Comparison of DNA extraction methods for drug susceptibility testing by allele-specific primer extension on a microsphere-based platform

Chelex-100 (in-house and commercialized) and MagPurix TB DNA Extraction Kit

Minji Kang, Jeong Seong Yang, Yoojeong Kim, Kyungjong Kim, Hongjo Choi, Seung Heon Lee

Research output: Contribution to journalArticle

Abstract

Tuberculosis (TB), caused by infections of the Mycobacterium tuberculosis (MTB) complex, is the ninth leading cause of death worldwide, and several molecular approaches for MTB species identification and the detection of mutations associated with drug resistance have been developed to date. We previously developed a diagnostic assay for drug susceptibility testing that can detect mutations conferring resistance to anti-TB drugs using allele-specific primer extension on a microsphere-based platform for multiplex polymerase chain reaction. The aim of the present study was to optimize this diagnostic assay based on the evaluation of three methods for extracting mycobacterial DNA from clinical samples. Mycobacterial DNA of 81 samples was digested and decontaminated by N-acetyl-L-cysteine-2% NaOH and then extracted using three methods: “in-house” 5% Chelex-100 chelating resin, InstaGene Matrix, and MagPurix TB DNA Extraction Kit. The former two methods are manual extraction methods, whereas the MagPurix TB DNA Extraction Kit is an automated extraction method used with the MagPurix 12 s automated nucleic acid purification system. The extracted DNA was then subjected to our diagnostic assay, and the results were compared among methods. The magnetic bead method exhibited a higher extraction efficiency and resulted in greater diagnostic efficacy than the two resin-based methods with respect to both target gene detection and acid-fast bacilli smear grades. Therefore, the MagPurix TB DNA Extraction Kit is the optimal MTB DNA extraction method for our diagnostic assay of TB drug susceptibility testing.

Original languageEnglish
Pages (from-to)105-108
Number of pages4
JournalJournal of Microbiological Methods
Volume152
DOIs
Publication statusPublished - 2018 Sep 1
Externally publishedYes

Fingerprint

Microspheres
Tuberculosis
Alleles
DNA
Pharmaceutical Preparations
Mycobacterium tuberculosis
Chelex 100
Mutation
Multiplex Polymerase Chain Reaction
Acetylcysteine
Drug Resistance
Nucleic Acids
Bacillus
Cause of Death
Acids
Infection
Genes

Keywords

  • DNA extraction
  • Molecular diagnostic method
  • Tuberculosis

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology
  • Microbiology (medical)

Cite this

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title = "Comparison of DNA extraction methods for drug susceptibility testing by allele-specific primer extension on a microsphere-based platform: Chelex-100 (in-house and commercialized) and MagPurix TB DNA Extraction Kit",
abstract = "Tuberculosis (TB), caused by infections of the Mycobacterium tuberculosis (MTB) complex, is the ninth leading cause of death worldwide, and several molecular approaches for MTB species identification and the detection of mutations associated with drug resistance have been developed to date. We previously developed a diagnostic assay for drug susceptibility testing that can detect mutations conferring resistance to anti-TB drugs using allele-specific primer extension on a microsphere-based platform for multiplex polymerase chain reaction. The aim of the present study was to optimize this diagnostic assay based on the evaluation of three methods for extracting mycobacterial DNA from clinical samples. Mycobacterial DNA of 81 samples was digested and decontaminated by N-acetyl-L-cysteine-2{\%} NaOH and then extracted using three methods: “in-house” 5{\%} Chelex-100 chelating resin, InstaGene Matrix, and MagPurix TB DNA Extraction Kit. The former two methods are manual extraction methods, whereas the MagPurix TB DNA Extraction Kit is an automated extraction method used with the MagPurix 12 s automated nucleic acid purification system. The extracted DNA was then subjected to our diagnostic assay, and the results were compared among methods. The magnetic bead method exhibited a higher extraction efficiency and resulted in greater diagnostic efficacy than the two resin-based methods with respect to both target gene detection and acid-fast bacilli smear grades. Therefore, the MagPurix TB DNA Extraction Kit is the optimal MTB DNA extraction method for our diagnostic assay of TB drug susceptibility testing.",
keywords = "DNA extraction, Molecular diagnostic method, Tuberculosis",
author = "Minji Kang and Yang, {Jeong Seong} and Yoojeong Kim and Kyungjong Kim and Hongjo Choi and Lee, {Seung Heon}",
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T2 - Chelex-100 (in-house and commercialized) and MagPurix TB DNA Extraction Kit

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AU - Yang, Jeong Seong

AU - Kim, Yoojeong

AU - Kim, Kyungjong

AU - Choi, Hongjo

AU - Lee, Seung Heon

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AB - Tuberculosis (TB), caused by infections of the Mycobacterium tuberculosis (MTB) complex, is the ninth leading cause of death worldwide, and several molecular approaches for MTB species identification and the detection of mutations associated with drug resistance have been developed to date. We previously developed a diagnostic assay for drug susceptibility testing that can detect mutations conferring resistance to anti-TB drugs using allele-specific primer extension on a microsphere-based platform for multiplex polymerase chain reaction. The aim of the present study was to optimize this diagnostic assay based on the evaluation of three methods for extracting mycobacterial DNA from clinical samples. Mycobacterial DNA of 81 samples was digested and decontaminated by N-acetyl-L-cysteine-2% NaOH and then extracted using three methods: “in-house” 5% Chelex-100 chelating resin, InstaGene Matrix, and MagPurix TB DNA Extraction Kit. The former two methods are manual extraction methods, whereas the MagPurix TB DNA Extraction Kit is an automated extraction method used with the MagPurix 12 s automated nucleic acid purification system. The extracted DNA was then subjected to our diagnostic assay, and the results were compared among methods. The magnetic bead method exhibited a higher extraction efficiency and resulted in greater diagnostic efficacy than the two resin-based methods with respect to both target gene detection and acid-fast bacilli smear grades. Therefore, the MagPurix TB DNA Extraction Kit is the optimal MTB DNA extraction method for our diagnostic assay of TB drug susceptibility testing.

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