Contents lists available at ScienceDirect Food Microbiology journal homepage: www.elsevier.com/locate/fm Simultaneous detection of four protozoan parasites on leafy greens using a novel multiplex PCR assay ☆ Karen Shapiro a,* , Minji Kim a,b , Veronica B. Rajal c,d , Michael J. Arrowood e , Andrea Packham a , Beatriz Aguilar a , Stefan Wuertz b,d,f a Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California, Davis, CA, 95616, USA b Department of Civil and Environmental Engineering, University of California, Davis, One Shields Avenue, Davis, CA, 95616, USA c Instituto de Investigaciones para la Industria Química (INIQUI), CONICET, Facultad de Ingeniería, Universidad Nacional de Salta (UNSa), Av. Bolivia 5150, Salta, 4400, Argentina d Singapore Centre for Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University (NTU), 60 Nanyang Drive, Singapore, 637551, Singapore e Waterborne Disease Prevention Branch, Division of Foodborne, Waterborne, and Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA f School of Civil and Environmental Engineering, NTU, 50 Nanyang Avenue, Singapore, 639798, Singapore 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. 1. Introduction Consumer demand for “ready to eat” fresh produce has been con- sistently rising as people modify dietary practices to eat more healthy foods without investing time in preparation. This economic reality has consequences for food safety for the simple reason that the shelf life of fresh foods like leafy vegetables is short, implying time from field to fork can facilitate transmission of pathogenic organisms present in soil or water that has contaminated crops. Water and foodborne pathogens currently represent the most important cause of enteric illness, with diarrhea representing the most prevalent infectious illness worldwide (Neira and Pruss-Ustun, 2016). Pathogens can be introduced on fresh produce during cultivation through contact with contaminated irriga- tion water (Amoros et al., 2010; Mota et al., 2009; Thurston-Enriquez et al., 2002), soil or fertilizer, as well as via handling and downstream processing and packaging steps. Current industry practices for monitoring microbial quality either focus on bacterial indicators that are poorly correlated with the presence of diverse classes of pathogens, or rely on the detection of specific bacterial pathogens (e.g., Salmonella and Listeria) after cultivation to ascertain food safety. Protozoan pa- thogens, on the other hand, are rarely tested for in fresh produce. Yet protozoan contamination of fresh produce is of growing importance – in both advanced and emerging market economies (Caradonna et al., 2017; Dixon, 2016; Lalonde and Gajadhar, 2016). Four key foodborne protozoan pathogens, including Cryptosporidium, Giardia, Cyclospora cayetanensis, and Toxoplasma gondii, were targeted in this study due to their current or projected capacity to cause significant illness in fresh produce consumers. Cyclospora cayetanensis and Cryptosporidium spp. are currently con- sidered the most important protozoan pathogens, based on the number of disease outbreaks in people that have been associated with con- taminated fresh produce (Ramos et al., 2013). A multistate outbreak of https://doi.org/10.1016/j.fm.2019.103252 Received 17 December 2018; Received in revised form 5 April 2019; Accepted 23 June 2019 ☆ Author Note: The findings and conclusions in this report are those of the author(s) and do not necessarily represent the views of the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry. * Corresponding author. 4206 VM3A, SVM: PMI, University of California, Davis, CA, 95616, USA. E-mail address: kshapiro@ucdavis.edu (K. Shapiro). Food Microbiology 84 (2019) 103252 Available online 24 June 2019 0740-0020/ © 2019 Elsevier Ltd. All rights reserved. T