title: Effect of PCR amplicon length on suppressing signals from membrane-compromised cells by propidium monoazide treatment.
abstract: Treatment of microbiological samples with viability dyes prior to extraction of DNA and PCR amplification for downstream analysis has evolved into a commonly applied method. The addition of this easy-to-perform step to the sample analysis procedure inhibits the amplification of DNA from dead cells with compromised cell membranes. The method is currently used both in combination with quantitative PCR (qPCR), end-point PCR, and isothermal amplification. We present here a detailed study of the effect of amplicon size on amplification signals from unstressed and heat-exposed cells after treatment with propidium monoazide (PMA). PMA treatment was shown to be more efficient in excluding dead cells from the analysis both in combination with qPCR (PMA-qPCR) and denaturing gradient gel electrophoresis (PMA-DGGE), when longer amplicons were used. When applied to pure cultures of the fish pathogens Vibrio anguillarum and Flavobacterium psychrophilum exposed to a heat gradient ranging from mild to lethal, qPCR product lengths did not influence PMA-qPCR results at low temperatures, whereas an increasingly strong impact was seen at higher temperatures. Membrane permeability as a result of heat exposure might however have to be considered a conservative parameter for cell death for these pathogens as culturability and redox activity were lost at lower stress intensities than membrane integrity. When applying PMA-DGGE to an environmental water sample which was either left untreated or was exposed to heat, differences to non-PMA treated samples tended to slightly increase when amplified fragments in the first round of the nested PCR were longer, whereas the impact of 1st-round cycle numbers remains unclear.
authors.affiliation: Laboratorio de Biopelículas y Microbiología Ambiental, Centro de Biotecnología, Universidad de Concepción, PO Box 160-C, Concepción, Chile.
authors.forename: Paz Jopia
authors.initials: PJ
authors.lastname: Contreras
authors.forename: Homero
authors.initials: H
authors.lastname: Urrutia
authors.forename: Katherine
authors.initials: K
authors.lastname: Sossa
authors.forename: Andreas
authors.initials: A
authors.lastname: Nocker
mesh_terms: D001386:Azides / Q000737:chemistry*; D002462:Cell Membrane / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D058645:Denaturing Gradient Gel Electrophoresis; D005417:Flavobacterium / Q000737:chemistry* / Q000254:growth & development; D006358:Hot Temperature; D050296:Microbial Viability; D010084:Oxidation-Reduction; D011419:Propidium / Q000031:analogs & derivatives* / Q000737:chemistry; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013312:Stress, Physiological; D014733:Vibrio / Q000737:chemistry* / Q000254:growth & development
chemical_list: D001386:Azides; D004269:DNA, Bacterial; C533957:propidium monoazide; D011419:Propidium