Abstract
Cyclosporiasis, caused by the coccidian parasite Cyclospora cayetanensis, has emerged as an increasing global public health concern, with the incidence of laboratory-confirmed domestically acquired cases in the US exceeding 10,000 since 2018. A recently published qPCR assay (Mit1C) based on a mitochondrial target gene showed high specificity and good sensitivity for the detection of C. cayetanensis in fresh produce. The present study shows the integration and verification of the same mitochondrial target into a fully automated and streamlined platform that performs DNA isolation, PCR, hybridization, results visualization, and reporting of results to simplify and reduce hands-on time for the detection of this parasite. By using the same primer sets for both the target of interest (i.e., Mit1C) and the internal assay control (IAC), we were able to rapidly migrate the previously developed Mit1C qPCR assay into the more streamlined and automated format Rheonix C. cayetanensisTM Assay. Once the best conditions for detection were optimized and the migration to the fully automated format was completed, we compared the performance of the automated platform against the original "bench top" Mit1C qPCR assay. The automated Rheonix C. cayetanensis Assay achieved equivalent performance characteristics as the original assay, including the same performance for both inclusion and exclusion panels, and it was able to detect as low as 5 C. cayetanensis oocysts in fresh produce while significantly reducing hands-on time. We expect that the streamlined assay can be used as a tool for outbreak and/or surveillance activities to detect the presence of C. cayetanensis in produce samples.