Location: Characterization and Interventions for Foodborne Pathogens
Title: Investigating the quantification capabilities of a nanopore-based sequencing platform for food safety application via external standards of lambda DNA and lambda spiked beefAuthor
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Harper, Sky |
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Counihan, Katrina |
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Kanrar, Siddhartha |
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Paoli, George |
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Tilman, Shannon |
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Gehring, Andrew |
Submitted to: Foods
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 10/11/2024 Publication Date: 10/18/2024 Citation: Harper, S.A., Counihan, K.L., Kanrar, S., Paoli, G., Tilman, S.M., Gehring, A.G. 2024. Investigating the quantification capabilities of a nanopore-based sequencing platform for food safety application via external standards of lambda DNA and lambda spiked beef. Foods. 13:3304. https://doi.org/10.3390/foods13203304. DOI: https://doi.org/10.3390/foods13203304 Interpretive Summary: There are 600 million cases of disease and roughly 420,000 deaths that occur globally each year due to foodborne pathogens. Current methods to screen and identify pathogens in swine, poultry, and cattle products include immunoassays, molecular methods such as PCR, and traditional culturing. However, these methods are often used in tandem to screen, quantify, and characterize samples, prolonging real-time comprehensive analysis. Next-generation sequencing (NGS) is a new technology that combines sequencing chemistry, matrices, and bioinformatics technology making it capable to rapidly sequence long strands of DNA as well as whole genomes. The goal of this project was to evaluate the quantitative capabilities of a commercial real-time NGS sequencer. This investigation explored the correlation between known amounts of the analyte (lambda DNA; pathogenic bacterial surrogate) with data output, in both the absence and presence of a background matrix (bovine DNA). This endeavor was successful in demonstrating a direct correlation between the concentration of analyte with the amount of data produced, number of bases sequenced, and number of reads generated in the absence and presence of a background matrix. In the presence of bovine DNA, the sequenced data successfully matched an NCBI reference to identify the analyte. Furthermore, the sample analysis took less than 3 hours, demonstrating the applicability of this NGS sequencer in food safety as a rapid method for screening, identification, and quantification. Technical Abstract: There are 600 million cases of disease and roughly 420,000 deaths that occur globally each year due to foodborne pathogens. Current methods to screen and identify pathogens in swine, poultry, and cattle products include immuno-based techniques (e.g., immunoassay integrated biosensors), molecular methods (e.g., DNA hybridization and PCR assays), and traditional culturing. However, these methods are often used in tandem to screen, quantify, and characterize samples, prolonging real-time comprehensive analysis. Next-generation sequencing (NGS) is a new technology that combines sequencing chemistry, matrices, and bioinformatics, which makes it capable of rapidly sequencing long strands of DNA as well as whole genomes. The goal of this project was to evaluate the quantitative capabilities of the real-time NGS Oxford Nanopore Technologies’ MinION sequencer. This investigation explored the correlation between known amounts of the analyte (lambda DNA; pathogenic bacterial surrogate) with data output, in both the absence and presence of a background matrix (Bos taurus DNA). This endeavor was successful in demonstrating a positive linear correlation between the concentration of analyte with the amount of data produced, number of bases sequenced, and number of reads generated in the absence and presence of a background matrix. In the presence of Bos taurus DNA, the sequenced data was successfully mapped to an NCBI lambda reference genome to identify the analyte. Furthermore, the workflow from pre-extracted DNA to target identification was executed in less than 3 hours, demonstrating the potential of long-read sequencing in food safety as a rapid method for screening, identification, and quantification. |