[ overview on DNA barcoding: a new horizon in fungal molecular diagnostics]

Total number of fungal species has been estimated over 1.5 million, of which less than 10 percent have formally been described. Advances made in molecular biology, development of gene and genome sequence technologies, discovery and description of new species among different clusters of life, especially fungi, has been accelerated. With respect to the failure of morphological-based species recognition methods for fungi identification, use and application of DNA based identification methods have assumed great importance for rapid and accurate identification of fungal species. DNA barcoding is of central importance among the new approaches for the identification of fungi and pseudo fungi and has been used during the last 10 years. For fungi, the internal transcribed spacer region of ribosomal DNA [ITS-rDNA] has become an appropriate barcode. Efficacy of ITS-rDNA sequence data in successful identification of fungal species has resulted in general agreement among mycologists to use ITS-rDNA region as a gold standard barcode for fungi. However, it seems that ITS-rDNA region will be used as the primary barcode for identification of fungal species and accurate identification will further be performed by use of sequence data from the other genomic loci as secondary barcodes. Considering the pleomorphic nature of the fungi, use of DNA barcoding has assumed higher importance. Hundreds of thousands of reference barcodes have been generated for a great number of species by DNA barcoding projects. Scientists now are facing new challenges for barcode data management, facilitation and acceleration in species identification process using DNA barcodes by the end-users through automation of DNA barcoding system

[ use of rolling circle amplification for molecular identification of cladophialophora carrionii and cladophialophora yegresii]

Epidemiological studies indicate that not only the incidence of fungal infections is dramatically on the rise, especially in the immunocompromised hosts, but also the sensitivity of etiological agents to antifungal drugs shows a remarkable reduction. Therefore, early detection at the species level is critically important for proper clinical management. Because standard diagnostic procedures are time consuming, expensive, and less sensitive, PCR-based molecular techniques have been developed. In the present study, we aim to describe a rapid and sensitive technique based on the rolling circle PCR amplification for accurate and fast identification of Cladophialophora carrionii vs. C. yegresii. Specific padlock probes were designed based on a single nucleotide polymorphism [SNP] difference in the internal transcribed spacer [ITS] rDNA region of Cladophialophora strains to differentiate between C. carrionii and C. yegresii. The probe sequences are complementary to the target DNA leading to the linker position that after hybridization with the target DNA will be joined together by DNA ligase, form a closed molecule and hybridize to the target DNA for replication at single-temperature conditions. We successfully amplified the target fungi DNA at the species level without any false and negative cross reactivity. The RCA product was visualized on 1.5% agarose gel to clarify the specificity of the probe-DNA template binding. These results demonstrate that RCA is a powerful and accurate tool for discrimination and identification of pathogenic fungi