Murphy Helen R, Lee Seulgi, da Silva Alexandre J
U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, Division of Food and Environmental Microbiology, Laurel, Maryland 20708, USA.
J Food Prot. 2017 Jul;80(7):1133-1144. doi: 10.4315/0362-028X.JFP-16-492.
Cyclospora cayetanensis is a protozoan parasite that causes human diarrheal disease associated with the consumption of fresh produce or water contaminated with C. cayetanensis oocysts. In the United States, foodborne outbreaks of cyclosporiasis have been linked to various types of imported fresh produce, including cilantro and raspberries. An improved method was developed for identification of C. cayetanensis in produce at the U.S. Food and Drug Administration. The method relies on a 0.1% Alconox produce wash solution for efficient recovery of oocysts, a commercial kit for DNA template preparation, and an optimized TaqMan real-time PCR assay with an internal amplification control for molecular detection of the parasite. A single laboratory validation study was performed to assess the method's performance and compare the optimized TaqMan real-time PCR assay and a reference nested PCR assay by examining 128 samples. The samples consisted of 25 g of cilantro or 50 g of raspberries seeded with 0, 5, 10, or 200 C. cayetanensis oocysts. Detection rates for cilantro seeded with 5 and 10 oocysts were 50.0 and 87.5%, respectively, with the real-time PCR assay and 43.7 and 94.8%, respectively, with the nested PCR assay. Detection rates for raspberries seeded with 5 and 10 oocysts were 25.0 and 75.0%, respectively, with the real-time PCR assay and 18.8 and 68.8%, respectively, with the nested PCR assay. All unseeded samples were negative, and all samples seeded with 200 oocysts were positive. Detection rates using the two PCR methods were statistically similar, but the real-time PCR assay is less laborious and less prone to amplicon contamination and allows monitoring of amplification and analysis of results, making it more attractive to diagnostic testing laboratories. The improved sample preparation steps and the TaqMan real-time PCR assay provide a robust, streamlined, and rapid analytical procedure for surveillance, outbreak response, and regulatory testing of foods for detection of C. cayetanensis.
卡耶塔环孢子球虫是一种原生动物寄生虫，可导致人类腹泻疾病，这与食用被卡耶塔环孢子球虫卵囊污染的新鲜农产品或水有关。在美国，食源性环孢子虫病暴发与各种进口新鲜农产品有关，包括香菜和树莓。美国食品药品监督管理局开发了一种改进方法，用于鉴定农产品中的卡耶塔环孢子球虫。该方法依靠0.1%的爱尔康诺克斯农产品清洗液高效回收卵囊，使用商业试剂盒制备DNA模板，并采用优化的TaqMan实时荧光定量PCR检测法及内部扩增对照，用于该寄生虫的分子检测。进行了一项单实验室验证研究，通过检测128个样本评估该方法的性能，并比较优化的TaqMan实时荧光定量PCR检测法和参考巢式PCR检测法。样本包括25克接种了0、5、10或200个卡耶塔环孢子球虫卵囊的香菜或50克接种了上述相同数量卵囊的树莓。接种5个和10个卵囊的香菜样本，实时荧光定量PCR检测法的检出率分别为50.0%和87.5%，巢式PCR检测法的检出率分别为43.7%和94.8%。接种5个和10个卵囊的树莓样本，实时荧光定量PCR检测法的检出率分别为25.0%和75.0%，巢式PCR检测法的检出率分别为18.8%和68.8%。所有未接种样本均为阴性，所有接种200个卵囊的样本均为阳性。两种PCR方法的检出率在统计学上相似，但实时荧光定量PCR检测法操作更简便，更不易发生扩增子污染，且能监测扩增过程并分析结果，使其对诊断检测实验室更具吸引力。改进后的样本制备步骤和TaqMan实时荧光定量PCR检测法为食品中卡耶塔环孢子球虫的监测、暴发应对及监管检测提供了一种强大、简化且快速的分析程序。
