[ 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

Optimization of reactive blue 19 decolorization by ganoderma SP. using response surface methodology

Synthetic dyes are extensively used in different industries. Dyes have adverse impacts such as visual effects, chemical oxygen demand, toxicity, mutagenicity and carcinogenicity characteristics. White rot fungi, due to extracellular enzyme system, are capable to degrade dyes and various xenobiotics. The aim of this study was to optimize decolorization of reactive blue 19 [RB19] dye using Ganoderma sp. fungus. Response Surface Methodology [RSM] was used to study the effect of independent variables, namely glycerol concentration [15, 20 and 25 g/L], temperature [27, 30 and 33 °C] and pH [5.5, 6.0 and 6.5] on color removal efficiency in aqueous solution. From RSM-generated model, the optimum conditions for RB19 decolorization were identified to be at temperature of 27°C, glycerol concentration of 19.14 mg/L and pH=6.3. At the optimum conditions, predicted decolorization was 95.3 percent. The confirmatory experiments were conducted and confirmed the results by 94.89% color removal. Thus, this statistical approach enabled to improve reactive blue 19 decolorization process by Ganoderma sp. up to 1.27 times higher than non-optimized conditions