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Molecular detection of Cyclospora cayetanensis in human stool specimens using UNEX-based DNA extraction and real-time PCR

Published online by Cambridge University Press:  08 November 2017

Yvonne Qvarnstrom Open the ORCID record for Yvonne Qvarnstrom [Opens in a new window] ,
Theresa Benedict ,
Paula L. Marcet ,
Ryan E. Wiegand ,
Barbara L. Herwaldt  and
Alexandre J. da Silva
Yvonne Qvarnstrom*
Affiliation:
Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, GA 30329, USA
Theresa Benedict
Affiliation:
Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, GA 30329, USA
Paula L. Marcet
Affiliation:
Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, GA 30329, USA
Ryan E. Wiegand
Affiliation:
Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, GA 30329, USA
Barbara L. Herwaldt
Affiliation:
Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, GA 30329, USA
Alexandre J. da Silva
Affiliation:
Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 8301 Muirkirk Rd, Laurel, MD 20708, USA
*
Author for correspondence: Yvonne Qvarnstrom, E-mail: [email protected]
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Abstract

Cyclospora cayetanensis is a coccidian parasite associated with diarrheal illness. In the USA, foodborne outbreaks of cyclosporiasis have been documented almost every year since the mid-1990s. The typical approach used to identify this parasite in human stools is an examination of acid-fast-stained smears under bright-field microscopy. UV fluorescence microscopy of wet mounts is more sensitive and specific than acid-fast staining but requires a fluorescence microscope with a special filter not commonly available in diagnostic laboratories. In this study, we evaluated a new DNA extraction method based on the Universal Nucleic Acid Extraction (UNEX) buffer and compared the performances of four published real-time polymerase chain reaction (PCR) assays for the specific detection of C. cayetanensis in stool. The UNEX-based method had an improved capability to recover DNA from oocysts compared with the FastDNA stool extraction method. The best-performing real-time PCR assay was a C. cayetanensis-specific TaqMan PCR that targets the 18S ribosomal RNA gene. This new testing algorithm should be useful for detection of C. cayetanensis in human stool samples.

Keywords

coccidiaCyclospora cayetanensismolecular diagnosticsstool DNA extraction
Type
Research Article
Information
Parasitology , Volume 145 , Issue 7 , June 2018 , pp. 865 - 870
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Copyright
Copyright © Cambridge University Press 2017 

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