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. 2018 Jun;145(7):865-870.
doi: 10.1017/S0031182017001925. Epub 2017 Nov 8.

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

Yvonne Qvarnstrom  1 , Theresa Benedict  1 , Paula L Marcet  1 , Ryan E Wiegand  1 , Barbara L Herwaldt  1 , Alexandre J da Silva  2
Affiliations

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

Yvonne Qvarnstrom et al. Parasitology. 2018 Jun.

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: Cyclospora cayetanensis; coccidia; molecular diagnostics; stool DNA extraction.

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Figures

Fig. 1
Fig. 1
High-resolution melt analysis curves using the SYBR Green assay. The first derivative of the fluorescence multiplied by −1 [−R’(T)] is plotted against the temperature (°C). Blue = simian Cyclospora spp. (C. cercopitheci, C. papionis, and C. colobi); red = C. cayetanensis; green = Eimeria acervulina.
Fig. 2
Fig. 2
High-resolution melt analysis curves using the EvaGreen assay. The first derivative of the fluorescence multiplied by −1 [−R’(T)] is plotted against the temperature (°C). Red = C. cayetanensis-positive stools (Tm = 85.0–85.5); green = C. cayetanensis-negative stools with Tm = 84.0–84.5; black = C. cayetanensis-negative stools with Tm = 85.0–85.5 (i.e., false positives).
See this image and copyright information in PMC

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