Optimization of the Mit1C real-time PCR assay using Bio-Rad CFX96 for the detection of Cyclospora cayetanensis in agricultural waters.

Increased cases of cyclosporiasis within the United States linked to domestically grown fresh produce highlight the need to identify sources of environmental contamination for Cyclospora cayetanensis. Recently, the Mit1C real-time PCR assay was validated by the U.S. FDA as the US-FDA's BAM 19c for molecular detection of C. cayetanensis Cox3 gene in agricultural water using the now discontinued ABI-7500 platform. This study optimized the Mit1C real-time PCR assay on the Bio-Rad CFX96 platform for molecular detection of C. cayetanensis in environmental water. Using the Mit1AA synthetic fragment suspended in a 5 µg/mL salmon sperm carrier for standard curves, various combinations of cycling times, annealing temperatures, and primer/probe concentrations were optimized to achieve reproducible, sensitive, specific, and efficient amplification signal. Cross-reactivity was addressed with an exclusivity panel alongside an agricultural water positive control spiked with 200 C. cayetanensis oocysts and com-pared on both platforms. Results suggested only decreasing the annealing temperature (66°C), increasing cycles (42), and increasing cycle cutoff (Cq ≤ 40) to achieve performance comparable to the ABI-7500. Directly utilizing the ABI-7500 protocol for the Bio-Rad CFX96 platform diminished assay sensitivity and efficiency. We adapted the Mit1C real-time PCR protocol for the Bio-Rad CFX96 platform and suggest that instrument differences can have considerable impact on assay performance.