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Applied and Environmental Microbiology, July 2008, p. 4354-4358, Vol. 74, No. 14
0099-2240/08/$08.00+0     doi:10.1128/AEM.00032-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Highly Sensitive and Specific PCR Assay for Reliable Detection of Cyclospora cayetanensis Oocysts{triangledown}

Laura F. Lalonde* and Alvin A. Gajadhar

Centre for Food-borne and Animal Parasitology, Canadian Food Inspection Agency, Saskatoon, Saskatchewan, Canada S7N 2R3

Received 4 January 2008/ Accepted 15 May 2008

Multiple outbreaks of food-borne gastroenteritis caused by the coccidian parasite Cyclospora cayetanensis have been reported annually in North America since 1995. Detection of C. cayetanensis contamination typically relies on laborious and subjective microscopic examination of produce washes. Molecular detection methods based on nested PCR, restriction fragment length polymorphism, or multiplex PCR have been developed for C. cayetanensis; however, they have not been adequately validated for use on food products. Further challenges include reliably extracting DNA from coccidian oocysts since their tough outer wall is resistant to lysis and overcoming PCR inhibitors in sample matrices. We describe preliminary validation of a reliable DNA extraction method for C. cayetanensis oocysts and a sensitive and specific novel PCR assay. The sensitivity and repeatability of the developed methods were evaluated by multiple DNA extractions and PCR amplifications using 1,000-, 100-, 10-, or 1-ooycst aliquots of C. cayetanensis oocysts in water or basil wash sediment. Successful PCR amplification was achieved on 15 and 5 replicates extracted from aliquots containing 1,000 oocysts in water and basil wash, respectively. All 45 replicates of the 100-oocyst aliquots in water and 5 in basil wash were amplified successfully, as were 43/45 and 41/45 of the 10- and 1-oocyst aliquots in water and 9/15 and 2/15 in basil wash, respectively. The developed primers showed no cross-reactivity when tested against bacteria, nematodes, and protozoans, including Eimeria, Giardia, and Cryptosporidium. Our results indicate that these methods are specific, can reliably detect a single oocyst, and overcome many of the limitations of microscopic diagnosis.

* Corresponding author. Mailing address: Canadian Food Inspection Agency, Saskatoon Laboratory, Centre for Food-borne and Animal Parasitology, 116 Veterinary Road, Saskatoon, Saskatchewan, Canada S7N 2R3. Phone: (306) 975-5996. Fax: (306) 975-5711. E-mail: lalondel{at}inspection.gc.ca

{triangledown} Published ahead of print on 23 May 2008.

Applied and Environmental Microbiology, July 2008, p. 4354-4358, Vol. 74, No. 14
0099-2240/08/$08.00+0     doi:10.1128/AEM.00032-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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