Belinda C Ferrari1, Duncan Veal. 1. Centre for Fluorometric Applications in Biotechnology, School of Biological Sciences, Macquarie University, Sydney, Australia. bferrari@rna.bio.mq.edu.au
Abstract
BACKGROUND: Giardia is a protozoan parasite of concern to water utilities. Giardia detection relies on cyst isolation and confirmation with the use of fluorescence microscopy. It is of interest to develop a flow cytometric (FCM) method that reliably detects one cyst in 10 L of water. To date all available antibodies have targeted the same epitope on the cyst wall. To achieve a reliable method, two independent probes are required. METHODS: Giardia cysts were spiked into a backwash water sample with and without prior hybridization to peptide nucleic acid (PNA) probes. Immunomagnetic separation (IMS) as a pre-enrichment step was compared with filtration of the water sample. Cysts were recovered with two-color FCM. Those cysts hybridized with PNA and fluorescein isothiocyanate (FITC) were dual stained with monoclonal antibody (mAb) conjugated to phycoerythrin (PE); those not hybridized to PNA were dual stained with mAb-FITC and mAb-PE. RESULTS: A fourfold increase in fluorescent signal intensity was obtained when combining the mAb-PE and PNA probe compared with two-color antibody staining. When combined with IMS, Giardia was successfully identified by FCM, with no false positives detected. CONCLUSIONS: Analysis-only FCM detection of Giardia in water is feasible. Further method development incorporating PNA probe hybridization after IMS is necessary. Copyright 2003 Wiley-Liss, Inc.
BACKGROUND:Giardia is a protozoan parasite of concern to water utilities. Giardia detection relies on cyst isolation and confirmation with the use of fluorescence microscopy. It is of interest to develop a flow cytometric (FCM) method that reliably detects one cyst in 10 L of water. To date all available antibodies have targeted the same epitope on the cyst wall. To achieve a reliable method, two independent probes are required. METHODS:Giardia cysts were spiked into a backwash water sample with and without prior hybridization to peptide nucleic acid (PNA) probes. Immunomagnetic separation (IMS) as a pre-enrichment step was compared with filtration of the water sample. Cysts were recovered with two-color FCM. Those cysts hybridized with PNA and fluorescein isothiocyanate (FITC) were dual stained with monoclonal antibody (mAb) conjugated to phycoerythrin (PE); those not hybridized to PNA were dual stained with mAb-FITC and mAb-PE. RESULTS: A fourfold increase in fluorescent signal intensity was obtained when combining the mAb-PE and PNA probe compared with two-color antibody staining. When combined with IMS, Giardia was successfully identified by FCM, with no false positives detected. CONCLUSIONS: Analysis-only FCM detection of Giardia in water is feasible. Further method development incorporating PNA probe hybridization after IMS is necessary. Copyright 2003 Wiley-Liss, Inc.
Authors: Denise Feder; Suzete A O Gomes; André A de Thomaz; Diogo B Almeida; Wagner M Faustino; Adriana Fontes; Cecília V Stahl; Jacenir R Santos-Mallet; Carlos L Cesar Journal: Parasitol Res Date: 2009-09-16 Impact factor: 2.289
Authors: Suzete A O Gomes; Cecilia Stahl Vieira; Diogo B Almeida; Jacenir R Santos-Mallet; Rubem F S Menna-Barreto; Carlos L Cesar; Denise Feder Journal: Sensors (Basel) Date: 2011-12-15 Impact factor: 3.576