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Manyun Yang

Multiplex identification of viable pathogens by nanopore metatranscriptome sequencing

Viable pathogenic bacteria are major biohazards that pose significant threats to food safety, public health, and industry stakeholders' economic well-being. Despite the recent developments in rapid detection platforms, quantification of viable pathogens in food remains a major challenge. In this study, we proposed to develop and validate metatranscriptome sequencing using the Oxford Nanopore MinION as a novel strategy to achieve real-time pathogen identification in food. Specifically, we developed a universal nanopore sample extraction and library preparation protocol applicable to all major foodborne pathogens. Nanopore sequencing of both bacteria transcriptome (RNA) and cDNA, produced by reverse transcription-polymerase chain reaction (RT-PCR), were evaluated and compared for their capacities. We were able to identify three viable bacterial targets from a community standard, comprised of E. coli O157:H7, Salmonella enterica and Listeria monocytogenes. Metatranscriptome sequencing requires a larger amount of RNA input, but it is RT-PCR free, while cDNA sequencing showed substantially better limit of detection and sensitivity. In this study, we demonstrated that nanopore sequencing could represent a significant breakthrough in multiplex detection of viable pathogens. They were readily deployable and easily transferable to many food safety & quality applications, including spoilage and pathogen monitoring, risk assessment, traceability, and outbreak inspection, food adulteration and integrity, quality assurance/quality control (QA/QC), and consumer point-of-care (POC) testing.