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Applied and Environmental Microbiology, August 2003, p. 4662-4669, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4662-4669.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

PCR-Restriction Fragment Length Polymorphism Method for Detection of Cyclospora cayetanensis in Environmental Waters without Microscopic Confirmation

Joan M. Shields and Betty H. Olson*

Department of Environmental Analysis and Design, School of Social Ecology, University of California, Irvine, California 92697-7070

Received 20 December 2002/ Accepted 27 May 2003

We developed an alternative nested-PCR-restriction fragment length polymorphism (RFLP) protocol for the detection of Cyclospora cayetanensis in environmental samples that obviates the need for microscopic examination. The RFLP method, with the restriction enzyme AluI, differentiates the amplified target sequence from C. cayetanensis from those that may cross-react. This new protocol was used to reexamine a subset (121 of 180) of surface water samples. Samples previously positive when the CYCF3E and CYCR4B primers (33) and RFLP with MnlI (20) were used were also PCR positive with the new primers; however, they were RFLP negative. We verified, by sequencing these amplicons, that while two were most likely other Cyclospora species, they were not C. cayetanensis. We can detect as few as one oocyst seeded into an autoclaved pellet flocculated from 10 liters of surface water. This new protocol should be of great use for environmental microbiologists and public health laboratories.

* Corresponding author. Mailing address: Department of Environmental Health, Science and Policy, School of Social Ecology, University of California, Irvine, CA 92697-7070. Phone: (949) 824-5281. Fax: (949) 824-2056. E-mail: bholson{at}uci.edu.

Applied and Environmental Microbiology, August 2003, p. 4662-4669, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4662-4669.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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