Zucol, F., Ammann, R.A., Berger, C., Aebi, C., Altwegg, M., Niggli, F.K., and Nadal, D.
Notes: A panel of 11 Gram-negative and 11 Gram-positive bacterial species was used to develop a real-time PCR detection method. Initially DNA was purified from 1ml of various dilutions of bacteria resuspended in a saline solution using either the QIAamp DNA blood mini kit or the Wizard® SV Genomic DNA Purification System. The results suggested that the Wizard® SV Genomic DNA Purification System extraction protocol was superior in disrupting the bacterial cell wall (especially of Gram-positive bacteria), to allow release of bacterial DNA. Using DNA purified with the Wizard® SV Genomic DNA Purification System, detection of S. aureus and E. coli at concentrations as low as 101 CFU per PCR was achieved. The Wizard® SV Genomic DNA Purification System purification protocol provided in eNotes online (www.promega.com/enotes/applications/ap0051_tabs.htm) was used with the following modifications: The bacterial pellet was resuspended in 400µl of enzymatic lysis solution (47mM EDTA, 25mg/ml lysozyme, 20µg/ml lysostaphin) and incubated for 2 hours at 37°C. Next, 19.2mg/ml proteinase K was added (final concentration 0.4mg/ml), and the mixture was incubated for 1 hour at 55°C. Finally, the Nuclei Lysis Solution and RNase Solution were added, mixed and incubated for 10 minutes at 80°C. For real-time PCR analysis, 2µl of genomic DNA was used. (3676)