Abstract
Three molecular techniques random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and restriction fragment length polymorphism (RFLP) were employed for identification and to study the genetic relationship among six species of dermatophytes and three species of yeasts isolated from Egyptian and Libyan patients with skin mycosis. Each species was represented by two isolates, one from Egyptian patients and the second from Libyan. RAPD in which four random 10-mer primers and two ISSR primers were used to amplify the DNA fragments of target fungi and RFLP in which two universal primers (ITS1 and ITS4) were used to amplify the internal transcribed spacer (ITS) regions of the ribosomal (rRNA) gene in fungal isolates followed by digestion with HinfI and HaeIII endonucleases was carried out. Three molecular marker techniques showed considerable potential for identifying and discriminating dermatophytes and Candida species and the achieved results confirmed identification based on conventional morphological methods. Results of RAPD and ISSR markers revealed 78.7% genetic similarity (GS) betweenMicrosporum canis and other tested fungi reflecting a relatively longer genetic distance from other isolates of dermatophytes and yeasts. Candida krusei andCandida tropicalis were closely related showing 93.3% GS. C. albicans showed 90.9% similarity with other species of Candida. Epidermophyton floccosum was easily separated from all Trichophyton species showing 87.3% similarity. Unique bands were displayed by certain fungi and can be taken as a positive marker for isolate identification and discrimination. RFLP technique revealed differences in the number (1 to 5) and size (8 to 378 base pairs) of DNA fragments depending on the fungal isolate and restriction enzyme used. Within each fungal species, different isolates of dermatophytes and Candida from Egypt and Libya showed close relationship. Seven isozyme systems namely esterase, peroxidase, malate dehydrogenase, acid phosphatase, glutamate-oxalo-acetate transaminase, Urease and protease were studied to detect the gene expression and genetic variability among the different isolates of dermatophytes and Candida. Key words: Random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), restriction fragment length polymorphism (RFLP), dermatophytes,Candida, isozymes.
Highlights
Conventional methods for identification of fungi rely on macro and micro morphological features of the colonies on general and specific culture media and on some biochemical and physiological complementary test
Phenotypic characteristic and can make the identification more difficult (Weitzman and Summerbell, 1995; Faggi et al, 2001; Kanbe, 2008; De Baere et al, 2010). Molecular marker approaches, such as nested-polymerase chain reaction (PCR) (Verrier et al, 2013), random amplified polymorphic DNA (RAPD)-PCR (Kim et al, 2001; Baeza et al, 2006; Leibner-Ciszak et al, 2010; Dobrowolska et al, 2011; Spesso et al, 2013), inter-simple sequence repeat (ISSR)-PCR (Cano et al, 2005; Khosrav et al, 2012), PCR- restriction fragment length polymorphism (RFLP) (Yang et al, 2008; RezaeiMatehkolaei et al, 2012; Samuel et al, 2013), real time PCR (Bergmans et al, 2010) and multiplex PCR assay (Arabatzis et al, 2007; Kim et al, 2011) and others have been adapted for detection of dermatophytes from clinical specimens
The number of bands per one primer ranged from 12 bands in the case of OPA06 RAPD primer to eight bands with 844A ISSR primer
Summary
Conventional methods for identification of fungi rely on macro and micro morphological features of the colonies on general and specific culture media and on some biochemical and physiological complementary test. Outside factors such as temperature variation, medium and chemotherapy, can greatly influence the. The RAPD approach which used to detect strain polymorphism depends on the application of short arbitrary primers that anneal to multiple genomic sites at appropriate temperature conditions and this technique does not depend on prior knowledge of species-specific sequence (Valério et al, 2006). Spesso et al (2013) used random amplified polymorphic DNA primers to recognise Microsporum canis, Microsporum gypseum, Trichophyton rubrum and Trichophyton interdigitale. For identification of Trichophyton mentagrophytes and Trichophyton tonsurans, Kim et al (1999 and 2001) proposed a RAPD method, in which T. mentagrophytes was divided into subtypes on the basis of amplification patterns in RAPD using three primers
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