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Food Microbiol. 2018 Feb;69:170-178. doi: 10.1016/j.fm.2017.08.008. Epub 2017 Aug 17.

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

Author information

1
U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, Division of Food and Environmental Microbiology, Laurel, MD 20708, USA. Electronic address: helen.murphy@fda.hhs.gov.
2
U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, Division of Food and Environmental Microbiology, Laurel, MD 20708, USA.
3
U.S. Food and Drug Administration, Southeast Food and Feed Laboratory, Atlanta, GA 30309, USA.
4
U.S. Food and Drug Administration, Pacific Northwest Laboratory, Bothell, WA 98021, USA.
5
U.S. Food and Drug Administration, Pacific Northwest Laboratory, Bothell, WA 98021, USA; IEH Laboratories & Consulting Group, Lake Forest Park, Washington 98155, USA.
6
Centers for Disease Control and Prevention, Center for Global Health, Division of Parasitic Diseases and Malaria, Parasitic Diseases Branch, Reference Diagnostic Laboratory, Atlanta, GA 30329, USA.
7
U.S. Department of Agriculture, Agricultural Research Service, Environmental Microbial and Food Safety Lab, Beltsville, MD 20705, USA.
8
IEH Laboratories & Consulting Group, Lake Forest Park, Washington 98155, USA.
9
Centers for Disease Control and Prevention, Division of Foodborne, Waterborne, and Environmental Diseases, Waterborne Disease Prevention Branch, Atlanta, GA 30329, USA.

Abstract

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.

KEYWORDS:

Cyclospora cayetanensis; Detection method; Produce; Real-time PCR; Validation

PMID:
28941898
DOI:
10.1016/j.fm.2017.08.008
[Indexed for MEDLINE]
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