Comparative evaluation of UNEX-based DNA extraction for molecular detection of Cyclospora cayetanensis, Toxoplasma gondii, and Cryptosporidium parvum as contaminants of berries

Tamirat Tefera Temesgen; Alessandra Barlaam; Kristoffer R Tysnes; Lucy J Robertson

doi:10.1016/j.fm.2020.103447

Comparative evaluation of UNEX-based DNA extraction for molecular detection of Cyclospora cayetanensis, Toxoplasma gondii, and Cryptosporidium parvum as contaminants of berries

Food Microbiol. 2020 Aug:89:103447. doi: 10.1016/j.fm.2020.103447. Epub 2020 Jan 26.

Authors

Tamirat Tefera Temesgen¹, Alessandra Barlaam², Kristoffer R Tysnes³, Lucy J Robertson³

Affiliations

¹ Laboratory of Parasitology, Department of Paraclinical Sciences, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, Adamstuen Campus, P.O. Box 369 Center, 0102, Oslo, Norway. Electronic address: temesgen.tamirat@nmbu.no.
² Department of Agriculture Science, Food and Environment, University of Foggia, 71122, Foggia, FG, Italy.
³ Laboratory of Parasitology, Department of Paraclinical Sciences, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, Adamstuen Campus, P.O. Box 369 Center, 0102, Oslo, Norway.

PMID: 32138995
DOI: 10.1016/j.fm.2020.103447

Abstract

The potential public health impact of foodborne parasites (FBP) transmitted via contaminated fresh produces indicates the necessity for robust and reliable laboratory methods for their detection and identification on this infection vehicle. Standardization of methods for detection of common FBP in fresh produce is to be expected and ensuring that the DNA extraction approach is most appropriate for the FBP of interest and for the matrix being analyzed is also important. Therefore, the aim of the present study was to compare the efficacy of two commercially available DNA extraction procedures, the UNEX-based method and DNeasy PowerSoil kit in the detection of three protozoan parasites, C. cayetanensis, C. parvum, and T. gondii, on contaminated berries. Oocysts of each parasite were spiked into the pellets of raspberry and blueberry washes. The spiked pellets were then randomly assigned to DNA extraction using either the PowerSoil or UNEX method, with DNA extraction with both methods performed by two independent analysts. The detection rate when berry washes were spiked with 20 oocysts of C. cayetanensis, T. gondii, and C. parvum was 95%, 85%, and 40%, respectively, when using the PowerSoil kit; whereas the equivalent results using the UNEX method were 55%, 60%, and 5%, respectively. In addition, significantly lower C_q values were achieved for each parasite in the samples spiked with 500 oocysts when the PowerSoil kit was used. Possible reasons for these results are discussed, and include the composition of both the beads and the buffers in each method.

Keywords: Berries; Cryptosporidium; Cyclospora; DNA extraction; DNeasy PowerSoil; Foodborne parasites; Fresh produce; Toxoplasma; UNEX.

MeSH terms

Cryptosporidium parvum / isolation & purification*
Cyclospora / isolation & purification*
DNA, Protozoan / isolation & purification
Food Contamination*
Fruit / parasitology*
Oocysts
Toxoplasma / isolation & purification*

Substances

DNA, Protozoan