title: Interlaboratory comparison of real-time PCR protocols for quantification of general fecal indicator bacteria.
abstract: The application of quantitative real-time PCR (qPCR) technologies for the rapid identification of fecal bacteria in environmental waters is being considered for use as a national water quality metric in the United States. The transition from research tool to a standardized protocol requires information on the reproducibility and sources of variation associated with qPCR methodology across laboratories. This study examines interlaboratory variability in the measurement of enterococci and Bacteroidales concentrations from standardized, spiked, and environmental sources of DNA using the Entero1a and GenBac3 qPCR methods, respectively. Comparisons are based on data generated from eight different research facilities. Special attention was placed on the influence of the DNA isolation step and effect of simplex and multiplex amplification approaches on interlaboratory variability. Results suggest that a crude lysate is sufficient for DNA isolation unless environmental samples contain substances that can inhibit qPCR amplification. No appreciable difference was observed between simplex and multiplex amplification approaches. Overall, interlaboratory variability levels remained low (<10% coefficient of variation) regardless of qPCR protocol.
authors: U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management Research Laboratory, Cincinnati, Ohio, United States. shanks.orin@epa.gov
authors: Orin C
authors: OC
authors: Shanks
authors: Mano
authors: M
authors: Sivaganesan
authors: Lindsay
authors: L
authors: Peed
authors: Catherine A
authors: CA
authors: Kelty
authors: A Denene
authors: AD
authors: Blackwood
authors: Monica R
authors: MR
authors: Greene
authors: Rachel T
authors: RT
authors: Noble
authors: Rebecca N
authors: RN
authors: Bushon
authors: Erin A
authors: EA
authors: Stelzer
authors: Julie
authors: J
authors: Kinzelman
authors: Tamara
authors: T
authors: Anan'eva
authors: Christopher
authors: C
authors: Sinigalliano
authors: David
authors: D
authors: Wanless
authors: John
authors: J
authors: Griffith
authors: Yiping
authors: Y
authors: Cao
authors: Steve
authors: S
authors: Weisberg
authors: Valarie J
authors: VJ
authors: Harwood
authors: Christopher
authors: C
authors: Staley
authors: Kevin H
authors: KH
authors: Oshima
authors: Manju
authors: M
authors: Varma
authors: Richard A
authors: RA
authors: Haugland
mesh_terms: D001419:Bacteria / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000145:classification* / Q000302:isolation & purification*; D004784:Environmental Monitoring / Q000379:methods; D005243:Feces / Q000382:microbiology*; D015588:Observer Variation; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D014871:Water Microbiology
chemical_list: D004269:DNA, Bacterial