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.