title: Peptide nucleic acid probe detection of mutations in Mycobacterium tuberculosis genes associated with drug resistance.
abstract: The emergence of drug-resistant strains of Mycobacterium tuberculosis is a serious public health problem. Many of the specific gene mutations that cause drug resistance in M. tuberculosis are point mutations. We are developing a PCR-peptide nucleic acid (PNA)-based ELISA as a diagnostic method to recognize point mutations in genes associated with isoniazid and rifampin resistance in M. tuberculosis. Specific point mutation-containing sequences and wild-type sequences of cloned mycobacterial genes were PCR-amplified, denatured, and hybridized with PNA probes bound to microplate wells. Using 15-base PNA probes, we established the hybridization temperatures (50 degrees C-55 degrees C) and other experimental conditions suitable for detecting clinically relevant point mutations in the katG and rpoB genes. Hybridization of PCR-amplified sequences that contained these point mutations with complementary mutation-specific PNAs resulted in significant increases in ELISA response compared with hybridization using wild-type-specific PNAs. Conversely, PCR-amplified wild-type sequences hybridized much more efficiently with wild-type PNAs than with the mutation-specific PNAs. Using the M. tuberculosis cloned genes and PCR-PNA-ELISA format developed here, M. tuberculosis sequences containing point mutations associated with drug resistance can be identified in less than 24 h.
authors.affiliation: US Food and Drug Administration, Rockville, MD 20857, USA. leb@cdrh.fda.gov
authors.forename: L E
authors.initials: LE
authors.lastname: Bockstahler
authors.forename: Z
authors.initials: Z
authors.lastname: Li
authors.forename: N Y
authors.initials: NY
authors.lastname: Nguyen
authors.forename: K A
authors.initials: KA
authors.lastname: Van Houten
authors.forename: M J
authors.initials: MJ
authors.lastname: Brennan
authors.forename: J J
authors.initials: JJ
authors.lastname: Langone
authors.forename: S L
authors.initials: SL
authors.lastname: Morris
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics*; D020135:Peptide Nucleic Acids / Q000235:genetics*; D017354:Point Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D014397:Tuberculosis, Pulmonary / Q000382:microbiology*
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D020135:Peptide Nucleic Acids