Detection by denaturing gradient gel electrophoresis of pncA mutations associated with pyrazinamide resistance in Mycobacterium tuberculosis isolates from the United States-Mexico border region

Mark T. McCammon, John S. Gillette, Derek P. Thomas, Srinivas V. Ramaswamy, Ishmael I. Rosas, Edward A. Graviss, Jan Vijg, Teresa N. Quitugua

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with pyrazinamide (PZA) resistance in the pncA gene of Mycobacterium tuberculosis. DGGE scans for mutations across large regions of DNA and rivals sequencing in its ability to detect DNA alterations. Specific mutations can often be recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint. Five PCR target fragments were designed to scan for DNA alterations across 600 bp of pncA in 181 M. tuberculosis isolates from patients residing in the U.S-Mexico border states of Texas and Tamaulipas, respectively. A region of pncA was observed with a high GC content and a melting temperature approaching 90°C that was initially refractory to denaturation, and a DGGE target fragment was specifically designed to detect mutations in this region. DGGE detected pncA mutations in 82 of 83 PZA-resistant isolates. By contrast, only 1 of 98 PZA-susceptible isolates harbored a detectable DNA alteration. The pncA gene was sequenced from 41 isolates, and 32 DNA alterations in 32 PZA-resistant isolates were identified, including 11 new mutations. DGGE also detected nine isolates whose susceptibility to PZA appeared to be incorrect, and DNA sequencing confirmed these apparent errors in drug susceptibility testing. These results demonstrate the power and usefulness of DGGE in detecting mutations associated with PZA resistance in M. tuberculosis.

Original languageEnglish (US)
Pages (from-to)2210-2217
Number of pages8
JournalAntimicrobial Agents and Chemotherapy
Volume49
Issue number6
DOIs
StatePublished - Jun 2005
Externally publishedYes

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Pyrazinamide
Denaturing Gradient Gel Electrophoresis
Mexico
Mycobacterium tuberculosis
Mutation
DNA
DNA Sequence Analysis
Base Composition
Dermatoglyphics
Freezing
Genes
Polymerase Chain Reaction
Temperature
Pharmaceutical Preparations

ASJC Scopus subject areas

  • Pharmacology (medical)

Cite this

Detection by denaturing gradient gel electrophoresis of pncA mutations associated with pyrazinamide resistance in Mycobacterium tuberculosis isolates from the United States-Mexico border region. / McCammon, Mark T.; Gillette, John S.; Thomas, Derek P.; Ramaswamy, Srinivas V.; Rosas, Ishmael I.; Graviss, Edward A.; Vijg, Jan; Quitugua, Teresa N.

In: Antimicrobial Agents and Chemotherapy, Vol. 49, No. 6, 06.2005, p. 2210-2217.

Research output: Contribution to journalArticle

McCammon, Mark T. ; Gillette, John S. ; Thomas, Derek P. ; Ramaswamy, Srinivas V. ; Rosas, Ishmael I. ; Graviss, Edward A. ; Vijg, Jan ; Quitugua, Teresa N. / Detection by denaturing gradient gel electrophoresis of pncA mutations associated with pyrazinamide resistance in Mycobacterium tuberculosis isolates from the United States-Mexico border region. In: Antimicrobial Agents and Chemotherapy. 2005 ; Vol. 49, No. 6. pp. 2210-2217.
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