title: Species-specific real-time PCR cell number quantification of the bloom-forming cyanobacterium Planktothrix agardhii.
abstract: A species-specific method to detect and quantify Planktothrix agardhii was developed by combining the SYBR Green I real-time polymerase chain reaction technique with a simplified DNA extraction procedure for standard curve preparation. Newly designed PCR primers were used to amplify a specific fragment within the rpoC1 gene. Since this gene exists in single copy in the genome, it allows the direct achievement of cell concentrations. The cell concentration determined by real-time PCR showed a linear correlation with the cell concentration determined from direct microscopic counts. The detection limit for cell quantification of the method was 8 cells μL(-1), corresponding to 32 cells per reaction. Furthermore, the real-time qPCR method described in this study allowed a successful quantification of P. agardhii from environmental water samples, showing that this protocol is an accurate and economic tool for a rapid absolute quantification of the potentially toxic cyanobacterium P. agardhii.
authors.affiliation: Laboratório de Biologia e Ecotoxicologia, Departamento de Saúde Ambiental, Instituto Nacional de Saúde Dr. Ricardo Jorge, Avenida Padre Cruz, 1649-016, Lisboa, Portugal.
authors.forename: Catarina
authors.initials: C
authors.lastname: Churro
authors.forename: Paulo
authors.initials: P
authors.lastname: Pereira
authors.forename: Vitor
authors.initials: V
authors.lastname: Vasconcelos
authors.forename: Elisabete
authors.initials: E
authors.lastname: Valério
mesh_terms: D058491:Bacterial Load / Q000379:methods*; D001426:Bacterial Proteins / Q000235:genetics; D052160:Benzothiazoles; D000458:Cyanobacteria / Q000235:genetics / Q000502:physiology*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000302:isolation & purification; D003959:Diamines; D009930:Organic Chemicals / Q000378:metabolism; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014871:Water Microbiology
chemical_list: D001426:Bacterial Proteins; D052160:Benzothiazoles; D017931:DNA Primers; D004269:DNA, Bacterial; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I