Identification of medically important pathogenic fungi by reference strand-mediated conformational analysis (RSCA)

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.

Use of Amplified Fragment Length Polymorphism Analysis To Identify Medically Important Candida spp., Including C . dubliniensis

“…Two other universally applicable methods for the identification of Candida species have been described: PCR fingerprinting and reference strand-mediated conformational analysis (20,21). However, whereas PCR fingerprinting uses mini-and microsatellite sequences as targets for the primers and reference strand-mediated conformational analysis is based on 18S rRNA sequences, AFLP patterns are a representation of the whole genome.…”

Multiplex PCR Amplimer Conformation Analysis for Rapid Detection of gyrA Mutations in Fluoroquinolone-Resistant Mycobacterium tuberculosis Clinical Isolates

“…The principle of this method is largely based on that of the traditional single-stranded conformation polymorphism technique (27) and a newly reported technique, reference strand-mediated conformation analysis (18). The involvement of multiple or multiplex PCR products in the analysis allowed much improved sensitivity and hence reliability in detecting mutations.…”

Use of PCR-DGGE Based Molecular Methods to Analyse Microbial Community Diversity and Stability during the Thermophilic Stages of an ATAD Wastewater Sludge Treatment Process as an Aid to Performance Monitoring

“…Advances in molecular tools can allow unprecedented opportunities for detailed studies of species population structure and dynamics within biotechnological processes [ 10 – 12 ]. Determination of microbial abundance has often been a key focus accomplished by direct DNA extraction followed by PCR amplification of genera or domain-specific genetic biomarkers [ 13 – 16 ] and recovery of 16S rDNA sequences of Bacteria and Achaea and 18S rDNA of active fungi and eukaryotic organisms [ 17 ], while internal transcribed spacer (ITS) sequences within ribosomal operons are also informative [ 18 ]. Molecular fingerprints of diversity can be achieved by coupling PCR amplification of taxonomical targets with sequence dissimilarities analyzed by denaturing gradient gel electrophoresis (DGGE) enabling the separation of DNA fragments of identical length but of different sequence [ 19 , 20 ], resulting in a DGGE fingerprint that is specific for the sample site.…”