Molecular mycology: DNA probes and applications of PCR technology.

Abstract

Future clinical mycology laboratories will increasingly utilize DNA-based methods for the recognition of pathogenic fungi in patient specimens and for the identification of fungal isolates. Although the reagents and protocols vary greatly, the polymerase chain reaction (PCR) and oligonucleotide probes are central to these procedures. This article will review concepts and strategies for the design of DNA probes and primers for the PCR, practical considerations for the implemention of DNA-based laboratory procedures, the application of DNA methods to population studies and epidemiology, and current identification of fungi in clinical material and in culture. Molecular evolutionary basis of DNA primer and probe design PCR, with or without subsequent oligonucleotide probe detection, can be used to detect and identify a group of target organisms to the exclusion of all other species [26]. The objective is to design PCR primers, which are usually oligonucleotides about 20-25 bases in length, that exactly complement DNA sequences shared by all members of the target group (the target group can consist of one or many species), but which contain at least one base mismatch to the species that must be excluded from detection. The greater the number of mismatches to the non-target organisms, the easier it is to discriminate them from the target group. These considerations are the basis for the design of both oligonucleotide probes and amplification primers. Any molecular diagnostic system should be designed to address clinically relevant questions to avoid spending effort on making fine but unnecessary distinctions. Understanding the kinds of questions that DNA-based systems can resolve will assist the physician and clinical laboratorian in discussions with the molecular researcher. The design of PCR primers and probes is fundamentally dependent on evolution at the DNA level. In general, the more time that has elapsed since two species shared a common ancestor, the more different they will be at the DNA level and the easier it will be to find a primer binding site that can distinguish between them. If species are close relatives (i.e. recently shared a common ancestor), it may be more difficult to detect one of them while excluding the other. A group of organisms that share a common ancestor will share mutations that arose during the descent of that common ancestor, assuming that at least some of those mutations have remained unchanged while the group diversified into separate species. The regions of these mutations, which distinguish the