US Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, Laurel, MD, USA.
JIFSAN, University of Maryland, College Park, MD, USA.
Food Microbiol. 2021 Jun;96:103719. doi: 10.1016/j.fm.2020.103719. Epub 2020 Dec 23.
Although multiple outbreak clusters of Cyclospora cayetanensis have been traced back to consumption of dishes in Mexican-style restaurants, the FDA Bacteriological Analytical Manual (BAM) does not currently provide methods to detect C. cayetanensis in dishes that contain multiple produce ingredients, such as salsas and guacamole. These complex food matrices also may contain high levels of fats, which can interfere with the detection. Several modifications to the BAM Chapter 19b method (washing produce, DNA extraction, and a TaqMan real-time PCR assay targeting the 18S rRNA gene of C. cayetanensis) were assessed with the goal to detect as few as 5 oocysts of C. cayetanensis in 25 g samples of commercial salsa/pico de gallo, guacamole, and salsa verde. Both freshly prepared and frozen versions of these foods were seeded with 5, 10 and 200 oocysts. For salsa samples, using a gentler washing step than recommended by BAM, we achieved detection of 5 oocysts in the samples (81.8%, n = 11). Increasing the amount of Alconox® in the wash solution to 1%, rather than the 0.1% used in BAM, and adjusting the DNA extraction protocol to process large wash pellets, enabled detection of 5 oocysts in guacamole. To reach the desired level of detection in salsa verde, two types of modifications were necessary: gentler washing and DNA extraction modifications. The use of these same method modifications on previously frozen food samples, provided levels of detection similar to those achieved with fresh dishes. Our modifications enabled robust and reproducible detection of C. cayetanensis in multi-ingredient Mexican dishes, detecting as few as 5 oocysts in 25 g samples. Validating and deploying effective methods to detect C. cayetanensis in high risk fresh produce and prepared dishes are critically important for prevalence studies and outbreak investigations of this parasite.
尽管已经追溯到食用墨西哥式餐厅菜肴导致环孢子虫（Cyclospora cayetanensis）多次暴发集群，但美国食品药品监督管理局（FDA）的细菌分析手册（BAM）目前并未提供在含有多种农产品成分的菜肴中检测环孢子虫的方法，例如萨尔萨酱和鳄梨酱。这些复杂的食物基质可能还含有高脂肪含量，这可能会干扰检测。对 BAM 第 19b 章方法（清洗农产品、DNA 提取以及针对环孢子虫 18S rRNA 基因的 TaqMan 实时 PCR 检测）进行了几项修改，目标是在 25 克商业萨尔萨酱/莎莎酱、鳄梨酱和萨尔萨酱绿中检测到低至 5 个环孢子虫卵囊。对这些食物的新鲜和冷冻版本都用 5、10 和 200 个卵囊进行接种。对于莎莎酱样品，使用比 BAM 建议的更温和的清洗步骤，我们在样品中检测到 5 个卵囊（81.8%，n=11）。在洗涤液中增加到 1%的 Alconox®用量，而不是 BAM 中使用的 0.1%，并调整 DNA 提取方案以处理大的洗涤球，从而在鳄梨酱中检测到 5 个卵囊。要在萨尔萨酱绿中达到预期的检测水平，需要进行两种类型的修改：更温和的清洗和 DNA 提取修改。对先前冷冻食品样品使用相同的方法修改，提供了与新鲜菜肴相似的检测水平。我们的修改使在多成分墨西哥菜肴中对环孢子虫进行稳健且可重复的检测成为可能，在 25 克样品中检测到低至 5 个卵囊。验证并部署在高风险新鲜农产品和制备菜肴中检测环孢子虫的有效方法，对于这种寄生虫的流行率研究和暴发调查至关重要。
