Optimization of DNA Extraction and PCR Conditions for Fungal Metagenome Analysis of Atmospheric Particulate Matter

Abstract

Several challenges arise in DNA extraction and gene amplification for airborne fungal metagenome analysis from a particulate matter (PM) samples. In this study, various conditions were tested to optimize the DNA extraction method from PM samples and polymerase chain reaction (PCR) conditions with primer set and annealing temperature. As a result of comparative evaluation of DNA extraction under various conditions, chemical cell lysis using buffer and proteinase K for 20 minutes and bead beating treatment were followed by using a commercial DNA extraction kit to efficiently extract DNA from the PM filter samples. To optimize the PCR conditions, PCR was performed using 10 primer sets for amplifying the ITS2 gene region. The concentration of the PCR amplicon was relatively high when the annealing temperature was 58°C with the ITS3tagmix3/ITS4 primer set. Even under these conditions, when the concentration of the PCR product was low, nested PCR was performed using the primary PCR amplicon as the template DNA to amplify the ITS2 gene at a satisfactory concentration. Using the methods optimized in this study, DNA extraction and PCR were performed on 15 filter samples that collected PM2.5 in Seoul, and the ITS2 gene was successfully amplified in all samples. The optimized methods can be used for research on analyzing and interpreting the fungal metagenome of atmospheric PM samples. © 2023, The Korean Society for Microbiology and Biotechnology.