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Diagnostic method for detection and identification of tuberculosis and non-tuberculosis mycobacteria and simultaneous confirmation of rifampin resistance of tuberculosis bacillus on basis of quanta matrix assay platform and kit therefor

A technology for Mycobacterium tuberculosis and non-tuberculosis, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, which can solve the problems of complex fragment characteristics and impossible identification of bacterial species, and reduce misdiagnosis. the effect of risk

Active Publication Date: 2019-08-23
OPTIPHARM +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, in the presence of two or more bacterial species (including M. tuberculosis and non-tuberculosis mycobacteria), the characterization of the fragments after treatment with restriction enzymes is complex, making it impossible to identify bacteria species

Method used

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  • Diagnostic method for detection and identification of tuberculosis and non-tuberculosis mycobacteria and simultaneous confirmation of rifampin resistance of tuberculosis bacillus on basis of quanta matrix assay platform and kit therefor
  • Diagnostic method for detection and identification of tuberculosis and non-tuberculosis mycobacteria and simultaneous confirmation of rifampin resistance of tuberculosis bacillus on basis of quanta matrix assay platform and kit therefor
  • Diagnostic method for detection and identification of tuberculosis and non-tuberculosis mycobacteria and simultaneous confirmation of rifampin resistance of tuberculosis bacillus on basis of quanta matrix assay platform and kit therefor

Examples

Experimental program
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Effect test

example 1

[0029] Example 1: Isolation and Characterization of Mycobacteria

[0030] Each sample was inoculated into 3% Ogawa medium and BACTEC MGIT 960 system and cultured at 37°C. A portion of the medium was collected, heated at 100°C for 20 minutes, and centrifuged at 13,000 rpm for 5 minutes. The collected supernatant was used for subsequent experiments. Differentiation of Mycobacterium tuberculosis from non-tuberculosis mycobacteria by molecular diagnostics using the MTB-ID PCR kit.

example 2

[0031] Example 2: REBA Myco-ID

[0032] For REBA Myco-ID, nucleic acids isolated from each sample were amplified by PCR using biotinylated primers. First, nucleic acids were pre-denatured at 94°C for 5 minutes. Then, the reaction product was denatured at 94 °C for 30 s, followed by annealing at 45 °C and an extension cycle at 65 °C for 30 s. The final cycle was performed at 72°C for 7 minutes to obtain a 270-bp PCR product. PCR products are amplified.

[0033] The amplified PCR product was used for REBA Myco-ID. Experimental conditions were according to the manufacturer's experimental guidelines. The experimental procedure is as follows. Mix the PCR product with the same amount of denaturing solution (0.2N NaOH, 0.2mM EDTA), let it stand at room temperature for 5 minutes, and add REBA Myco-ID strips (M&D, Korea), which have been pre-coated with 2X SSPE / 0.1 % SDS dilutions were prepared. The reaction was allowed to proceed at 50°C for 30 minutes. The reaction mixture wa...

example 3

[0034] Example 3: QMAP based dual ID

[0035] For QMAP-based Myco-ID, nucleic acids were isolated from each sample by PCR amplification using biotinylated primers. First, nucleic acids were pre-denatured at 94°C for 5 minutes. Then, the reaction product was denatured at 94 °C for 30 s, followed by annealing at 45 °C and an extension cycle at 65 °C for 30 s. The final cycle was performed at 72°C for 7 minutes to obtain a 250-bp PCR product. PCR products are amplified.

[0036] The amplified PCR product was mixed with the same amount of denaturing solution (0.2N NaOH, 0.2mM EDTA), allowed to stand at room temperature for 5 min, and added to a coupled disk (Quantamatrix, Seoul, Korea), which had previously been hybridized with Buffer dilution preparation. The reaction was allowed to proceed at 40°C for 30 minutes. The reaction mixture was washed three times with washing solution (WS) at 25 °C for 1 min, and washed with streptavidin-R-phycoerythrin conjugate diluted to 1:2000...

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Abstract

The present invention relates to a diagnostic method for the detection and identification of tuberculosis and non-tuberculosis mycobacteria and the simultaneous confirmation of rifampin and isoniazidresistances of tuberculosis bacillus on the basis of a quanta matrix assay platform and a kit therefor.

Description

technical field [0001] The application relates to a diagnostic method and a reagent group for simultaneously detecting and identifying Mycobacterium tuberculosis and non-tuberculosis Mycobacterium and judging the rifampicin resistance of Mycobacterium tuberculosis based on the QuantaMatrix assay platform. Background technique [0002] The prevalence of tuberculosis, a chronic infectious disease caused by Mycobacterium tuberculosis (MTB), is steadily decreasing in South Korea, but remains higher than in other countries. Despite the use of effective anti-TB drugs, incurable forms of TB, such as MDR-TB and XDR-TB, have increased. Worldwide, about 8 million people develop tuberculosis each year, and about 2 million people die from the disease each year. Therefore, rapid judgment of drug resistance in difficult-to-treat M. tuberculosis is essential for effective treatment and survival of patients. Tuberculosis is also caused by nontuberculous mycobacteria (NTM). In countries o...

Claims

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Application Information

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IPC IPC(8): C12Q1/689G16B5/00G16B45/00C12R1/32
CPCC12Q1/68G16B5/00G16B45/00Y02A50/30C12Q1/6813C12Q1/689C12Q2563/107
Inventor 李惠泳王惠英
Owner OPTIPHARM
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