Quantitative Measurement of Fungal DNA Extracted by Three Different Methods Using Real-Time Polymerase Chain Reaction

Abstract

Quantification of fungal populations in the environment is important for gaining a better understanding of various microbial processes. Recently, the development of real-time quantitative PCR (RTQ-PCR) has eliminated the variability associated with conventional quantitative PCR, thereby allowing the routine and reliable quantification of PCR products. Thus, in this present study, RTQ-PCR was used to quantify the fungal target DNA extracted by three commonly used DNA extraction protocols (bead mill homogenization, grinding in the presence of liquid nitrogen, and hot detergent SDS based enzymatic lysis combined with bead beating) to determine the suitability of the quantification of target DNA. For the purpose of this study, pure culture of Aspergillus fumigatus (model organism), sterilized soil seeded with a known amount of A. fumigatus (model soil system), and woodland and grassland soil samples (environmental samples) were chosen to extract DNA by the above three different protocols. The extracted DNA was then quantified by spectroscopy and a RTQ-PCR system. 18S rDNA specific universal fungal primers were used to quantify the target part and then amplification products were verified by agarose gel electrophoresis. Standard curves used for the quantification by RTQ-PCR revealed strong linear relationships ( R 2 =0.9994 for the primer pair NS1 and NS2 and 0.9938 for the primer pair nu-SSU-0817 and nu-SSU-1196) with a higher amplification efficiency, e =0.983 for the primer pair NS1 and NS2 and 0.956 for the primer pair nu-SSU-0817 and nu-SSU-1196. Although for pure culture the hot detergent SDS based enzymatic lysis combined with bead beating method showed the highest target DNA copy number (1.5 × 10 9 copies/μl), for the model soil system and both environmental samples the bead beating method was found to be suitable on the basis of the high target DNA copy numbers (6.16 × 10 8 and 2.7 × 10 8 copies/μl for woodland and grassland, respectively), high DNA yield (6.4 μg/g and 1.8 μg/g of soil for woodland and grassland, respectively), and high recovery on the basis of the target DNA copy number (39.2%), suggesting an overall high extraction efficiency.