applied in the directions of botanical equipment and methods, biochemical equipment and methods, and applications, can solve the problem of low detection sensitivity and unfavorable early detection of Mycobacterium tuberculosis complex and Mycobacterium genus, complex experimental steps for pre-cell culture, etc.

Inactive Publication Date: 2011-08-10
LG LIFE SCI LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0022] The above methods show low detection sensitivity and thus disadvantageously require prior cell culture prior to actual testing or should involve complicated experimental steps
Thus, these traditional methods are not conducive to the early detection of M. tuberculosis complex and Mycobacterium spp.

Method used

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  • Composition for detection of m. tuberculosis complex or mycobacteria genus and simultaneous detection method for m. tuberculosis complex and mycobacteria genus with multiplex real time PCR using the same
  • Composition for detection of m. tuberculosis complex or mycobacteria genus and simultaneous detection method for m. tuberculosis complex and mycobacteria genus with multiplex real time PCR using the same
  • Composition for detection of m. tuberculosis complex or mycobacteria genus and simultaneous detection method for m. tuberculosis complex and mycobacteria genus with multiplex real time PCR using the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0131] Example 1: Construction of primers and probes for multiplex real-time PCR

[0132] Deposited under accession number NC000962 in GenBank ( www.ncbi.nlm.nih.gov ) DNA sequence, select the appropriate sequence, and then use BLAST ( www.ncbi.nlm.nih.gov / BLAST / ) analyzed the DNA sequence again, and it was confirmed that the IS6110-specific primers and probes used herein were primers and probe sequences capable of amplifying only the Mycobacterium tuberculosis complex.

[0133] In addition, by obtaining the mycobacterial rpoB gene from the GenBank / Entrez protein database, the conserved region of the rpoB gene was analyzed using the software Clustal-X (see figure 1 ), select the appropriate sequence, and then use BLAST ( www.ncbi.nlm.nih.gov / BLAST / ) DNA sequences were analyzed again, and it was confirmed that the rpoB-specific primers and probes used herein were those capable of amplifying only mycobacteria.

[0134] Thereafter, other unknown base sequences were obta...

Embodiment 2

[0136] Embodiment 2: the synthesis of primer

[0137] According to methods such as "Synthesis of Oligonucleotide" described in paragraph 10.42 of Molecular Cloning 3rd Edition (Sambrook and Russell, Cold Spring Harbor Laboratory Press, New York, USA, 2001), according to Metabion (Germany) requirements synthetic implementation Primers analyzed in Example 1.

Embodiment 3

[0138] Example 3: Extract Mycobacterium DNA from Clinical Samples or Standard Strain Medium

[0139] Put 1-2mL of suspected tuberculosis patient saliva or standard strain culture medium and the same amount of 4NNaOH into a 15mL test tube, stir well, and then centrifuge at 4,000rpm for 20 minutes. Discard the supernatant and add 10 mL of PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM NaCl, 2 HPO 4 , 2mM KH 2 PO 4 ), and the resulting mixture was stirred thoroughly and centrifuged at 4,000 rpm for 20 minutes. Discard the supernatant again. The precipitate was transferred to a 1.5 mL test tube, and 1 mL of PBS buffer was added thereto. The resulting mixture was stirred and centrifuged at 13,000 rpm for 5 minutes. Discard the supernatant again. 50-100 μl of 5% (w / v) Chelex 100 resin (Bio-Rad) was added to the pellet, and the resulting mixture was heated at 100° C. for 20 minutes and centrifuged at 13,000 rpm for 3 minutes. The supernatant with the extracted DNA was used as a...

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Abstract

Provided is a composition for detection of M. tuberculosis complex or Mycobacterium genus, comprising (i) a primer and / or probe targeting IS6110 that is an M tuberculosis complex-specific gene, (ii) a primer and / or probe targeting rpoB that is a Mycobacterium genus-specific gene, and optionally (iii) a primer and / or probe targeting a plant-derived gene as an internal control. When multiplex real-time PCR is carried out using the composition of the present invention, it is possible to detect M tuberculosis complex as well as Mycobacterium genus by a single roundPCR assay. Therefore, the present invention enables rapid and easy clinical diagnosis with high reliability.

Description

technical field [0001] The present invention relates to a composition for detecting Mycobacterium tuberculosis complex or Mycobacterium genus and a method for simultaneously detecting Mycobacterium tuberculosis complex by multiplex real-time PCR using said composition Groups and methods of Mycobacteria. More specifically, the present invention relates to a composition for detecting Mycobacterium tuberculosis complex or Mycobacterium, said composition comprising: (i) primers and / or probes targeting IS6110, said IS6110 is a Mycobacterium tuberculosis complex-specific gene; (ii) primers and / or probes targeting rpoB, which is a Mycobacterium-specific gene; and optionally (iii) a target as an internal control Primers and / or probes to genes of plant origin. Background technique [0002] Tuberculosis has been the leading infectious disease causing death in the world since ancient times. The causative bacterial pathogens of tuberculosis are tuberculosis bacteria, which belong to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12Q1/68C12N15/10
CPCC12Q1/689C12Q2600/16C12Q2600/166
Inventor 姜镇锡朴荣石李在成
Owner LG LIFE SCI LTD
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