Live / Dead discrimination of bacteria via Dnase/Proteinase treatment
Varela Villarreal, J. / Jungfer, C. / Ursula, O. / Schwartz, T. (2011)
How Dead is Dead 2011, Tübingen, Deutschland, 16.-17. Juni, 2011
- Datum: 2011
Varela Villarreal, J. / Jungfer, C. / Ursula, O. / Schwartz, T. (2011): „Live / Dead discrimination of bacteria via Dnase/Proteinase treatment“. In: How Dead is Dead 2011, Tübingen, Deutschland, 16.-17. Juni, 2011
DNase I is an endonuclease that non-specifically cleaves single and double stranded DNA. This enzyme was tested in order to analyze its capacity of digesting eDNA and DNA coming from dead/injured cells with damaged cell membranes, leaving DNA from living and VBNC cells unaffected and available for DNA-based detection methods.
For this, an optimized DNase I/Proteinase K (DNase/PK) protocol was established using mixtures of living cells, dead cells and free genomic DNA. The efficiency of the DNase/PK method was compared with the already established propidium monoazide (PMA) technique. In both approaches RealTime PCR was used to assess the efficiencies of the DNase/PK and PMA application.
Additionally, the DNase/PK method was applied to natural drinking water biofilm samples from a German waterworks where ozone/ClO2 was used for disinfection. Shifts in the DNA patterns observed after PCR-DGGE analysis, demonstrated: (i) the applicability of PMA and DNase I treatment in natural biofilm investigation; (ii) detection of DNA from dead bacteria and eDNA was blocked by treatment with PMA or DNase I; and (iii) DNase/PK treatment demonstrated a discrimination of live and dead bacteria. Conventional cultivation methods, staining techniques and qPCR completed the biofilm analysis.