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Identification of medically important pathogenic fungi by reference strand-mediated conformational analysis (RSCA)
Brian P McILHATTON1, Caitriona Keating1, Martin D Curran1
1*Northern Ireland Regional Histocompatibility and Immunogenetics Laboratory, City Hospital, Belfast, †School of Medicine and ‡School of Biology and Biochemistry, Queen's University of Belfast, Belfast, §Department of Haematology, Royal Victoria Hospital, Belfast, ∥Department of Haematology, Queen's University of Belfast, Belfast, **Department of Bacteriology and Mycology, Royal Victoria Hospital, Belfast, Northern Ireland, ††Anthony Nolan Research Institute, Royal Free Hospital, Hampstead, London and ‡‡School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland.
Abstract:
This report describes the application of reference strand-mediated conformational analysis (RSCA), a novel DNA typing technique, for the identification of clinically significant fungal pathogens. RSCA is a heteroduplex-based conformational method which relies on detecting differences in the DNA conformation of heteroduplexes generated in this study by the annealing of different fungal 18S rRNA amplicons to a common fluorescent-labelled reference (FLR). These heteroduplexes are then observed with laser-based instrumentation and computer software to detect differences in the DNA conformation reproducibly. This technique was shown to generate unique and reproducible profiles for the 18S rRNA gene sequences of a number of medically important fungi, distinguishing different Candida species (C. albicans, C. kefyr, C. dubliniensis, C. lusitaniae, C. guilliermondii, C. tropicalis, C. krusei, C. glabrata, C. sake and C. parapsilosis), and in some cases detecting single nucleotide differences between 18S rRNA sequences. The RSCA technique was further evaluated with 50 human clinical isolates of Candida spp., previously identified by culture techniques, and was shown to identify the isolates correctly. This technique displays enormous potential as an alternative to DNA sequence determination and has the potential to become an automated technique that can be implemented in the routine setting.