Multi-laboratory validation of a modified real-time PCR assay (Mit1C) for the detection of Cyclospora cayetanensis in fresh produce.

Cyclospora cayetanensis is a foodborne protozoan parasite that causes the human diarrheal disease cyclosporiasis. Recently, the US FDA developed a modified real-time PCR method based on a specific mitochondrial target gene (Mit1C) to detect C. cayetanensis in fresh produce. The method was validated by single laboratory validation (SLV) studies in Romaine lettuce, cilantro, and raspberries. The present study aimed to evaluate the performance of the new real-time Mit1C (Mit1C qPCR) method by comparing it with the current BAM Chapter 19b qPCR (18S qPCR) as the reference method for the detection of the protozoan parasite C. cayetanensis in fresh produce in a multi-laboratory validation (MLV) setting with the participation of 13 collaborating laboratories. Each laboratory analyzed twenty-four blind-coded Romaine lettuce DNA test samples that included: two unseeded samples, three samples seeded with five oocysts, and one sample seeded with 200 oocysts in the first round and five unseeded samples, eight samples seeded with five oocysts, and five samples seeded with 200 oocysts in the second round. The overall detection rates across laboratories for Romaine lettuce samples inoculated with 200 and 5 oocysts and un-inoculated samples were 100% (78/78), 69.23% (99/143), and 1.1% (1/91), respectively, for Mit1C qPCR, and 100% (78/78), 61.54% (88/143) and 0% (0/91), respectively, for 18S qPCR. The relative level of detection (RLOD = LOD50, Mit1C/LOD50, 18S) was 0.81 with a 95% confidence interval (0.600, 1.095), which included 1. Thus, Mit1C qPCR and 18S qPCR had statistically similar levels of detection. Mit1C qPCR was highly reproducible as the between-laboratory variance in the test results was nearly zero (0) and showed a high specificity at 98.9%. In conclusion, this study demonstrated that the new, more specific Mit1C qPCR method is an effective alternative analytical tool for detection of C. cayetanensis in fresh produce.