title: The use of propidium monoazide in conjunction with qPCR and Illumina sequencing to identify and quantify live yeasts and bacteria.
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.
authors.affiliation: The University of British Columbia, Okanagan, Biology and Physical Geography Unit, 3333 University Way, Kelowna, BC VIV 1V7, Canada. Electronic address: mansak.ca@hotmail.com.
authors.forename: Mansak
authors.initials: M
authors.lastname: Tantikachornkiat
authors.affiliation: Okanagan College, Department of Biology, 1000 KLO Rd, Kelowna, BC V1Y 4X8, Canada.
authors.forename: Stacey
authors.initials: S
authors.lastname: Sakakibara
authors.affiliation: The University of British Columbia, Okanagan, Biology and Physical Geography Unit, 3333 University Way, Kelowna, BC VIV 1V7, Canada.
authors.forename: Marissa
authors.initials: M
authors.lastname: Neuner
authors.affiliation: The University of British Columbia, Okanagan, Biology and Physical Geography Unit, 3333 University Way, Kelowna, BC VIV 1V7, Canada.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Durall
mesh_terms: D001386:Azides / Q000494:pharmacology*; D001419:Bacteria / Q000145:classification* / Q000235:genetics; D015373:Bacterial Typing Techniques / Q000379:methods*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000187:drug effects / Q000235:genetics*; D004271:DNA, Fungal / Q000187:drug effects / Q000235:genetics*; D005658:Fungi / Q000145:classification* / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D050296:Microbial Viability; D016533:Mycological Typing Techniques / Q000379:methods*; D011419:Propidium / Q000031:analogs & derivatives* / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA
chemical_list: D001386:Azides; D004269:DNA, Bacterial; D004271:DNA, Fungal; C533957:propidium monoazide; D011419:Propidium
keywords: Culture-independent; Microbial populations; Miseq; Propidium monoazide; Propidium monoazide (PubChem CID: 3035529); Viable cells; qPCR