Simultaneous detection of four protozoan parasites on leafy greens using a novel multiplex PCR assay. (December 2019)

• Record Type: Journal Article
• Authors: Shapiro, Karen; Kim, Minji; Rajal, Veronica B.; Arrowood, Michael J.; Packham, Andrea; Aguilar, Beatriz; Wuertz, Stefan
• Is Part Of: Food microbiology. Volume 84(2019)
• Publication Date: 2019-12
• Subjects: Food Microbiology -- Periodicals; Aliments -- Microbiologie -- Périodiques; Food -- Microbiology; Periodicals; Food -- Microbiology -- Periodicals; Food contamination -- Periodicals; 664.001579
• DOI: 10.1016/j.fm.2019.103252
• Languages: English
• ISSNs: 0740-0020

Abstract

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. Graphical abstract: Image 1 Highlights: Multiplex PCR was developed for easy and affordable protozoan parasite detection. Limits of detection were 1–10 (oo)cysts per gram spinach. Compared with a stomacher, manual wash of spinach yielded higher parasite recovery. Multiplex PCR performed better than qPCR when applied on spiked spinach.