Peptide Nucleic Acid Probe Detection of Mutations in Mycobacterium tuberculosis Genes Associated with Drug Resistance
| Autor: | N Y Nguyen, M J Brennan, K A Van Houten, Sheldon L. Morris, J J Langone, L E Bockstahler, Z M Li |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Genetics
Mutation Peptide nucleic acid biology Point mutation Drug resistance Gene mutation rpoB biology.organism_classification medicine.disease_cause Molecular biology General Biochemistry Genetics and Molecular Biology Mycobacterium tuberculosis chemistry.chemical_compound chemistry medicine Molecular probe Biotechnology |
| Zdroj: | BioTechniques. 32:508-514 |
| ISSN: | 1940-9818 0736-6205 |
| DOI: | 10.2144/02323st01 |
| Popis: | 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°C–55°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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|