Method for detecting mycobacterium tuberculosis and nontuberculous mycobacteria using duplex real-time polymerase chain reaction and melting curve analysis

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

Inactive Publication Date: 2013-04-10
UNIV OF ULSAN FOUND FOR IND COOPERATION
View PDF3 Cites 2 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for detecting mycobacterium tuberculosis and nontuberculous mycobacteria using duplex real-time polymerase chain reaction and melting curve analysis
  • Method for detecting mycobacterium tuberculosis and nontuberculous mycobacteria using duplex real-time polymerase chain reaction and melting curve analysis
  • Method for detecting mycobacterium tuberculosis and nontuberculous mycobacteria using duplex real-time polymerase chain reaction and melting curve analysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1: Isolation and Detection of Mycobacterium Tuberculosis Complex and Nontuberculous Mycobacterium 1

[0073] 1. Detection target and primer design

[0074] The target genes to be detected by PCR and melting curve analysis were the IS6110 gene of the Mycobacterium tuberculosis complex (Mycobacterium tuberculosis, M. 16S rRNA gene. 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.

[0075] (1) Mycobacterium tuberculosis complex (MTC)

[0076] 1) Target gene: IS6110

[0077] 2) Primers

[0078] a. Forward primer: 5'-cgaactcaaggagcacatca-3' (SEQ ID NO: 1)

[0079] b. Reverse primer: 5'-agtttggtcatcagccgttc-3' (SEQ ID NO: 2)

[0080] 3) PCR product size: 135bp

[0081] 4) Average melting temperature (Tm) of PCR products: about 86...

Embodiment 1-1

[0091]

[0092] (1) DNA separation

[0093] DNA was isolated from 186 mycobacterial species and 78 nontuberculous mycobacterial species, all identified in clinical subjects, and 7 standard ATCC mycobacterial species, including Mycobacterium tuberculosis (ATCC 25177) , Mycobacterium intracellulare (ATCC 13950), Mycobacterium scrofula (ATCC 19981), Mycobacterium kansasii (ATCC 12478), Mycobacterium fortuitum (ATCC 6841), Mycobacterium abscessus (ATCC 19977) and Mycobacterium avium bacilli (ATCC 25291)).

[0094] Species identified in clinical subjects (including 186 mycobacterial species and 78 nontuberculous mycobacterial species) in liquid medium (MGIT Mycobacterium medium) or solid medium (Ogawa medium) tested, or isolated directly from a sputum sample. ATCC standard species were grown in broth.

[0095] DNA was isolated from mycobacteria grown in broth as follows. Transfer 500 μL of MGIT broth cultured with mycobacteria into a 1.5 mL tube and centrifuge at 14,000 rpm f...

Embodiment 1-2

[0103]

[0104] Double real-time PCR and melting curve analysis were 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.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The present invention provides: primers for detecting mycobacterium tuberculosis and nontuberculous mycobacteria, the primers being specific respectively to a mycobacterium tuberculosis-specific IS6110 gene and a nontuberculous mycobacteria-specific 16S rRNA; a mycobacterium tuberculosis and nontuberculous mycobacteria detection kit including the primers; and a method for detecting mycobacterium tuberculosis and nontuberculous mycobacteria by means of a duplex real-time polymerase chain reaction using the primers and by means of a melting curve analysis. The present invention can provide a clinical diagnostic means that can quickly detect mycobacterium tuberculosis and / or nontuberculous mycobacteria simultaneously and more effectively, at a lower cost, since the invention does not use a sequence-specific probe.

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-tuberculous mycobacteria by using the primer set through double real-time PCR and melting curve analysis. 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 th...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68C12R1/32
CPCC12Q2600/16C12Q1/689C12Q1/6851C12R2001/32
Inventor 金廷昱
Owner UNIV OF ULSAN FOUND FOR IND COOPERATION
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap