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Method for detecting mycobacterium tuberculosis and nontuberculous mycobacteria using duplex polymerase chain reaction

A technology of mycobacterium tuberculosis and mycobacteria, which is applied in the field of detection of mycobacterium tuberculosis and non-tuberculosis mycobacteria by using double polymerase chain reaction to achieve the effect of efficient clinical diagnosis means

Inactive Publication Date: 2013-04-10
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 detecting mycobacterium tuberculosis and nontuberculous mycobacteria using duplex polymerase chain reaction
  • Method for detecting mycobacterium tuberculosis and nontuberculous mycobacteria using duplex polymerase chain reaction
  • Method for detecting mycobacterium tuberculosis and nontuberculous mycobacteria using duplex polymerase chain reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Isolation and Detection of Mycobacterium Tuberculosis Complex and Nontuberculous Mycobacterium 1

[0055] 1. Detection target and primer design

[0056]The target genes to be detected are the IS6110 gene of Mycobacterium tuberculosis complex and the 16S rRNA gene of nontuberculous mycobacteria.

[0057] The universal primer was used as the forward primer to amplify the 16S rRNA gene of mycobacteria. NTM-1 and NTM-2, which are characteristic of nontuberculous mycobacteria, were used as reverse primers. These primers for target gene detection were designed using the Primer3 program.

[0058] (1) Mycobacterium tuberculosis complex (MTC)

[0059] 1) Target gene: IS6110

[0060] 2) Primers

[0061] a. Forward primer: 5'-cgaactcaaggagcacatca-3' (SEQ ID NO: 1)

[0062] b. Reverse primer: 5'-gtcgaggaccatggaggtg-3' (SEQ ID NO: 2)

[0063] 3) PCR product size: 385bp

[0064] (2) Non-tuberculous mycobacteria (NTM)

[0065] 1) Target gene: 16S rRNA

[0066] 2) ...

Embodiment 1-1

[0072]

[0073] (1) DNA separation

[0074] M. tuberculosis ATCC25177 and M. abscessus ATCC19977 were grown in the proprietary MGIT mycobacterial growth medium using the automated mycobacterial growth system BACTEC MGIT960 (Becton, Dickinson and Company, Maryland, USA). Transfer 500 μL of MGIT broth cultured with mycobacteria into a 1.5 mL tube and centrifuge at 14,000 rpm for 5 min. Remove the supernatant, dissolve the pellet in 300 μL sterile distilled water, and heat in a boiling water bath for 10 min. After centrifugation at 14,000 rpm for 5 min, the supernatant was used as template in PCR. A mixture of M. tuberculosis ATCC25177 and M. abscessus ATCC19977 was used as combined MTC and NTM species.

[0075] (2) Double PCR

[0076] Using the GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA), the duplex PCR starts with pre-denaturation at about 94°C for 2 min; then runs 40 cycles under the following conditions: denaturation at about 94°C for 30 seconds, a...

Embodiment 1-2

[0079]

[0080] Duplex PCR was performed in the same manner as in Example 1-1, except that the nucleotide sequence of SEQ ID NO: 5 was used as the reverse primer NTM-1.

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Abstract

Provided is a method for detecting mycobacterium tuberculosis and nontuberculous mycobacteria through a duplex polymerase chain reaction, the method using a kit for detecting mycobacterium tuberculosis and nontuberculous mycobacteria, the kit including a primer set for detecting mycobacterium tuberculosis and nontuberculosis mycobacteria specific to the mycobacterium tuberculosis-specific IS6110 gene or 16s rRNA gene and the nontuberculous mycobacteria-specific 16s rRNA gene. The present invention provides a clinical diagnosis means for more efficiently detecting mycobacterium tuberculosis and / or nontuberculous mycobacteria simultaneously using inexpensive general PCR equipment.

Description

technical field [0001] The invention relates to the detection of Mycobacterium tuberculosis and nontuberculous mycobacteria. More specifically, the present invention relates to a primer set capable of detecting specific nucleotide sequences of Mycobacterium tuberculosis and non-tuberculous A kit for detecting mycobacteria, and a method for simultaneously detecting tuberculosis mycobacteria and non-tuberculosis mycobacteria using the primer set. 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 identified as opportunistic pathogens of lung disease in patients with acquired immunodeficiency syndrome (AIDS). Additionally, the bacteria are also known to cause disease in other patients. With the reporting of the pathogenic mechanism of nontuberculous ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68C12R1/32
CPCC12Q1/689C12Q2600/16C12Q1/686
Inventor 金廷昱
Owner UNIV OF ULSAN FOUND FOR IND COOPERATION
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