Generation of DNA probes for detection of microorganisms by polymerase chain reaction fingerprinting

Abstract

Identification of medically relevant microorganisms is important for diagnosis, treatment and prevention of infectious diseases. This has initiated the development of a large number of identification and typing techniques based on phenotypic and genetic characteristics. In general, these last mentioned nucleic acid-mediated techniques provide more detailed and consistent information on strain-specific characteristics. However, the development of clinically useful microbial DNA/RNA probes requires nucleotide sequence information and a set of well defined reference organisms for test validation in comparison with the current gold standard. This is a requirement for the development of accurate nucleic acid hybridisation and/or amplification tests. Recently, it has been demonstrated that polymerase chain reaction (PCR)-mediated genetic typing of microorganisms can lead to the immediate isolation of species-specific DNA probes by comparison of DNA fingerprints. This combines the sensitivity of PCR with the specificity of DNA probing without the need to generate nucleic acid sequence information prior to probe development. The implications of this procedure for clinical microbiology and epidemiological surveillance will be discussed. It is shown that specific probes can be developed for various taxonomic levels and that detection and identification can be combined into a single, fast procedure. The versatility and widely applicable principles of this procedure will be highlighted and exemplified by some newly developed tests and a review of the current literature.