[Molecular methods in the diagnosis of infectious diseases]

In the long term, many of the conventional diagnostic approaches to detection and characterisation of infectious diseases is complemented or even replaced by recognition of DNA/RNA specific sequences. The molecular procedures are based on two distinct types of methods--nucleic acid hybridization techniques with specific molecular probes, and DNA amplification by the polymerase chain reaction PCR. In the first method DNA fragments generated by restriction endonucleases are separated by electrophoresis, transferred to proper membrane and annealed with specific oligonucleotides which are labelled with 32P or with a non-radioactive marker. PCR reaction utilizes a DNA extension enzyme (polymerase) which can add nucleotide bases to primers once a template is provided. Oligonucleotides primers have to recognize target molecule. The single cycle is then repeated and each time DNA segments are doubled. Multiplex polymerase chain reactions have been designed to identify an organism as well as its virulence-specific sequences or antibiotic resistance plasmids simultaneously. The major drawback of PCR procedure which result from its exquisite sensitivity have to be consider. The problem is contamination and many strategies have been developed to avoid it. To these research tools were added probes for in situ hybridization. Advantages provided by this technique include the ability to detect latent (non-replicating) viruses and to localize their genomes to nuclear or cytoplasmic regions within cells. Nucleic acid probes or the hybridization conditions can be manipulated so that a broad spectrum of genotypes could be detectable. This is particularly valuable in those emerging infections where the individual serotypes are unknown. Recombinant DNA approaches have now been described for detection of a wide range of infectious agents. Some remain research activities, others are more appropriate to the routine diagnostic laboratory. An understanding of a pathogen's life cycle and the host's responses to the infectious agent is enhanced by characterisation of the former's genome using molecular technology. The spread of epidemics or hospital-acquired (nonsocomial) infections is followed and characterised with more accuracy by the identification of unique DNA fingerprints for individual pathogens. The prospects for molecular medicine in microbiology are vast and will have profound effects in laboratory and clinical practice.