title: Method to detect only viable cells in microbial ecology.
abstract: Propidium monoazide can limit the analysis of microbial communities derived from genetic fingerprints to viable cells with intact cell membranes. However, PMA treatment cannot completely suppress polymerase chain reaction (PCR) amplification when the targeted gene is too short. PMA treatment in combination with two-step nested PCR was designed to overcome this problem. Four experiments were performed to determine the limitation of PMA treatment and to evaluate the suitability of the method by applying the following samples: (1) pure cultures of Escherichia coli O157:H7, Enterobacter aerogenes, and Alcaligenes faecalis; (2) pond water samples spiked with heat-killed E. coli O157:H7 and E. aerogenes; (3) anaerobic sludge samples exposed to increasing heat stress; and (4) selected natural samples of estuarine sediment and lake mud. Results from the first two experiments show that PMA treatment cannot efficiently suppress dead cells from PCR amplification when the targeted gene is as short as 190 bp, however, the two-step nested PCR can overcome this problem. The last two experiments indicate the method that PMA treatment in combination with two-step nested PCR is useful for viable cells detection in microbial ecology.
authors.affiliation: College of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, People's Republic of China.
authors.forename: Jian-Fei
authors.initials: JF
authors.lastname: Luo
authors.forename: Wei-Tie
authors.initials: WT
authors.lastname: Lin
authors.forename: Yong
authors.initials: Y
authors.lastname: Guo
mesh_terms: D042481:Alcaligenes faecalis / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification; D000693:Anaerobiosis; D001386:Azides / Q000494:pharmacology*; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D017753:Ecosystem; D021902:Enterobacter aerogenes / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification; D019453:Escherichia coli O157 / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification; D005618:Fresh Water / Q000382:microbiology; D019015:Geologic Sediments / Q000382:microbiology; D050296:Microbial Viability / Q000187:drug effects*; D016133:Polymerase Chain Reaction / Q000379:methods; D011419:Propidium / Q000031:analogs & derivatives* / Q000494:pharmacology; D012722:Sewage / Q000382:microbiology; D014871:Water Microbiology
chemical_list: D001386:Azides; D004269:DNA, Bacterial; D012722:Sewage; C533957:propidium monoazide; D011419:Propidium