Comparison of freeze-thaw cycles for nucleic acid extraction and molecular detection of Cryptosporidium parvum and Toxoplasma gondii oocysts in environmental matrices

Anna J W Manore; Sherilee L Harper; Beatriz Aguilar; J S Weese; Karen Shapiro

doi:10.1016/j.mimet.2018.11.017

Comparison of freeze-thaw cycles for nucleic acid extraction and molecular detection of Cryptosporidium parvum and Toxoplasma gondii oocysts in environmental matrices

J Microbiol Methods. 2019 Jan:156:1-4. doi: 10.1016/j.mimet.2018.11.017. Epub 2018 Nov 22.

Authors

Anna J W Manore¹, Sherilee L Harper¹, Beatriz Aguilar², J S Weese³, Karen Shapiro⁴

Affiliations

¹ Department of Population Medicine, Ontario Veterinary College, University of Guelph, 50 Stone Rd E, Guelph, ON N1G 2W1, Canada.
² Department of Pathology, Microbiology & Immunology, School of Veterinary Medicine, One Shields Ave, 4206 VM3A, University of California, Davis, CA 95616-5270, USA.
³ Centre for Public Health and Zoonoses, University of Guelph, 50 Stone Rd E, Guelph, ON N1G 2W1, Canada.
⁴ Department of Population Medicine, Ontario Veterinary College, University of Guelph, 50 Stone Rd E, Guelph, ON N1G 2W1, Canada; Department of Pathology, Microbiology & Immunology, School of Veterinary Medicine, One Shields Ave, 4206 VM3A, University of California, Davis, CA 95616-5270, USA. Electronic address: kshapiro@ucdavis.edu.

PMID: 30468750
DOI: 10.1016/j.mimet.2018.11.017

Abstract

Freeze-thaw DNA extraction methods and PCR primers were compared to optimize detection of Cryptosporidium parvum and Toxoplasma gondii oocysts in different matrices. Increasing FT cycles did not increase parasite DNA detection, and primers targeting the 18S ssrRNA gene yielded the most sensitive detection of C. parvum oocysts.

Keywords: Cryptosporidium parvum; DNA extraction; Detection; Freeze-thaw; PCR; Toxoplasma gondii.

Publication types

Comparative Study
Research Support, Non-U.S. Gov't

MeSH terms

Analytic Sample Preparation Methods / methods*
Cryptosporidium parvum / genetics
Cryptosporidium parvum / isolation & purification*
DNA Primers / genetics
DNA, Protozoan / isolation & purification*
Freezing
Oocysts / genetics
Oocysts / isolation & purification*
RNA, Protozoan / isolation & purification*
RNA, Ribosomal, 18S / genetics
Toxoplasma / genetics
Toxoplasma / isolation & purification*

Substances

DNA Primers
DNA, Protozoan
RNA, Protozoan
RNA, Ribosomal, 18S