Detection of Mycobacterium tuberculosis DNA with thermophilic strand displacement amplification and fluorescence polarization

• Published in: Clinical chemistry (Baltimore, Md.) Vol. 42; no. 10; pp. 1604 - 1608
• Main Authors: Walker, GT, Linn, CP
• Format: Journal Article
• Language: English
• Published: Washington, DC Am Assoc Clin Chem 01-10-1996; American Association for Clinical Chemistry
• Subjects: Bacterial diseases; Bacterial diseases of the respiratory system; Biological and medical sciences; Deoxyribonuclease EcoRI; DNA Primers; DNA Restriction Enzymes; DNA, Bacterial - analysis; DNA-Directed DNA Polymerase; Fluorescein; Fluoresceins; Fluorescence Polarization; Human bacterial diseases; Infectious diseases; Medical sciences; Mycobacterium tuberculosis - genetics; Nucleic Acid Amplification Techniques; Nucleic Acid Hybridization; Oligonucleotide Probes; Sensitivity and Specificity; Respiratory disease; Enzyme; Transferases; Mycobacterial infection; Infection; Nucleotidyltransferases; Gene amplification; Tuberculosis; Mycobacterium tuberculosis; Investigation method; Mycobacteriales; Fluorescent labelling; Clinical biology; Restriction endonuclease; Bacteriosis; Mycobacteriaceae; Bacteria; Actinomycetes; Diagnosis; DNA-directed DNA polymerase
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1093/clinchem/42.10.1604

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.