title: Combination of competitive quantitative PCR and constant-denaturant capillary electrophoresis for high-resolution detection and enumeration of microbial cells.
abstract: A novel quantitative PCR (QPCR) approach, which combines competitive PCR with constant-denaturant capillary electrophoresis (CDCE), was adapted for enumerating microbial cells in environmental samples using the marine nanoflagellate Cafeteria roenbergensis as a model organism. Competitive PCR has been used successfully for quantification of DNA in environmental samples. However, this technique is labor intensive, and its accuracy is dependent on an internal competitor, which must possess the same amplification efficiency as the target yet can be easily discriminated from the target DNA. The use of CDCE circumvented these problems, as its high resolution permitted the use of an internal competitor which differed from the target DNA fragment by a single base and thus ensured that both sequences could be amplified with equal efficiency. The sensitivity of CDCE also enabled specific and precise detection of sequences over a broad range of concentrations. The combined competitive QPCR and CDCE approach accurately enumerated C. roenbergensis cells in eutrophic, coastal seawater at abundances ranging from approximately 10 to 10(4) cells x ml(-1). The QPCR cell estimates were confirmed by fluorescent in situ hybridization counts, but estimates of samples with <50 cells x ml(-1) by QPCR were less variable. This novel approach extends the usefulness of competitive QPCR by demonstrating its ability to reliably enumerate microorganisms at a range of environmentally relevant cell concentrations in complex aquatic samples.
authors.affiliation: Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: E L
authors.initials: EL
authors.lastname: Lim
authors.forename: A V
authors.initials: AV
authors.lastname: Tomita
authors.forename: W G
authors.initials: WG
authors.lastname: Thilly
authors.forename: M F
authors.initials: MF
authors.lastname: Polz
mesh_terms: D000818:Animals; D017931:DNA Primers; D016054:DNA, Protozoan / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000379:methods; D056890:Eukaryota / Q000235:genetics* / Q000254:growth & development*; D005618:Fresh Water / Q000469:parasitology; D017404:In Situ Hybridization, Fluorescence; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan