Simultaneous detection of four protozoan parasites on leafy greens using a novel multiplex PCR assay

Pathogen contamination of fresh produce presents a health risk for consumers; however, the produce industry still lacks adequate tools for simultaneous detection of protozoan parasites. Here, a simple multiplex PCR (mPCR) assay was developed for detection of protozoan (oo)cysts and compared with pre...

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Bibliographic Details
Published in:Food microbiology 2019-12, Vol.84, p.103252, Article 103252
Main Authors: Shapiro, Karen, Kim, Minji, Rajal, Veronica B., Arrowood, Michael J., Packham, Andrea, Aguilar, Beatriz, Wuertz, Stefan
Format: Article
Language:English
Subjects:
Animals
Cryptosporidium - isolation & purification
DNA, Protozoan - genetics
Food Parasitology - methods
Giardia - isolation & purification
Limit of Detection
Multiplex Polymerase Chain Reaction - methods
Oocysts - isolation & purification
Parasites - isolation & purification
Real-Time Polymerase Chain Reaction
Sensitivity and Specificity
Spinacia oleracea - parasitology
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Summary:Pathogen contamination of fresh produce presents a health risk for consumers; however, the produce industry still lacks adequate tools for simultaneous detection of protozoan parasites. Here, a simple multiplex PCR (mPCR) assay was developed for detection of protozoan (oo)cysts and compared with previously published real-time PCR assays and microscopy methods. The assay was evaluated for simultaneous detection of Cryptosporidium, Giardia, Cyclospora cayetanensis, and Toxoplasma gondii followed by parasite differentiation via either a nested specific PCR or a restriction fragment length polymorphism (RFLP) assay. Spiking experiments using spinach as a model leafy green were performed for assay validation. Leaf-washing yielded higher recoveries and more consistent detection of parasites as compared with stomacher processing. Lowest limits of detection using the nested mPCR assay were 1–10 (oo)cysts/g spinach (in 10 g samples processed), and this method proved more sensitive than qPCR for parasite detection. Microscopy methods were more reliable for visual detection of parasites in lower spiking concentrations, but are more costly and laborious, require additional expertise, and lack molecular confirmation essential for accurate risk assessment. Overall, the nested mPCR assay provides a rapid (
ISSN:0740-0020
1095-9998
DOI:10.1016/j.fm.2019.103252