| Autor: |
Schurig S; Institute of Animal Hygiene and Veterinary Public Health, Leipzig University, 04103 Leipzig, Germany.; Xpedite Diagnostics GmbH, 80687 Munich, Germany., Kobialka R; Institute of Animal Hygiene and Veterinary Public Health, Leipzig University, 04103 Leipzig, Germany., Wende A; Xpedite Diagnostics GmbH, 80687 Munich, Germany., Ashfaq Khan MA; Institute of Animal Hygiene and Veterinary Public Health, Leipzig University, 04103 Leipzig, Germany., Lübcke P; Institute of Pharmacy, University of Greifswald, 17489 Greifswald, Germany., Eger E; Institute of Infection Medicine, Christian-Albrecht University Kiel, 24105 Kiel, Germany.; University Medical Center Schleswig-Holstein, 24105 Kiel, Germany., Schaufler K; Institute of Pharmacy, University of Greifswald, 17489 Greifswald, Germany.; Institute of Infection Medicine, Christian-Albrecht University Kiel, 24105 Kiel, Germany.; University Medical Center Schleswig-Holstein, 24105 Kiel, Germany., Daugschies A; Institute of Parasitology, Centre for Infectious Disease, Leipzig University, 04103 Leipzig, Germany., Truyen U; Institute of Animal Hygiene and Veterinary Public Health, Leipzig University, 04103 Leipzig, Germany., Abd El Wahed A; Institute of Animal Hygiene and Veterinary Public Health, Leipzig University, 04103 Leipzig, Germany. |
| Abstrakt: |
Wastewater monitoring became a promising solution in the early detection of outbreaks. Despite the achievements in the identification of pathogens in wastewater using real-time PCR, there is still a lack of reliable rapid nucleic acid extraction protocols. Therefore, in this study, samples were subjected to alkali, proteinase K and/or bead-beating followed by reverse purification magnetic beads-based separation. Wastewater samples spiked with S. aureus , E. coli and C. parvum were used as examples for Gram-positive and -negative bacteria and protozoa, respectively. All results were compared with a spin column technology as a reference method. Proteinase K with bead beating (vortexing with 0.1 mm glass beads for three minutes) was particularly successful for bacterial DNA extraction (three- to five-fold increase). The most useful extraction protocol for protozoa was pre-treatment with proteinase K (eight-fold increase). The selected methods were sensitive as far as detecting one bacterial cell per reaction for S. aureus , ten bacterial cells for E. coli and two oocysts for C. parvum . The extraction reagents are cold chain independent and no centrifuge or other large laboratory equipment is required to perform DNA extraction. A controlled validation trial is needed to test the effectiveness at field levels. |
