A new, sensitive polynucleotide probe for distinguishingCandida albicansstrains and its use with a computer assisted archiving and pattern comparison system

Abstract

The repetitive DNA sequence poly[d(GT).d(CA)], (polyGT) can be used to generate DNA fingerprints that distinguish different yeast genera. In this study we demonstrate that the probe can also be used to distinguish individual strains of clinical isolates of Candida albicans. Isolates were fingerprinted by probing Southern blots of restriction enzyme-cleaved DNA samples with radioactively labelled polyGT. The discrimination between strains was clearer than can be achieved by direct visualization of ethidium bromide stained gels and was comparable to that achieved with previous DNA probes. However, the advantage of this probe is that it is not limited to Candida species since polyGT sequences appear to be ubiquitous in eukaryotes. Fingerprints were also generated from Southern blots using a commercial (AMBIS) radioanalytical imaging computer system. A proprietary software package (MICRO PM) was effective in discriminating between the C. albicans strains. These preliminary results indicate the potential value of this probe for discrimination between Candida isolates in epidemiological studies of candidosis.The increased incidence of systemic forms of candidosis in immunocompromised patients, and of mucosal and oesophageal candidosis in AIDS patients, has emphasized the need for a rapid typing system for epidemiological studies. DNA fingerprinting techniques are particularly attractive as the assessment of genotype generally involves less subjective judgement than the assessment of phenotype.Conserved repetitive sequences present in the genome of Candida species, such as the ribosomal RNA genes [5] and mitochondrial DNA [8] have already been shown to give rise to recognizably different patterns in ethidium bromide stained gels of restriction enzyme-cleaved DNA. For example, ethidium bromide stained gels of EcoRi digested DNA have been successfully used to follow outbreaks of systemic candidosis [6]; other studies have suggested that Hinfl can be an even more effective restriction enzyme for identifying Candida albicans strains by this type of restriction fragment length polymorphism (RFLP) analysis [12]. There are, however drawbacks to this approach; first, the patterns can be difficult to distinguish against the background smear of other genomic sequences and second, RFLPs detectable within all copies of a repeated sequence will reflect relatively ancient strain divergences where mutations have been fixed. When strains have diverged more recently, for example within hospi-

The Ins and Outs of DNA Fingerprinting the Infectious Fungi

“…A variety of other probes have been developed for C. albicans over the past several years that might be useful for DNA fingerprinting studies. Wilkinson et al (422) first used poly(G-T)asaSouthernblothybridizationprobeanddemonstrated variation among C. albicans isolates and differences between C. albicans and C. glabrata. Noting in 1993 that there were no good, accessible probes for Candida species other than for C. albicans, Sullivan et al tested five oligonucleotides, (GGAT)4, (GGTG)5, (GATA)4, (GACA)4 and (GT)8, on a variety of Candida species and selected (CT)8 as the most effective for strain discrimination based on pattern complexity and variability (374).…”

Molecular genetic approaches to identification, epidemiology and taxonomy of non-albicans Candida species

“…has been extended by the application of synthetic oligonucleotides composed of short repetitive sequence motifs as molecular fingerprinting probes [33]. All eukaryotic genomes contain multiple copies of short tandemly repeated microsatellite sequences which are excellent markers for the detection of genomic variation [7,[63][64][65][66]. By using short oligonucleotide probes complimentary to these sequences, including informative DNA fingerprint profiles consisting of multiple hybridisation bands-ranging in size from c.100 bp to c.20 kb for isolates of C. tropicalis, C. glabrata, C. krusei, C. dubliniensis and Cryptococcus neoformans-can be obtained [ 18, 33,67].…”

Application of molecular typing methods to dermatophyte species that cause skin and nail infections

“…Poly (dG-dT) has been used successfully to differentiate strains of C. albicans [23], C. glabrata [24] and Malassezia pachydermatis [ 121 and appeared to be an ideal probe for the epidemiology of the dermatophytes. Hybridisation to blots of Eco RIdigested DNA demonstrated minimal variation in 28 isolates of 7: mentagrophytes around 9 kb, and in 12 isolates of II: rubrum between 3 and 4 kb (Fig.…”