Detection of Cyclospora cayetanensis in Food and Water Samples: Optimized Protocols for Specific and Sensitive Molecular Methods from a Regulatory Agency Perspective
IF 2.1 4区 农林科学Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Mauricio Durigan , Laura Ewing-Peeples , Sonia Almeria , Kannan V. Balan , John Grocholl , Sachi Irizawa , Mark Mammel
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引用次数: 0
Abstract
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.