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 IS 6110 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 probed. 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 IS 6110 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.