Evaluation of nucleic acid extraction methods for recovery of Cyclospora cayetanensis, Salmonella enterica, and murine norovirus from water and sludge

The coccidian parasite Cyclospora cayetanensis is the causative agent for foodborne outbreaks of cyclosporiasis and multiple fresh produce recalls annually. In recent years, this organism has been reported in the water near produce growing operations during outbreak investigations, prompting a call for more research on its environmental prevalence in the United States. Currently, there is a lack of performance data available on methods for conducting this research, including the performance of DNA extraction methods for molecular testing. Extraction methods for environmental samples must be efficient due to the often-limited amount of target nucleic acid and the potential for molecular inhibitors present in an environmental sample. This study assessed the performance of C. cayetanensis nucleic acid extraction seeded into surface water, produce wash water, and tap water by two methods designed for use with environmental samples: the PowerViral and UNEX methods. The PowerSoil extraction method (2 g) was assessed for C. cayetanensis extraction from seeded sewage sludge - an environmental sample type used to evaluate parasite carriage within communities. Extraction performance of the PowerViral and UNEX methods were also assessed for the detection of the foodborne bacterial pathogen Salmonella and a surrogate for foodborne viruses, murine norovirus (MNV) seeded into surface water, produce wash water, and tap water. The PowerViral method resulted in consistent detection (83-100 %) of C. cayetanensis, S. enterica, and MNV across all water types. Detection rates for the UNEX method ranged from 56 to 100 % prevalence for tap water and wash water, but there were no detections for any microbe from surface water. The PowerSoil method resulted in poor recovery of C. cayetanensis from sludge (≤1 % recovery), while both the PowerViral and UNEX methods effectively recovered C. cayetanensis from sludge (4-36 % recovery).