The protozoan parasites Cryptosporidium spp., Giardia duodenalis and Toxoplasma gondii can be transmitted to humans through shellfish consumption. No standardized methods are available for their detection in these foods, and the performance of the applied methods are rarely described in occurrence studies. Through spiking experiments, we characterized different performance criteria (e.g. sensitivity, estimated limit of detection (eLD95_METH), parasite DNA recovery rates (DNA-RR)) of real-time qPCR based-methods for the detection of the three protozoa in mussel's tissues and hemolymph. Digestion of mussels tissues by trypsin instead of pepsin and the use of large buffer volumes was the most efficient for processing 50g-sample. Trypsin digestion followed by lipids removal and DNA extraction by thermal shocks and a BOOM-based technique performed poorly (e.g. eLD95_METH from 30 to >3000 parasites/g). But trypsin digestion and direct DNA extraction by bead-beating and FastPrep homogenizer achieved higher performance (e.g. eLD95_METH: 4–400 parasites/g, DNA-RR: 19–80%). Direct DNA recovery from concentrated hemolymph, by thermal shocks and cell lysis products removal was not efficient to sensitively detect the protozoa (e.g. eLD95_METH: 10–1000 parasites/ml, DNA-RR ≤ 24%). The bead-beating DNA extraction based method is a rapid and simple approach to sensitively detect the three protozoa in mussels using tissues, that can be standardized to different food matrices. However, quantification in mussels remains an issue.

原生动物寄生虫隐孢子虫、十二指肠贾第鞭毛虫和弓形虫可通过食用贝类传播给人类。没有标准化的方法可用于检测这些食物中的它们，并且在发生研究中很少描述所应用方法的性能。通过加标实验，我们表征了不同的性能标准（例如灵敏度、估计检测限（eLD95 _METH)、寄生虫 DNA 回收率 (DNA-RR)) 的实时 qPCR 方法检测贻贝组织和血淋巴中的三种原生动物。用胰蛋白酶代替胃蛋白酶消化贻贝组织，使用大缓冲液体积对处理 50g 样品是最有效的。胰蛋白酶消化，然后通过热冲击和基于 BOOM 的技术去除脂质和 DNA 提取效果不佳（例如，eLD95 _METH从 30 到 >3000 寄生虫/g）。但是胰蛋白酶消化和通过珠打和 FastPrep 匀浆器直接提取 DNA 获得了更高的性能（例如 eLD95 _METH: 4–400 寄生虫/g, DNA-RR: 19–80%)。通过热冲击和细胞裂解产物去除从浓缩血淋巴中直接回收 DNA 不能有效地灵敏检测原生动物（例如 eLD95 _METH：10–1000 寄生虫/ml，DNA-RR ≤ 24%）。基于珠子跳动 DNA 提取的方法是一种使用组织灵敏检测贻贝中三种原生动物的快速简单方法，可以标准化为不同的食物基质。然而，贻贝的量化仍然是一个问题。