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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 (R2=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 × 109 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 × 108 and 2.7 × 108 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. [Copyright &y& Elsevier]

To determine the extent of interference of co-extracted DNA contamination in the quantification of the tod gene transcript, two different concentrations of RNA (high, 500 ng/μl; low, 250 ng/μl) from a toluene-induced culture of Pseudomonas putida were treated with different amounts of DNase (2, 4, 6 and 8 U) and incubated for 30 and 60 min. The highly sensitive and reproducible TaqMan system was used to quantify the transcript of the tod gene, the tod gene in contaminating DNA and the 16S rRNA gene in DNase-treated RNA samples. For the high RNA concentration, the shorter incubation time (30 min) lowered the level of contaminating DNA as evidenced by the presence of 2.5 × 106 copies of the tod gene before treatment to 1.4 × 105 copies/μl (8 U), whereas, irrespective of the DNase units used, the longer incubation time (60 min) considerably lowered the level of DNA contamination (2.5 × 106 to 6.5 × 102 copies of the tod gene/μl). However, for the low RNA concentration, DNase treatment was found to be equally effective in lowering the level of contaminating DNA (106 to 102 copies of the tod gene/μ), irrespective of the incubation time and the amount of DNase used. Although the results of gel electrophoresis of conventional PCR amplification of the low RNA concentration revealed the absence of the target gene in contaminating DNA, the results of the TaqMan PCR indicated that a very low amount of contaminating DNA (less than 103 copies of the tod gene/μl) was still present in RNA samples, even after the DNase treatment. The number of copies of the tod gene transcript in RNA samples did not show any marked variation because of the DNase treatment. However, the proportion of contaminating DNA in RNA samples considerably decreased due to the treatment (0.01 to 0.000001). Furthermore, these results suggested that the extent of the removal of contaminating DNA from RNA samples depends on the concentration of RNA, the amount of DNase used and the incubation time. It is also suggested that the copies of the catabolic genes in contaminating DNA have to be quantified along with the target genes in RNA samples to have a more accurate quantification of the target genes for better understanding of their roles in many microbial processes. [Copyright &y& Elsevier]