title: PCR-based quantitation of Cryptosporidium parvum in municipal water samples.
abstract: A PCR method for the quantitation of Cryptosporidium parvum oocysts in municipal drinking water samples was investigated. Quantitative PCR uses an internal standard (IS) template with unknown target numbers to compare to standards of known concentrations in a standard curve. The IS template was amplified using the same primers used to amplify a portion of a 358 bp gene fragment that encodes a repetitive oocyst wall protein in C. parvum. Municipal water samples spiked with known numbers of C. parvum oocysts were tested by quantitative PCR using the IS and the Digene SHARP Signal System Assay for PCR product detection. The absorbance readings for target DNA and IS templates versus the number of molecules of the target DNA were plotted to generate standard curves for estimating oocyst numbers. The method allowed the quantitation of oocysts from log 3 to log 5 spiked into municipal water samples.
authors.affiliation: Department of Environmental Biology, University of Guelph, Ontario, Canada.
authors.forename: E
authors.initials: E
authors.lastname: Chung
authors.forename: J E
authors.initials: JE
authors.lastname: Aldom
authors.forename: R A
authors.initials: RA
authors.lastname: Carreno
authors.forename: A H
authors.initials: AH
authors.lastname: Chagla
authors.forename: M
authors.initials: M
authors.lastname: Kostrzynska
authors.forename: H
authors.initials: H
authors.lastname: Lee
authors.forename: G
authors.initials: G
authors.lastname: Palmateer
authors.forename: J T
authors.initials: JT
authors.lastname: Trevors
authors.forename: S
authors.initials: S
authors.lastname: Unger
authors.forename: R
authors.initials: R
authors.lastname: Xu
authors.forename: S A
authors.initials: SA
authors.lastname: De Grandis
mesh_terms: D000818:Animals; D001483:Base Sequence; D016785:Cryptosporidium parvum / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000032:analysis; D008969:Molecular Sequence Data; D010270:Parasite Egg Count; D016133:Polymerase Chain Reaction / Q000379:methods; D011933:Reagent Kits, Diagnostic; D014871:Water Microbiology; D014881:Water Supply
chemical_list: D016054:DNA, Protozoan; D011933:Reagent Kits, Diagnostic