Use of fluorescence resonance energy transfer for rapid detection of isoniazid resistance in Mycobacterium tuberculosis clinical isolates

Z. Saribas, P. Yurdakul, A. Alp, A. Gunalp

Research output: Contribution to journalArticle

Abstract

SETTING: Rapid detection of drug resistance in Mycobacterium tuberculosis is important to select effective treatment and prevent transmission of resistant isolates. OBJECTIVE: To evaluate the use of fluorescence resonance energy transfer (FRET) for rapid detection of isoniazid (INH) resistance in M. tuberculosis clinical isolates. DESIGN: One hundred INH-resistant and 50 INH-susceptible isolates of M. tuberculosis were included in the study. The drug susceptibility of all isolates was determined by the standard agar proportion method, and all isolates were then tested by FRET. Three genes associated with INH resistance, katG, inhA and ahpC, were analysed. All isolates were amplified with three pairs of primers. Three pairs of fluorescently labelled DNA probes specific to codon 315 of katG, nucleotide 209 in the regulatory region of inhA and a frequent mutation site in the intergenic region of oxyR-ahpC, were used for mutation detection. RESULTS: The results obtained using FRET were compared with those from the proportion method. The sensitivity and specificity of FRET were respectively 76% and 100%. The frequencies of mutations were 48% in katG, 17% in inhA, 8% in ahpC, 2% in inhA-ahpC and 1% in inhA-katG. CONCLUSION: FRET is a rapid, specific method that can be useful to detect INH resistance in M. tuberculosis clinical isolates.

Original languageEnglish (US)
Pages (from-to)181-187
Number of pages7
JournalInternational Journal of Tuberculosis and Lung Disease
Volume9
Issue number2
StatePublished - Feb 2005
Externally publishedYes

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Fluorescence Resonance Energy Transfer
Isoniazid
Mycobacterium tuberculosis
Mutation
Intergenic DNA
Nucleic Acid Regulatory Sequences
DNA Probes
Mutation Rate
Drug Resistance
Codon
Agar
Nucleotides
Sensitivity and Specificity
Pharmaceutical Preparations
Genes

Keywords

  • Fluorescence resonance energy transfer
  • Isoniazid
  • Mycobacterium tuberculosis
  • Real time PCR

ASJC Scopus subject areas

  • Pulmonary and Respiratory Medicine

Cite this

Use of fluorescence resonance energy transfer for rapid detection of isoniazid resistance in Mycobacterium tuberculosis clinical isolates. / Saribas, Z.; Yurdakul, P.; Alp, A.; Gunalp, A.

In: International Journal of Tuberculosis and Lung Disease, Vol. 9, No. 2, 02.2005, p. 181-187.

Research output: Contribution to journalArticle

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AU - Alp, A.

AU - Gunalp, A.

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N2 - SETTING: Rapid detection of drug resistance in Mycobacterium tuberculosis is important to select effective treatment and prevent transmission of resistant isolates. OBJECTIVE: To evaluate the use of fluorescence resonance energy transfer (FRET) for rapid detection of isoniazid (INH) resistance in M. tuberculosis clinical isolates. DESIGN: One hundred INH-resistant and 50 INH-susceptible isolates of M. tuberculosis were included in the study. The drug susceptibility of all isolates was determined by the standard agar proportion method, and all isolates were then tested by FRET. Three genes associated with INH resistance, katG, inhA and ahpC, were analysed. All isolates were amplified with three pairs of primers. Three pairs of fluorescently labelled DNA probes specific to codon 315 of katG, nucleotide 209 in the regulatory region of inhA and a frequent mutation site in the intergenic region of oxyR-ahpC, were used for mutation detection. RESULTS: The results obtained using FRET were compared with those from the proportion method. The sensitivity and specificity of FRET were respectively 76% and 100%. The frequencies of mutations were 48% in katG, 17% in inhA, 8% in ahpC, 2% in inhA-ahpC and 1% in inhA-katG. CONCLUSION: FRET is a rapid, specific method that can be useful to detect INH resistance in M. tuberculosis clinical isolates.

AB - SETTING: Rapid detection of drug resistance in Mycobacterium tuberculosis is important to select effective treatment and prevent transmission of resistant isolates. OBJECTIVE: To evaluate the use of fluorescence resonance energy transfer (FRET) for rapid detection of isoniazid (INH) resistance in M. tuberculosis clinical isolates. DESIGN: One hundred INH-resistant and 50 INH-susceptible isolates of M. tuberculosis were included in the study. The drug susceptibility of all isolates was determined by the standard agar proportion method, and all isolates were then tested by FRET. Three genes associated with INH resistance, katG, inhA and ahpC, were analysed. All isolates were amplified with three pairs of primers. Three pairs of fluorescently labelled DNA probes specific to codon 315 of katG, nucleotide 209 in the regulatory region of inhA and a frequent mutation site in the intergenic region of oxyR-ahpC, were used for mutation detection. RESULTS: The results obtained using FRET were compared with those from the proportion method. The sensitivity and specificity of FRET were respectively 76% and 100%. The frequencies of mutations were 48% in katG, 17% in inhA, 8% in ahpC, 2% in inhA-ahpC and 1% in inhA-katG. CONCLUSION: FRET is a rapid, specific method that can be useful to detect INH resistance in M. tuberculosis clinical isolates.

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