Water Supply Vol 2 No 3 pp 9–15 © IWA Publishing 2002
A sensitive, semi-quantitative direct PCR-RFLP assay for simultaneous detection of five Cryptosporidium species in treated drinking waters and mineral waters
R.A.B. Nichols*, C.A. Paton**, B.M. Campbell***, J. Wastling**** and H.V. Smith*****
*Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow G21 3UW, UK (E-mail: rosely.nichols@spdl.org.uk)
**Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow G21 3UW, UK
***Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow G21 3UW, UK
****Division of Infection and Immunity, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, UK
*****Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow G21 3UW, UK
ABSTRACT
We describe a semi-quantitative PCR-RFLP method for detecting low densities of Cryptosporidium spp. oocysts present in final drinking water samples and natural mineral waters. UK Standard Operating Protocols were used to concentrate oocysts from drinking water samples. Oocysts were concentrated from mineral waters by membrane filtration. Cryptosporidium oocysts identified by epifluorescence microscopy on slides or filters were subjected to DNA extraction and PCR-RFLP analysis. Oocysts were disrupted by freeze-thawing in lysis buffer. Amplicons were readily detected from 2 to 5 intact oocysts on ethidium bromide stained gels following 1 round of PCR. DNA extracted from C. parvum, C. muris, C. baileyi, human-derived C. meleagridis, and C. felis were used to confirm species identity by PCR-RFLP following simultaneous digestion with DraI and VspI.
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