Ligation amplification and fluorescence detection of Mycobacterium tuberculosis DNA

Abstract

Current methods for the identification of Mycobacterium tuberculosis are dependent upon culture of the bacteria and are necessarily lengthy due to the slow growth of this agent. The development of DNA probe technology offers rapid, accurate and cost effective alternatives for the identification of such fastidious organisms. A technique for detecting specific DNA sequences, known as oligonucleotide ligation assay (OLA) involves the ligation of two adjacent oligonucleotides annealed to target DNA, and has been previously described. Amplification of the target sequences can be accomplished by including complementary pairs of oligonucleotides and a thermal stable ligase in a reaction which cycles between annealing/ligation and denaturing temperatures. Using a cloned portion of an insertion sequence, IS6110, which has been reported to be specific for M. tuberculosis complex as target DNA, we demonstrate the ligation dependent amplification of a 40 base pair region of plasmid bearing IS6110. By employing oligonucleotides which are each labelled with a different fluorescent dye, the reaction can be followed by fluorescence detection on an Applied Biosystems model 373A DNA sequencer. Using this approach, we have optimized conditions for the detection of 100 target molecules in a mixture containing 4 micrograms of unrelated DNA. Since the insertion sequence is repeated on average 12-14 times in the genome of M. tuberculosis, this corresponds to a theoretical detection level of 7-8 organisms. Completion of this entire assay can be accomplished in less than 8 h and serves as a basis for further studies in the development of a rapid clinical diagnostic test for tuberculosis.