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A novel protocol to isolate, detect and differentiate taeniid eggs in leafy greens and berries using real-time PCR with melting curve analysis
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A novel protocol to isolate, detect and differentiate taeniid eggs in leafy greens and berries using real-time PCR with melting curve analysis
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Journal Article
A novel protocol to isolate, detect and differentiate taeniid eggs in leafy greens and berries using real-time PCR with melting curve analysis
2019
Overview
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.
Publisher
BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
Subject
/ Animals
/ Berries
/ Biomedical and Life Sciences
/ DNA
/ Echinococcus granulosus - classification
/ Echinococcus granulosus - genetics
/ Echinococcus granulosus - isolation & purification
/ Echinococcus multilocularis - isolation & purification
/ Eggs
/ Food contamination & poisoning
/ Food Contamination - analysis
/ Fruits
/ Health
/ Melting
/ Methods
/ PCR
/ Protocol
/ Protozoa
/ quantitative polymerase chain reaction
/ Real-Time Polymerase Chain Reaction - methods
/ risk
/ Taenia
/ Taenia - isolation & purification
/ Veterinary Medicine/Veterinary Science
/ Virology
