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Method for the detection of Mycobacterium tuberculosis and nontuberculous mycobacteria by the use of dual real-time polymerase chain reaction

A technology of mycobacterium tuberculosis and mycobacteria, applied in the direction of microorganism-based methods, biochemical equipment and methods, biological testing, etc.

Inactive Publication Date: 2014-10-29
UNIV OF ULSAN FOUND FOR IND COOPERATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional reagents have problems with the accuracy of detection and diagnosis

Method used

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  • Method for the detection of Mycobacterium tuberculosis and nontuberculous mycobacteria by the use of dual real-time polymerase chain reaction
  • Method for the detection of Mycobacterium tuberculosis and nontuberculous mycobacteria by the use of dual real-time polymerase chain reaction
  • Method for the detection of Mycobacterium tuberculosis and nontuberculous mycobacteria by the use of dual real-time polymerase chain reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0169] Example 1: Isolation and Detection of Mycobacterium Tuberculosis Complex and Nontuberculous Mycobacterium 1

[0170] 1. Detection target and primer design

[0171] The target genes to be detected are the IS6110 gene of Mycobacterium tuberculosis complex (MTC: Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, and Mycobacterium microti) and the 16S rRNA of nontuberculous mycobacteria (NTM) Gene. Primers and Taqman probes designed using the Primer3 program were used in the detection of the target gene.

[0172] (1)MTC

[0173] 1) Target gene: IS6110

[0174] 2) Primers

[0175] a. Forward primer: 5'-cgaactcaaggagcacatca-3' (SEQ ID NO: 1)

[0176] b. Reverse primer: 5'-agtttggtcatcagccgttc-3' (SEQ ID NO: 2)

[0177] 3) Taqman probe

[0178] 5'-VIC-agtgtggctaaccctgaa-MGB-3' (SEQ ID NO: 3)

[0179] 4) PCR product size: 135bp

[0180] (2)NTM

[0181] 1) Target gene: 16S rRNA

[0182] 2) Primers

[0183] a. Forward primer: 5'-ggyrayctgccctgca...

Embodiment 1-1

[0190]

[0191] (1) DNA separation

[0192]DNA was isolated from 186 mycobacterial species and 78 nontuberculous mycobacterial species (all identified in clinical subjects), as well as 7 standard ATCC mycobacterial species (including Mycobacterium tuberculosis (ATCC25177), Mycobacterium intracellulare (ATCC13950), Mycobacterium scrofula (ATCC19981), Mycobacterium kansasii (ATCC12478), Mycobacterium fortuitum (ATCC6841), Mycobacterium abscessus (ATCC19977) and Mycobacterium avium (ATCC25291)) .

[0193] DNA was isolated from 186 mycobacterial species and 78 nontuberculous mycobacterial species (all identified in clinical subjects), as well as 7 standard ATCC mycobacterial species (including Mycobacterium tuberculosis (ATCC25177), Mycobacterium intracellulare (ATCC13950), Mycobacterium scrofula (ATCC19981), Mycobacterium kansasii (ATCC12478), Mycobacterium fortuitum (ATCC6841), Mycobacterium abscessus (ATCC19977) and Mycobacterium avium (ATCC25291)) .

[0194] Species ident...

Embodiment 1-2

[0203]

[0204] Duplex real-time PCR was performed in the same manner as in Example 1-1, except that the nucleotide sequence of SEQ ID NO: 6 was used as the reverse primer NTM-1.

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Abstract

Disclosed are a primer set and / or a probe capable of detecting specific nucleotide sequences of MTC and NTM, a kit for the detection of MTC and NTM, comprising the same, and a method for detecting MTC and NTM by duplex real-time PCR using the same. Useful in detecting genes characteristic of MTC and NTM, the primer sets and / or probes, detection kits, and detection methods can be applied as the clinical diagnosis of diseases caused by MTC and NTM, and therefore find applications in the medical fields including hospitals, research institutes, etc.

Description

technical field [0001] The invention relates to the detection of Mycobacterium tuberculosis and nontuberculous mycobacteria. More specifically, the present invention relates to primer sets and / or probes capable of detecting the specific nucleotide sequences of Mycobacterium tuberculosis and non-tuberculosis Mycobacteria, and a use comprising said primer sets and / or probes. A kit for detecting mycobacterium tuberculosis and non-tuberculosis mycobacteria, and a method for simultaneously detecting mycobacterium tuberculosis and non-tuberculosis mycobacteria through double real-time PCR using the primer set and / or probe. Background technique [0002] Nontuberculous mycobacteria are widely distributed in the environment, especially in wet soils, swamps and rivers, and were considered non-pathogenic until their opportunistic character was discovered. In the 1980s, nontuberculous mycobacteria were discovered as opportunistic pathogens of lung disease in patients with acquired immu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68C12R1/32G01N33/52
CPCC12Q2600/16C12Q1/689C12Q1/6851C12R2001/32
Inventor 金廷昱
Owner UNIV OF ULSAN FOUND FOR IND COOPERATION
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