Mauricio Durigan , Laura Ewing-Peeples , Sonia Almeria , Kannan V. Balan , John Grocholl , Sachi Irizawa , Mark Mammel
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In this study, new more specific molecular markers for the detection of <em>C. cayetanensis</em> were developed based on updated genomic databases of Apicomplexa mitochondrial sequences. Novel alternative reagents and supplies, as well as optimization protocols, were tested in spiked produce and agricultural water samples. The selected Mit1C primers and probe combined showed at least 13 mismatches to other related species. The new optimized qualitative real-time PCR assay with modifications to sample processing and replacement of discontinued items produced results comparable to the previously validated methods. In conclusion, the new optimized qualitative Mit1C real-time PCR assay demonstrated an increase in its specificity in comparison to other detection methods previously published, while it showed to be robust and as sensitive as the previously validated method at the FDA. 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引用次数: 0
摘要
卡耶坦环孢子虫是吸虫门的一种球形寄生虫,可引起环孢子虫病,这是一种人类特有的胃肠道疾病。与大多数肠道病原体不同,卡耶坦球虫不会通过人与人之间的直接粪-口传播进行感染,因为脱落的卵囊必须暴露在环境诱因下才具有传染性。开发针对卡耶坦球虫的特异性和灵敏性检测方法对于有效解决数据缺口和在疫情调查期间提供监管支持至关重要。在这项研究中,基于最新的线粒体蚁属基因组数据库,开发了用于检测卡耶坦蚁的更特异的新分子标记。在农产品和农业用水样本中测试了新的替代试剂和耗材以及优化方案。所选的 Mit1C 引物和探针与其他相关物种至少有 13 处错配。新的优化定性实时 PCR 检测方法对样品处理进行了修改,并替换了停用的项目,其结果与之前验证的方法相当。总之,与之前公布的其他检测方法相比,新的优化 Mit1C 实时 PCR 定性检测方法提高了特异性,同时也显示出其稳健性和灵敏度不亚于 FDA 之前验证的方法。这项研究还扩大了可用于检测农产品和农业用水样本中 C. cayetanensis 的 PCR 试剂阵列,并对农业用水检测方法进行了多项改进,包括替换已停产的试剂和用于水过滤的新型透析过滤器。
Detection of Cyclospora cayetanensis in Food and Water Samples: Optimized Protocols for Specific and Sensitive Molecular Methods from a Regulatory Agency Perspective
Cyclospora cayetanensis is a coccidian parasite of the phylum Apicomplexa that causes cyclosporiasis, a human-specific gastrointestinal disease. Unlike most enteric pathogens, C. cayetanensis does not infect via direct fecal–oral transmission between humans because shed oocysts must be exposed to environmental triggers prior to becoming infectious. The development of specific and sensitive detection methods for C. cayetanensis is crucial to effectively address data gaps and provide regulatory support during outbreak investigations. In this study, new more specific molecular markers for the detection of C. cayetanensis were developed based on updated genomic databases of Apicomplexa mitochondrial sequences. Novel alternative reagents and supplies, as well as optimization protocols, were tested in spiked produce and agricultural water samples. The selected Mit1C primers and probe combined showed at least 13 mismatches to other related species. The new optimized qualitative real-time PCR assay with modifications to sample processing and replacement of discontinued items produced results comparable to the previously validated methods. In conclusion, the new optimized qualitative Mit1C real-time PCR assay demonstrated an increase in its specificity in comparison to other detection methods previously published, while it showed to be robust and as sensitive as the previously validated method at the FDA. This study has also expanded the array of PCR reagents that can be used to detect C. cayetanensis in produce and agricultural water samples and provided several improvements to the method for detection in agricultural water including replacements for discontinued items and a new dialysis filter for water filtration.
期刊介绍:
The Journal of Food Protection® (JFP) is an international, monthly scientific journal in the English language published by the International Association for Food Protection (IAFP). JFP publishes research and review articles on all aspects of food protection and safety. Major emphases of JFP are placed on studies dealing with:
Tracking, detecting (including traditional, molecular, and real-time), inactivating, and controlling food-related hazards, including microorganisms (including antibiotic resistance), microbial (mycotoxins, seafood toxins) and non-microbial toxins (heavy metals, pesticides, veterinary drug residues, migrants from food packaging, and processing contaminants), allergens and pests (insects, rodents) in human food, pet food and animal feed throughout the food chain;
Microbiological food quality and traditional/novel methods to assay microbiological food quality;
Prevention of food-related hazards and food spoilage through food preservatives and thermal/non-thermal processes, including process validation;
Food fermentations and food-related probiotics;
Safe food handling practices during pre-harvest, harvest, post-harvest, distribution and consumption, including food safety education for retailers, foodservice, and consumers;
Risk assessments for food-related hazards;
Economic impact of food-related hazards, foodborne illness, food loss, food spoilage, and adulterated foods;
Food fraud, food authentication, food defense, and foodborne disease outbreak investigations.