Live / Dead discrimination of bacteria via Dnase/Proteinase treatment
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chair:
Varela Villarreal, J. / Jungfer, C. / Ursula, O. / Schwartz, T. (2011)
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place:
How Dead is Dead 2011, Tübingen, Deutschland, 16.-17. Juni, 2011
- Date: 2011
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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
Abstract
DNA-based molecular biology techniques are very sensitive, but have some limitations to discriminate DNA coming from live, injured and dead cells as well as extracellular DNA (eDNA) in natural and technical systems. Regarding this dilemma, DNase I combined with proteinase K treatment was used for a better discrimination between live and dead bacteria.
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.
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.