Amplification-based nucleic acid scanning techniques to assess genetic polymorphism in Candida

Opportunistic pathogen Candida causes common fungal infections that manifest both superficially and systemically, especially in compromised patients. Although C. albicans is by far the main etiological agent of candidosis, the frequency of isolation of other non-albicans species such as C. glabrata and C. krusei is increasing at an alarming rate. Therefore, the epidemiology, pathogenicity, and diagnosis of infections due to these organisms are of great importance. Of a variety of genotyping methods utilized for strain delineation of these Candida species, amplification-based techniques such as randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), restriction digestion-mediated PCR (RFLP-PCR), and single-stranded conformational polymorphism (SSCP) and microsatellite PCR (interrepeat PCR, IR-PCR) are the most popular and widely used. In the last decade or so these techniques have helped unravel the clinical epidemiological features of pathogenicity, diversity, microevolution, and natural heterozygosity in Candida species. Here we review in detail the basic principles of RAPD, the nature of the primer and factors influencing its selection, and the limitations of RAPD assays as well as analysis and interpretation of banding profiles generated using the software programs. In addition, the principles of other RAPD-based amplification techniques (AFLP, RFLP-PCR, SSCP, and IR-PCR) and their application in molecular epidemiologic studies of Candida species in particular and other fungi in general are also reviewed. It is concluded that these methods have wide applicability in genotyping fungi, although they differ greatly in their resolution and have advantages and drawbacks depending on the task in question.