Western Kentucky University
Development of Rapid Quantitative Real-Time PCR Method to Detect Total Environmental Fungi
Institution
Western Kentucky University
Faculty Advisor/ Mentor
Shivendra Sahi
Abstract
Fungi are nearly ubiquitous in the environment. There are currently accepted methods to quantify fungi from environmental samples; however, most methods are based on culturing fungal organisms, they are time consuming, and not representative of total fungal abundance. The downfall of such methods is failure to represent species that are difficult to culture or species that have specialized growth requirements. These pitfalls can be avoided by utilizing polymerase chain reaction (PCR) to detect environmental fungi. More advantageous than standard PCR is quantitative real-time PCR (qRT-PCR), which allows measurement of initial DNA concentration. For fungal detection, previously described fungal specific primers for 18s rDNA were compared and a primer pair that previously demonstrated a high degree of fungal specificity was chosen. Primer annealing temperatures were optimized using a temperature gradient qRT-PCR experiment; the optimal annealing temperature was confirmed through analysis of PCR product melting curves and polyacrylamide gel electrophoresis. Fungal primers were used in qRT-PCR reactions with genomic DNA extracts from several different cultured fungi species, genomic bacterial DNA, genomic mammalian DNA, genomic invertebrate DNA, and genomic plant DNA in order to test primer specificity. To prove PCR products were generated from the intended targets, successful PCR reactions were sequenced and confirmed to be the suspected fungal targets. Saccharomyces cerevisiae genomic DNA was chosen as the qRT-PCR DNA concentration standard; the S. cerevisiae standards were used to quantify genomic DNA extracted from cultured fungi in order to prove the viability of this method in amplifying and quantifying a broad spectrum fungal species.
Development of Rapid Quantitative Real-Time PCR Method to Detect Total Environmental Fungi
Fungi are nearly ubiquitous in the environment. There are currently accepted methods to quantify fungi from environmental samples; however, most methods are based on culturing fungal organisms, they are time consuming, and not representative of total fungal abundance. The downfall of such methods is failure to represent species that are difficult to culture or species that have specialized growth requirements. These pitfalls can be avoided by utilizing polymerase chain reaction (PCR) to detect environmental fungi. More advantageous than standard PCR is quantitative real-time PCR (qRT-PCR), which allows measurement of initial DNA concentration. For fungal detection, previously described fungal specific primers for 18s rDNA were compared and a primer pair that previously demonstrated a high degree of fungal specificity was chosen. Primer annealing temperatures were optimized using a temperature gradient qRT-PCR experiment; the optimal annealing temperature was confirmed through analysis of PCR product melting curves and polyacrylamide gel electrophoresis. Fungal primers were used in qRT-PCR reactions with genomic DNA extracts from several different cultured fungi species, genomic bacterial DNA, genomic mammalian DNA, genomic invertebrate DNA, and genomic plant DNA in order to test primer specificity. To prove PCR products were generated from the intended targets, successful PCR reactions were sequenced and confirmed to be the suspected fungal targets. Saccharomyces cerevisiae genomic DNA was chosen as the qRT-PCR DNA concentration standard; the S. cerevisiae standards were used to quantify genomic DNA extracted from cultured fungi in order to prove the viability of this method in amplifying and quantifying a broad spectrum fungal species.