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  • Interlaboratory validation of an improved method for detection of Cyclospora cayetanensis in produce using a real-time PCR assay.

Interlaboratory validation of an improved method for detection of Cyclospora cayetanensis in produce using a real-time PCR assay.

Food microbiology (2017-09-25)
Helen R Murphy, Hediye Nese Cinar, Gopal Gopinath, Kathy E Noe, Lacresha D Chatman, Nancy E Miranda, June H Wetherington, Jason Neal-McKinney, Gabrielle S Pires, Elizabeth Sachs, Kristopher J Stanya, Cynthia L Johnson, Fernanda S Nascimento, Monica Santin, Aleksey Molokin, Mansour Samadpour, Harish Janagama, Amy Kahler, Candace Miller, Alexandre J da Silva
RESUMO

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.

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Sigma-Aldrich
Antibiotic Antimycotic Solution (100×), Stabilized, with 10,000 units penicillin, 10 mg streptomycin and 25 μg amphotericin B per mL, 0.1 μm filtered, BioReagent, suitable for cell culture