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.
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.
Impact and interest:
Citation counts are sourced monthly from and citation databases.
These databases contain citations from different subsets of available publications and different time periods and thus the citation count from each is usually different. Some works are not in either database and no count is displayed. Scopus includes citations from articles published in 1996 onwards, and Web of Science® generally from 1980 onwards.
Citations counts from theindexing service can be viewed at the linked Google Scholar™ search.
|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: firstname.lastname@example.org|
|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 00:00|
|Last Modified:||29 Feb 2012 13:21|
Repository Staff Only: item control page