Document Detail


Detection of viable oocysts of Cryptosporidium parvum following nucleic acid sequence based amplification.
MedLine Citation:
PMID:  11305648     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
A reliable method using nucleic acid sequence based amplification (NASBA) with subsequent electrochemiluminescent detection for the specific and sensitive detection of viable oocysts of Cryptosporidium parvum in environmental samples was developed. The target molecule was a 121-nt sequence from the C. parvum heat shock protein hsp70 mRNA. Oocysts of C. parvum were isolated from environmental water via vortex flow filtration and immunomagnetic separation. A brief heat shock was applied to the oocysts and the nucleic acid purified using an optimized very simple but efficient nucleic acid extraction method. The nucleic acid was amplified in a water bath for 60-90 min with NASBA, an isothermal technique that specifically amplifies RNA molecules. Amplified RNA was hybridized with specific DNA probes and quantified with an electrochemiluminescence (ECL) detection system. We optimized the nucleic acid extraction and purification, the NASBA reaction, amplification, and detection probes. We were able to amplify and detect as few as 10 mRNA molecules. The NASBA primers as well as the ECL probes were highly specific for C. parvum in buffer and in environmental samples. Our detection limit was approximately 5 viable oocysts/sample for the assay procedure, including nucleic acid extraction, NASBA, and ECL detection. Nonviable oocysts were not detected.
Authors:
A J Baeumner; M C Humiston; R A Montagna; R A Durst
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Analytical chemistry     Volume:  73     ISSN:  0003-2700     ISO Abbreviation:  Anal. Chem.     Publication Date:  2001 Mar 
Date Detail:
Created Date:  2001-04-17     Completed Date:  2001-05-21     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0370536     Medline TA:  Anal Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1176-80     Citation Subset:  IM    
Affiliation:
Department of Food Science and Technology, Cornell University, Geneva, New York 14456-0462, USA. ajb23@cornell.edu
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MeSH Terms
Descriptor/Qualifier:
Animals
Chemiluminescent Measurements
Cryptosporidium parvum / chemistry*
Heat-Shock Proteins / chemistry
Nucleic Acid Amplification Techniques
Water Microbiology*
Chemical
Reg. No./Substance:
0/Heat-Shock Proteins

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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