A novel protocol to isolate, detect and differentiate taeniid eggs in leafy greens and berries using real-time PCR with melting curve analysis.

Frey, Caroline F.; Oakley, Jenna R.; Lobanov, Vladislav A.; Marreros, Nelson; Schurer, Janna M.; Lalonde, Laura F. (2019). A novel protocol to isolate, detect and differentiate taeniid eggs in leafy greens and berries using real-time PCR with melting curve analysis. Parasites & Vectors, 12(1), p. 590. BioMed Central 10.1186/s13071-019-3834-8

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BACKGROUND: Zoonotic taeniid cestodes are amongst the most important food-borne parasites affecting human health worldwide. Contamination of fresh produce with the eggs of Echinococcus granulosus (s.l.), Echinococcus multilocularis, and some Taenia species pose a potential food safety risk. However, very few studies have attempted to investigate the potential contamination of fresh produce with taeniid eggs and the available methods are not standardized for this purpose. Established protocols do exist for testing leafy greens and berries for contamination with protozoan parasites and are used in national surveillance programmes. This methodology could be suitable for the detection of taeniids. The objective of this project was to develop and standardize a sensitive and reliable method to detect contamination of leafy greens and berries with eggs of zoonotic taeniids and to differentiate between E. multilocularis, E. granulosus (s.l.) and Taenia spp. METHODS: We compared the efficacy of different wash solutions to remove Taenia spp. eggs from spiked produce, assessed two DNA extraction kits for their performance on Taenia spp. eggs, and adapted a published conventional multiplex PCR into a real-time PCR with fluorescence melting curve analysis (MCA) that was optimized for use on produce washes. Analytical specificity of this protocol was assessed using non-spiked produce washes as well as a variety of other potentially contaminating parasites. RESULTS: The protocol as established in this study had an analytical sensitivity of detecting five eggs per spiked sample for both romaine lettuce and strawberries. Unequivocal identification of E. multilocularis, E. granulosus (s.l.) and Taenia spp. was possible through MCA. Amplicon sequencing allowed identification of Taenia to the species level. The real-time PCR also amplified DNA from Dicrocoelium sp., but with a clearly discernable melting curve profile. CONCLUSION: The new protocol for screening produce for taeniid contamination was highly sensitive. Melting curve analysis and the possibility of amplicon sequencing made this assay very specific. Once further validated, this method could be employed for surveillance of produce for contamination with taeniid parasites to assess potential risks for consumers.

Item Type:

Journal Article (Original Article)

Division/Institute:

05 Veterinary Medicine > Department of Infectious Diseases and Pathobiology (DIP) > Institute of Parasitology

UniBE Contributor:

Frey Marreros Canales, Caroline

Subjects:

600 Technology > 630 Agriculture

ISSN:

1756-3305

Publisher:

BioMed Central

Funders:

[UNSPECIFIED] Canadian Food Inspection Agency

Language:

English

Submitter:

Caroline Franziska Frey Marreros Canales

Date Deposited:

28 Feb 2020 14:46

Last Modified:

01 Mar 2020 02:50

Publisher DOI:

10.1186/s13071-019-3834-8

PubMed ID:

31849342

BORIS DOI:

10.7892/boris.141032

URI:

https://boris.unibe.ch/id/eprint/141032

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