美国食品药品局细菌分析手册（BAM）第19b章：使用实时PCR对新鲜农产品中的环孢子虫进行分子检测 （2024年12月版）BAM Chapter 19b Molecular Detection of Cyclospora cayetanensis in Fresh Produce Using Real Time PCR

Cyclospora cayetanensis is a protozoan parasite that causes a human diarrheal disease called cyclosporiasis (1). Infected individuals shed spherical (8-10 μm in diameter) unsporulated oocysts into the environment with feces. Person-to-person transmission is considered unlikely because oocysts require a week or longer in the environment to sporulate and become infective. C. cayetanensis oocysts are not culturable, which makes the application of viability assays impractical. According to the latest surveillance data, 2,272 cases of cyclosporiasis were reported from 40 states in 2023 without travel history outside the US indicating that they were acquired domestically. Illnesses and outbreaks caused by C. cayetanensis are mainly associated with the consumption of fresh produce such as herbs, berries, and leafy greens (2).
The present FDA BAM Chapter 19b method for detection of C. cayetanensis in fresh produce was validated under the FDA Guidelines for the Validation of Analytical Methods for the Detection of Microbial Pathogens in Foods and Feeds, third Edition (4). The method includes three steps: 1) Produce wash procedure; 2) Isolation of DNA from produce wash, and 3) Detection by qPCR amplification based on the detection of the C. cayetanensis mitochondrial Cox3 gene (Mit1C qPCR).
The Mit1C target provides a region on the mitochondrial genome of C. cayetanensis that is specific to C. cayetanensis based on current multiple sequence alignment using BLAST searches against C. cayetanensis and other genera/species (e.g., Eimeria spp, and Isospora spp.) in the Apicomplexa phylum. The real-time PCR was optimized using Prime TimeTM Gene Expression master mix from Integral DNA technologies (IDT) and the method was developed and validated for use in the ABI 7500 Fast platform. The method was validated by a single validation study that included exclusivity, inclusivity, and Mit1C qPCR detection in C. cayetanensis inoculated food matrices in three commodities (raspberries, cilantro and Romaine lettuce) compared to the previous 18S qPCR method (4). Then, the new method was validated for Romaine lettuce in a multi-laboratory validation study (5) approved by the Microbiology Methods Validation Subcommittee in November 2024.

更多内容请查看附件。