BACKGROUND Zoonotic taeniid cestodes are amongst the most important food-borne parasites affecting human health worldwide. Contamination of fresh produce with the eggs of Echinococcus granulosus (s.l.), Echinococcus multilocularis, and some Taenia species pose a potential food safety risk. However, very few studies have attempted to investigate the potential contamination of fresh produce with taeniid eggs and the available methods are not standardized for this purpose. Established protocols do exist for testing leafy greens and berries for contamination with protozoan parasites and are used in national surveillance programmes. This methodology could be suitable for the detection of taeniids. The objective of this project was to develop and standardize a sensitive and reliable method to detect contamination of leafy greens and berries with eggs of zoonotic taeniids and to differentiate between E. multilocularis, E. granulosus (s.l.) and Taenia spp. METHODS We compared the efficacy of different wash solutions to remove Taenia spp. eggs from spiked produce, assessed two DNA extraction kits for their performance on Taenia spp. eggs, and adapted a published conventional multiplex PCR into a real-time PCR with fluorescence melting curve analysis (MCA) that was optimized for use on produce washes. Analytical specificity of this protocol was assessed using non-spiked produce washes as well as a variety of other potentially contaminating parasites. RESULTS The protocol as established in this study had an analytical sensitivity of detecting five eggs per spiked sample for both romaine lettuce and strawberries. Unequivocal identification of E. multilocularis, E. granulosus (s.l.) and Taenia spp. was possible through MCA. Amplicon sequencing allowed identification of Taenia to the species level. The real-time PCR also amplified DNA from Dicrocoelium sp., but with a clearly discernable melting curve profile. CONCLUSION The new protocol for screening produce for taeniid contamination was highly sensitive. Melting curve analysis and the possibility of amplicon sequencing made this assay very specific. Once further validated, this method could be employed for surveillance of produce for contamination with taeniid parasites to assess potential risks for consumers.

中文翻译：

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使用实时PCR和熔解曲线分析技术来分离，检测和区分叶类蔬菜和浆果中的鸟卵的新方法。

背景技术人畜共患病的eni虫是全世界范围内影响人类健康的最重要的食源性寄生虫。新鲜农产品被颗粒棘球oc虫卵，多囊棘球Ta虫卵和某些Ta虫物种污染，构成潜在的食品安全风险。然而，极少有研究试图研究棕褐色鸡蛋对新鲜农产品的潜在污染，目前尚无标准化的方法可用于此目的。确实存在用于测试绿叶和浆果中是否存在原生动物寄生虫污染的已建立规程，并且已在国家监视计划中使用。该方法可能适合于检测ta虫。该项目的目的是开发和标准化一种灵敏而可靠的方法，以检测人畜共患病的eni蛇类卵对绿叶蔬菜和浆果的污染，并区分多叶大肠杆菌，粒状大肠杆菌和Ta虫。方法我们比较了不同洗涤液去除Ta虫的功效。加标农产品中的卵，评估了两种DNA提取试剂盒在Taenia spp上的表现。鸡蛋，然后将已发布的常规多重PCR修改为具有荧光熔解曲线分析（MCA）的实时PCR，该荧光熔解曲线分析已针对在农产品洗涤中的使用进行了优化。使用未加标的农产品洗涤液以及各种其他可能污染的寄生虫评估了该方案的分析特异性。结果本研究建立的方案具有分析灵敏度，可以检测到每个加标样品中的生菜和草莓都含有五个鸡蛋。明确鉴定多叶E. granulosus（sl）和Taenia spp。通过MCA是可能的。扩增子测序允许在物种水平上鉴定of虫。实时PCR还扩增了Dicrocoelium sp。的DNA，但具有清晰可辨的熔解曲线。结论筛选农产品中的烯污染的新方案是高度敏感的。熔解曲线分析和扩增子测序的可能性使该测定非常特异性。一旦进一步验证，该方法可用于监视农产品中是否被棕褐色寄生虫污染，以评估对消费者的潜在风险。