title: Fluorescence resonance energy transfer (FRET)-based specific labeling of Cryptosporidium oocysts for detection in environmental samples.
abstract: BACKGROUND
  Accurate detection and quantification of Cryptosporidium oocysts in water are a challenge to the water industry. This article demonstrates a way to fluorescently label Cryptosporidium oocysts, based on fluorescence resonance energy transfer (FRET). Labeled oocysts can then be applied to environmental waters and their movement followed by flow cytometric detection and enumeration of the FRET-labeled oocysts, as demonstrated here with environmental water samples.
  
  
  METHODS
  Cryptosporidium oocysts were labeled with three fluorochromes, FITC, Texas red, and Cy7, that through FRET yielded a Stokes shift of approximately 272 nm with excitation from a standard argon laser emitting at 488 nm. Defined flow cytometric settings and gatings were used to select FITC/green (530-nm), Texas red/red (650-nm), and Cy7/infrared (780-nm) fluorescing particles with light scatter properties similar to oocysts. Water concentrates were seeded with 10 tri-labeled oocysts and were analyzed using flow cytometry. Unseeded water concentrates were also analyzed.
  
  
  RESULTS
  Analysis of unseeded water concentrates detected no autofluorescent particle similar to the labeled oocysts. Labeled oocysts were detected successfully with up to 85% recovery in water concentrates spiked with 10 tri-labeled oocysts.
  
  
  CONCLUSIONS
  Low numbers of FRET-labeled oocysts can be quantified and clearly distinguished from autofluorescing background in environmental water concentrates.
authors.affiliation: School of Civil and Environmental Engineering, University of New South Wales, Sydney, New South Wales, Australia. jin@civeng.unsw.edu.au
authors.forename: J
authors.initials: J
authors.lastname: Chung
authors.forename: G
authors.initials: G
authors.lastname: Vesey
authors.forename: M
authors.initials: M
authors.lastname: Gauci
authors.forename: N J
authors.initials: NJ
authors.lastname: Ashbolt
mesh_terms: D000818:Animals; D003458:Cryptosporidium / Q000737:chemistry* / Q000166:cytology; D004777:Environment; D004784:Environmental Monitoring / Q000379:methods*; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D033661:Oocysts / Q000737:chemistry*; D014867:Water / Q000032:analysis / Q000469:parasitology
chemical_list: D005456:Fluorescent Dyes; D014867:Water