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Determination of Cryptosporidium parvum oocyst viability by fluorescence in situ hybridization (FISH) using a ribosomal RNA-directed probes

Smith, James J., Gunasekera, Thusitha S., Barardi, Celia R. M., Veal, Duncan, & Vesey, Graham (2004) Determination of Cryptosporidium parvum oocyst viability by fluorescence in situ hybridization (FISH) using a ribosomal RNA-directed probes. Journal of Applied Microbiology, 96(2), pp. 409-417.

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Abstract

AIMS: Fluorescence in situ hybridization (FISH) has been proposed for species-specific detection, and viability determination of Cryptosporidium parvum oocysts. FISH-based viability determination depends on rRNA decay after loss of viability. We examined the effects of RNase(s) and RNase inhibitors on FISH of C. parvum. METHODS AND RESULTS: FISH was performed using a 5'-Texas red-labelled DNA oligonucleotide probe at 1 pM microl(-1). Intact and heat-permeabilized oocysts were treated with 1-100 microg ml(-1) RNase. FISH of intact oocysts appeared unaffected by exogenous RNase if this was neutralized before permeabilization. FISH fluorescence of heat-killed oocysts stored in phosphate-buffered saline at room temperature decayed by 1/2 after 55 h, but remained detectable after 6 days. Addition of vanadyl ribonucleoside complex (VRC) extended rRNA half-life of heat-permeabilized oocysts to 155 h. CONCLUSIONS: Extended rRNA half-life may result in viability overestimation using FISH. RNase pretreatment before FISH is recommended to destroy residual rRNA in recently killed oocysts. Incorporation of 1-10 mM l(-1) VRC before FISH permeabilization steps should neutralize RNase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Elimination of FISH fluorescence of nonviable C. parvum is desirable. Use of RNase and VRC is suggested to reduce numbers of false-positive 'viable' oocysts.

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27 citations in Scopus
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25 citations in Web of Science®

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ID Code: 8514
Item Type: Journal Article
Additional Information: For more information, please refer to the journal’s website (see hypertext link) or contact the author. Author contact details: jj.smith@qut.edu.au
DOI: 10.1046/j.1365-2672.2004.02150.x
ISSN: 1364-5072
Subjects: Australian and New Zealand Standard Research Classification > TECHNOLOGY (100000) > MEDICAL BIOTECHNOLOGY (100400) > Medical Biotechnology Diagnostics (incl. Biosensors) (100402)
Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > MICROBIOLOGY (060500) > Infectious Agents (060502)
Divisions: Past > QUT Faculties & Divisions > Faculty of Science and Technology
Current > Institutes > Institute of Health and Biomedical Innovation
Current > Research Centres > Science Research Centre
Copyright Owner: Copyright 2004 Blackwell Publishing
Deposited On: 11 Jul 2007
Last Modified: 29 Feb 2012 23:21

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