Metagenomic detection of protozoan parasites on leafy greens aided by a rapid and efficient DNA extraction protocol

Sohail Naushad ¹ Ruimin Gao ² Marc-Olivier Duceppe ¹ Andree Ann Dupras ¹ Sarah Julia Reiling ³ Harriet Merks ⁴ Brent Dixon ⁴ Dele Ogunremi ^1*

• ¹ Ottawa Laboratory Fallowfield, Canadian Food Inspection Agency (CFIA), Ottawa, Ontario, Canada
• ² Respiratory Viruses and Coronaviruses Section, National Microbiology Laboratory, Public Health Agency of Canada (PHAC), Winnipeg, Manitoba, Canada
• ³ McGill University Health Centre, Montreal, Quebec, Canada
• ⁴ Bureau of Microbial Hazards, Health Canada, Ottawa, Ontario, Canada

Infections with protozoan parasites associated with the consumption of fresh produce is an on-going issue in developed countries but mitigating the risk is hampered by the lack of adequate methods for their detection and identification. We developed a metagenomic next-generation sequencing (mNGS) method using a MinION sequencer for the identification of parasites in intentionally contaminated lettuce to achieve a more accurate and rapid method than the traditional molecular and microscopy methods commonly used for regulatory purposes. The efficient lysis of oocysts and cysts was a prerequisite for the sensitive detection of parasite DNA and was rapidly achieved within 3 min. Amplification of extracted DNA led to the generation of 0.16 -8.25 μg of DNA (median = 4.10 μg), sufficient to perform mNGS. Nanopore sequencing followed by data analysis using the CosmosID webserver led to the consistent identification of as few as 100 Cryptosporidium parvum oocysts in 25 g of fresh lettuce. The assay proved useful for the simultaneous detection of Cryptosporidium parvum, C. hominis, C. muris, Giardia duodenalis and Toxoplasma gondii. This novel mNGS assay, therefore, has the potential to be used as a single universal test for diagnostics of foodborne parasites, and subtyping of parasites for foodborne outbreak investigations and surveillance studies.