Experimental design for the optimization of propidium monoazide treatment to quantify viable and non-viable bacteria in piggery effluents

BackgroundDistinguishing between viable and dead bacteria in animal and urban effluents is a major challenge. Among existing methods, propidium monoazide (PMA)-qPCR is a promising way to quantify viable cells. However, its efficiency depends on the composition of the effluent, particularly on total suspended solids (TSS)) and on methodological parameters. The aim of this study was evaluate the influence of three methodological factors (concentration of PMA, incubation time and photoactivation time) on the efficiency of PMA-qPCR to quantify viable and dead cells of Listeria monocytogenes used as a microorganism model, in two piggery effluents (manure and lagoon effluent containing 20 and 0.4 TSS g.kg−1, respectively). An experimental design strategy (Doehlert design and desirability function) was used to identify the experimental conditions to achieve optimal PMA-qPCR results.ResultsThe quantification of viable cells of L. monocytogenes was mainly influenced by the concentration of PMA in the manure and by the duration of photoactivation in the lagoon effluent. Optimal values differed with the matrix: 55 μM PMA, 5 min incubation and 56 min photoactivation for manure and 20 μM PMA, 20 min incubation and 30 min photoactivation for lagoon effluent. Applied to five manure and four lagoon samples, these conditions resulted in satisfactory quantification of viable and dead cells.ConclusionPMA-qPCR can be used on undiluted turbid effluent with high levels of TSS, provided preliminary tests are performed to identify the optimal conditions.

• Publication year

  2015

• Venue

  BMC Microbiology

• Publication date

  2015-08-16

• Fields of study

  Biology, Medicine, Environmental Science

• Identifiers
  DOI 10.1186/s12866-015-0505-6PMID 26276157PMCID 4537567
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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