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

Karen Shapiro, M. Kim, Veronica B. Rajal, Michael J. Arrowood, Andrea Packham, Beatriz Aguilar, Stefan Wuertz

Research output: Contribution to journalArticle

Abstract

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 (<24 h), inexpensive ($10 USD/sample), and simple approach for simultaneous detection of protozoan pathogens on fresh produce.

Original languageEnglish (US)
Article number103252
JournalFood Microbiology
Volume84
DOIs
StatePublished - Dec 1 2019

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Multiplex Polymerase Chain Reaction
green leafy vegetables
Protozoa
Parasites
parasites
assays
Spinacia oleracea
Polymerase Chain Reaction
Cysts
Microscopy
fresh produce
Cyclospora
spinach
Giardia
Cryptosporidium
microscopy
Cyclospora cayetanensis
Toxoplasma
Restriction Fragment Length Polymorphisms
Limit of Detection

ASJC Scopus subject areas

  • Food Science
  • Microbiology

Cite this

Simultaneous detection of four protozoan parasites on leafy greens using a novel multiplex PCR assay. / Shapiro, Karen; Kim, M.; Rajal, Veronica B.; Arrowood, Michael J.; Packham, Andrea; Aguilar, Beatriz; Wuertz, Stefan.

In: Food Microbiology, Vol. 84, 103252, 01.12.2019.

Research output: Contribution to journalArticle

Shapiro, Karen ; Kim, M. ; Rajal, Veronica B. ; Arrowood, Michael J. ; Packham, Andrea ; Aguilar, Beatriz ; Wuertz, Stefan. / Simultaneous detection of four protozoan parasites on leafy greens using a novel multiplex PCR assay. In: Food Microbiology. 2019 ; Vol. 84.
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