title: Microarray and allele specific PCR detection of point mutations in Mycobacterium tuberculosis genes associated with drug resistance.
abstract: Global public health is threatened by the emergence of potentially dangerous antibiotic drug-resistant strains of Mycobacterium tuberculosis. Point mutations in certain M. tuberculosis genes are associated with the resistance of M. tuberculosis strains to antibiotic drugs. The purpose of this study was to develop a suitable microarray-based protocol for the detection of point mutations in M. tuberculosis genes associated with drug resistance. We initially developed a conventional, oligonucleotide microarray protocol and used it to detect and identify on a single microarray slide a number of point mutation-containing rpoB and katG gene target sequences. However, the occurrence of some non-specific hybridization led us to the development of an improved protocol based on allele specific PCR combined with tags/anti-tags and microarrays. This protocol was evaluated by detecting point mutations in M. tuberculosis katG and rpoB gene templates produced by recombinant PCR. The methodology allowed sequences containing single point mutations to be readily distinguished from wild type sequences. The data obtained with the improved protocol had strong and specific signals and relatively low amounts of non-specific hybridization. We successfully used this protocol to detect and identify (<8 h) a number of clinically relevant point mutations in the rpoB, katG and rpsL genes of M. tuberculosis clinical isolates. Our allele specific PCR/tags and anti-tags/microarray protocol has several advantages over our conventional oligonucleotide microarray protocol, and it may have broad applications for point mutation detection.
authors.affiliation: Center for Devices and Radiological Health, U.S. Food and Drug Administration, Rockville, MD 20850, USA. xxt4@cdrh.fda.gov
authors.forename: Xing
authors.initials: X
authors.lastname: Tang
authors.forename: Sheldon L
authors.initials: SL
authors.lastname: Morris
authors.forename: John J
authors.initials: JJ
authors.lastname: Langone
authors.forename: Larry E
authors.initials: LE
authors.lastname: Bockstahler
mesh_terms: D000483:Alleles; D001426:Bacterial Proteins / Q000235:genetics; D001431:Bacteriological Techniques; D001483:Base Sequence; D002374:Catalase / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D024881:Drug Resistance, Bacterial / Q000235:genetics; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis