Detection of Mycobacterium tuberculosis DNA with thermophilic strand displacement amplification and fluorescence polarization.
Strand displacement amplification (SDA) is an isothermal, in vitro method for diagnostics that amplifies a target DNA sequence by using a restriction enzyme and DNA polymerase. We have combined a new thermophilic form of SDA that involves restriction enzyme BsoBI and polymerase exo-Bca with fluorescence polarization for detection of Mycobacterium tuberculosis DNA by using the IS6110 insertion element as the target sequence. A 5'-fluorescein-labeled oligodeoxynucleotide detector probe hybridizes to the amplified product as it rises in concentration during SDA, and the single- to double-stranded conversion is monitored through an increase in fluorescence polarization. The associated change in polarization upon amplification of the target sequence is enhanced by specific polymerase binding to the double-stranded detector probe. Fewer than 10 M. tuberculosis genomes can be amplified and detected with an extremely simple protocol that takes only 20 min and uses relatively simple instrumentation and reagents, all of which can be purchased off-the-shelf.
Walker
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Becton Dickinson Research Center, Research Triangle Park, NC 27709-2016, USA. walker@bdrc.bd.com
Linn
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D017931:DNA Primers; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000032:analysis*; D004259:DNA-Directed DNA Polymerase; D015246:Deoxyribonuclease EcoRI; D019793:Fluorescein; D005452:Fluoresceins; D005454:Fluorescence Polarization; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004269:DNA, Bacterial; D005452:Fluoresceins; D015345:Oligonucleotide Probes; D004259:DNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI; D019793:Fluorescein