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Fluorescence resonance energy transfer (FRET)‐based specific labeling of Cryptosporidium oocysts for detection in environmental samples
Author(s) -
Chung J.,
Vesey G.,
Gauci M.,
Ashbolt N.J.
Publication year - 2004
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.20020
Subject(s) - förster resonance energy transfer , cryptosporidium , fluorescence , flow cytometry , enumeration , chemistry , biology , microbiology and biotechnology , physics , optics , mathematics , combinatorics , feces
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 ∼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. © 2004 Wiley‐Liss, Inc.