Microorganisms, regardless of whether pathogenic or not, may cause enormous economic losses due to adverse effects on human and animal health, or by damaging the quality of the agricultural and food products. Based on...Microorganisms, regardless of whether pathogenic or not, may cause enormous economic losses due to adverse effects on human and animal health, or by damaging the quality of the agricultural and food products. Based on these effects, the development of prompt molecular methods and their involvement in the practical pathogen diagnostic diagnostics is more than actual. This paper is focused on the evaluation of easy-to-perform and highly budget-friendly, PCR-related DNA purification protocols for diagnostic purposes especially in water or similar simple matrices. The slight modifications of earlier described DNA isolation methods, which rely on chelate exchange resin and/or ethanol-sodium-based heat lysis, we reevaluated in comparison with a widely used commercial kit. The efficiency of DNA purification techniques was assessed from Gramnegative as well as Gram-positive bacteria and yeast using quantitative PCR. The effectivity of different methods tested may vary depending on the bacterial or yeast species in question. Nevertheless, in our hands, the chelate exchange resin-based methods were found to be the most robust and/or satisfying at least by an acceptable reproducibility rate. Our presented results support the potential of low-cost but still sensitive molecular microbe detection procedures consisting of only a few pipetting steps resulting in good reproducibility and the least possible environmental burden, serving as a good starting point for developments of matrix-specific processes and methods.展开更多
文摘Microorganisms, regardless of whether pathogenic or not, may cause enormous economic losses due to adverse effects on human and animal health, or by damaging the quality of the agricultural and food products. Based on these effects, the development of prompt molecular methods and their involvement in the practical pathogen diagnostic diagnostics is more than actual. This paper is focused on the evaluation of easy-to-perform and highly budget-friendly, PCR-related DNA purification protocols for diagnostic purposes especially in water or similar simple matrices. The slight modifications of earlier described DNA isolation methods, which rely on chelate exchange resin and/or ethanol-sodium-based heat lysis, we reevaluated in comparison with a widely used commercial kit. The efficiency of DNA purification techniques was assessed from Gramnegative as well as Gram-positive bacteria and yeast using quantitative PCR. The effectivity of different methods tested may vary depending on the bacterial or yeast species in question. Nevertheless, in our hands, the chelate exchange resin-based methods were found to be the most robust and/or satisfying at least by an acceptable reproducibility rate. Our presented results support the potential of low-cost but still sensitive molecular microbe detection procedures consisting of only a few pipetting steps resulting in good reproducibility and the least possible environmental burden, serving as a good starting point for developments of matrix-specific processes and methods.
