O15 Use of molecular identification techniques to study oral microbes and microcosms

Abstract

Over the last 100 years traditional culturing techniques have shaped our understanding of the oral microflora. However it is generally accepted that around 50% of the oral microbiota is unculturable. Recent advances in molecular biology have given rise to numerous techniques which form the basis of a number of strategies that can be used to detect and identify micro‐organisms to levels and specificities unattainable by conventional culture techniques. The molecular biology basis of these techniques can be conveniently split in to two broad groups. (1) DNA hybridization techniques: these are used for slot blot and checkerboard analysis and when combined with microscopy form a powerful visualization technique – fluorescent in situ hybridization (FISH). (2) PCR based approach: development of the PCR concept has lead to techniques such as global PCR, specific PCR, nested PCR, PCR‐cloning, reverse transcriptase PCR, real time PCR and PCR‐DGGE. The above techniques can usually be used on their own however they provide even more power when used in conjunction with each other. For example PCR‐cloning or PCR‐DGGE can be used to analyse a whole community. From this, un‐named or unculturable taxa can be ‘identified’ and targeted for further characterisation. This may involve the use of PCR specific for the taxon in question, to survey a large number of samples to obtain some prevalence data or in a real time setting to quantify the taxon. Indeed, a fluorescently labelled probe could be synthesized and used to visualize the taxon in an appropriate environmental sample or microcosm using confocal laser scanning microscopy. All the above techniques add up to a powerful set of tools which if selected appropriately have the potential to revolutionize our understanding of the microbial world.