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Interlaboratory validation of an improved method for detection of Cyclospora cayetanensis in produce using a real-time PCR assay
- Murphy, Helen R., Cinar, Hediye Nese, Gopinath, Gopal, Noe, Kathy E., Chatman, Lacresha D., Miranda, Nancy E., Wetherington, June H., Neal-McKinney, Jason, Pires, Gabrielle S., Sachs, Elizabeth, Stanya, Kristopher J., Johnson, Cynthia L., Nascimento, Fernanda S., Santin, Monica, Molokin, Aleksey, Samadpour, Mansour, Janagama, Harish, Kahler, Amy, Miller, Candace, da Silva, Alexandre J.
- Food microbiology 2018 v.69 pp. 170-178
- Cyclospora cayetanensis, Protozoa, cilantro, genes, oocysts, parasites, quantitative polymerase chain reaction, raspberries, ribosomal DNA, washing, United States
- A collaborative validation study was performed to evaluate the performance of a new U.S. Food and Drug Administration method developed for detection of the protozoan parasite, Cyclospora cayetanensis, on cilantro and raspberries. The method includes a sample preparation step in which oocysts are recovered from produce using an enhanced produce washing solution containing 0.1% Alconox and a commercially available method to disrupt the C. cayetanensis oocysts and extract DNA. A real-time PCR assay targeting the C. cayetanensis 18S rDNA gene with an internal amplification control to monitor PCR inhibition provides species-specific identification. Five laboratories blindly analyzed a total of 319 samples consisting of 25 g of cilantro or 50 g of raspberries which were either uninoculated or artificially contaminated with C. cayetanensis oocysts. Detection rates for cilantro inoculated with 200, 10, and 5 oocysts, were 100%, 80%, and 31%, respectively. For raspberries, the detection rates for samples inoculated with 200, 10, and 5 oocysts were 100%, 90% and 50%, respectively. All uninoculated samples, DNA blank extracts, and no-template PCR controls were negative. Reproducibility between laboratories and analysts was high and the method was shown to be an effective analytical tool for detection of C. cayetanensis in produce.