Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California, Davis, CA, 95616, USA.
Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California, Davis, CA, 95616, USA; Department of Civil and Environmental Engineering, University of California, Davis, One Shields Avenue, Davis, CA, 95616, USA.
Food Microbiol. 2019 Dec;84:103252. doi: 10.1016/j.fm.2019.103252. Epub 2019 Jun 24.
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.
新鲜农产品上的病原体污染对消费者的健康构成威胁；然而，农产品行业仍然缺乏同时检测原生动物寄生虫的足够工具。在这里，开发了一种简单的多重 PCR（mPCR）检测方法，用于检测原生动物（oo）囊，并与之前发表的实时 PCR 检测方法和显微镜方法进行了比较。该检测方法用于同时检测隐孢子虫、贾第虫、环孢子虫和刚地弓形虫，然后通过嵌套特异性 PCR 或限制性片段长度多态性（RFLP）检测进行寄生虫分化。使用菠菜作为模型叶菜进行了接种实验，以验证检测方法的有效性。与胃管处理相比，叶片洗涤可提高回收率并更一致地检测寄生虫。使用嵌套 mPCR 检测方法的最低检测限为 1-10（oo）个囊/克菠菜（在处理的 10 克样品中），该方法比 qPCR 更灵敏，可用于寄生虫检测。显微镜方法在较低的接种浓度下更可靠地用于寄生虫的视觉检测，但成本更高，更费力，需要额外的专业知识，并且缺乏准确风险评估所需的分子确认。总体而言，嵌套 mPCR 检测方法为新鲜农产品中同时检测原生动物病原体提供了一种快速（<24 小时）、廉价（$10 美元/样本）且简单的方法。
