Abstract
The multicopy and specific insertion element IS6110 provide a good target for the detection of M. tuberculosis complex by amplification techniques such as Polymerase Chain Reaction (PCR). However, the emergence of IS6110-negative strains suggests that false negative results may occur if only IS6110 is used as the target for detection. In this report, a multiplex PCR system was established using primers derived from the insertion sequence IS6110 and a recently discovered IS-like element, designated as B9 (accession number 78639) which has been shown to be potentially useful for the detection of M tuberculosis including IS6110-negative strains (Musa, 1996). The multiplex PCR was then evaluated using adult and paediatric clinical samples. The results shows that the multiplex system is both sensitive (93.1% and 76% in adult and paediatric cases respectively) and specific (89.6% and 72.1% % in adult and paediatric cases respectively) and exceeds that of the conventional methods of microscopy and culture. The discovery of IS6110 single copy strains has triggered many questions regarding transposability of the IS element in these strains, their relationship to BCG and their epidemiological significance. The lack of transposability in these strains and in BCG are hypothesised to be due to mutation or mutations within this element particularly in the region designated as ORFa which is thought to regulate the expression of the transposase protein. In order to examine this hypothesis, the element in 8 single copy strains, chosen at random from clinical isolates were characterised. The sequence results indicated that the insertion sequence of the single copy strains of M. tuberculosis were identical to ISP57 which was isolated from a single copy BCG strain. However data of a recently transposed element and a resequence of the original 1S986 cloned copy showed that all the sequences were identical (B. Plikaytis and J. Dale, personal communications) and the differences between the published sequences of IS986 and 1S987 were due to sequencing errors. This implies that the mobility of the IS elements is dependent on the transcriptional activity of the flanking chromosomal region and not within the IS itself further research in the evolutionary relationship of the strains and variants is needed to understand the transposability of IS6110 in these strains.