Water Supply Vol 2 No 3 pp 95–100 © IWA Publishing 2002
A fast and sensitive nucleic acid extraction method for the detection of Cryptosporidium by PCR in environmental water samples
J. Dellundé*, S. Pina**, J. Jofre*** and F. Lucena****
*Departament of Microbiology, University of Barcelona, Spain (E-mail: dellunde@porthos.bio.ub.es)
**Departament of Microbiology, University of Barcelona, Spain (E-mail: spina@porthos.bio.ub.es)
***Departament of Microbiology, University of Barcelona, Spain (E-mail: joan@porthos.bio.ub.es)
****Departament of Microbiology, University of Barcelona, Spain (E-mail: lucena@porthos.bio.ub.es)
ABSTRACT
A new protocol based on a combination of an excystation process followed by a nucleic acid extraction that combines the lysing and nuclease-inactivating properties of the chaotropic agent guanidinium thiocyanate and the nucleic acid-binding properties of silica particles is described for the extraction and purification of nucleic acids from Cryptosporidium. The application of nested and/or semi-nested PCR using different external and internal primers to DNA extracted by this method from seeded and naturally occurring Cryptosporidium oocysts concentrated and purified from environmental samples detects numbers of oocysts ranging from 20 to 50. The method is feasible, detects mostly excystable oocysts and no problems of inhibition of PCR were observed when applied to environmental samples.
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