The use of propidium monoazide in conjunction with qPCR and Illumina sequencing to identify and quantify live yeasts and bacteria

Int J Food Microbiol. 2016 Oct 3;234:53-59. doi: 10.1016/j.ijfoodmicro.2016.06.031. Epub 2016 Jun 24.

Abstract

Culture-independent methods of microbial identification have been developed, which allow for DNA extraction directly from environmental samples without subjecting microbes to growth on nutrient media. These methods often involve next generation DNA sequencing (NGS) for identifying microbes and qPCR for quantifying them. Despite the benefits of extracting all DNA from the sample, results may be compromised by amplifying DNA from dead cells. To address this short-coming, the use of propidium monoazide (PMA) has been used to deactivate DNA in non-viable cells. Nevertheless, its optimization has not been fully explored under a variety of conditions. In this study, we optimized the PMA method for both yeasts and bacteria. Specifically, we explored the effect different PMA concentrations and different cell densities had on DNA amplification (as part of next generation DNA sequencing) from both dead and viable bacterial and yeast cells. We found PMA was effective in eliminating DNA that was associated with dead yeast and bacterial cells for all cell concentrations. Nevertheless, DNA (extracted from viable yeast and bacterial cells) amplified most abundantly when PMA concentration was at 6μM and when yeast densities ranged between 10(6) to 10(7)CFU/mL and bacterial densities were approximately 10(8)CFU/mL.

Keywords: Culture-independent; Microbial populations; Miseq; Propidium monoazide; Propidium monoazide (PubChem CID: 3035529); Viable cells; qPCR.

MeSH terms

  • Azides / pharmacology*
  • Bacteria / classification*
  • Bacteria / genetics
  • Bacterial Typing Techniques / methods*
  • Base Sequence
  • DNA, Bacterial / drug effects
  • DNA, Bacterial / genetics*
  • DNA, Fungal / drug effects
  • DNA, Fungal / genetics*
  • Fungi / classification*
  • Fungi / genetics
  • High-Throughput Nucleotide Sequencing / methods
  • Microbial Viability*
  • Mycological Typing Techniques / methods*
  • Propidium / analogs & derivatives*
  • Propidium / pharmacology
  • Real-Time Polymerase Chain Reaction / methods
  • Sequence Analysis, DNA

Substances

  • Azides
  • DNA, Bacterial
  • DNA, Fungal
  • propidium monoazide
  • Propidium