Chemiluminescent detection of strand displacement amplified DNA from species comprising the Mycobacterium tuberculosis complex.
Strand displacement amplification, a new isothermal in vitro DNA amplification technique, was used to amplify target DNA contained within the IS6110 insertion element of the species within the Mycobacterium complex (Mycobacterium tuberculosis, M. bovis, M. bovis-BCG, M. africanum and M. microti). The target nucleic acid sequence is present in approximately ten, two, one, five and five copies in M. tuberculosis, M. bovis, M. bovis-BCG, M. africanum and M. microti, respectively. Amplified products were detected using a non-isotopic microtitre plate assay employing a biotinylated oligodeoxynucleotide probe and an alkaline phosphatase conjugated oligodeoxynucleotide probe. Lumiphos 530 was the chemiluminescent substrate for alkaline phosphatase. The combination of the strand displacement amplification method with this sensitive and rapid (less than 2 h) detection system resulted in the specific detection of as few as 1-25 initial IS6110 targets in the five Mycobacterium complex species based on signal/noise criteria. Negative results were obtained with eight other Mycobacterium species as well as with 32 non-Mycobacterium species.
Spargo
C A
CA
Becton Dickinson Research Center, Research Triangle Park, NC 27709.
Haaland
P D
PD
Jurgensen
S R
SR
Shank
D D
DD
Walker
G T
GT
D001483:Base Sequence; D001710:Biotin; D004256:DNA Polymerase I; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D005189:False Positive Reactions; D005784:Gene Amplification; D006801:Humans; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000235:genetics* / Q000302:isolation & purification; D009163:Mycobacterium bovis / Q000235:genetics / Q000302:isolation & purification; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D012680:Sensitivity and Specificity; D014376:Tuberculosis / Q000175:diagnosis*
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D001710:Biotin; D004256:DNA Polymerase I