Novel Reagents and Approaches for Portable Sample Preparation and Detection of Foodborne Pathogens
| dc.contributor.advisor | Moore, Matthew | |
| dc.contributor.advisor | Brehm-Stecher, Byron | |
| dc.contributor.advisor | He, Lili | |
| dc.contributor.author | Stoufer, Sloane | |
| dc.contributor.department | Food Science | |
| dc.date.accessioned | 2025-08-15T19:40:39Z | |
| dc.date.available | 2025-08-15T19:40:39Z | |
| dc.date.issued | 2025-05 | |
| dc.description.abstract | Significant advances have been made in recent years to develop portable endpoint detection methods for foodborne virus detection, particularly in the form of isothermal nucleic acid amplification methods. However, these methods have significant drawbacks; namely, they can only analyze a very small sample volume, and are vulnerable to matrix-associated amplification inhibitors. If these isothermal amplification methods are to be effective in in-field settings, there is a need for equally portable sample preparation methods. The first step in any diagnostic assay is disinfection. Both intact microbes and residual nucleic acid from previous samples can contaminate nucleic acid-based detection assays, leading to false positive results. However, few surface disinfectants have been validated against free nucleic acid. For this reason, we tested the capacity of several commercial surface disinfectants to degrade three types of nucleic acid (viral ssRNA, eukaryotic DNA, PCR product). Only hypochlorite-based disinfectants were effective (dilute chlorine bleach and a commercial hypochlorite-based disinfectant). However, the bleach must be diluted fresh in distilled water before each use for best results, while the commercial disinfectant gave consistent results over several months without the need for extra preparation steps. Therefore, the commercial hypochlorite-based disinfectant would work better as part of a portable microbial detection kit. The next step is sample preparation, specifically target separation and concentration from the sample matrix. For this purpose, we evaluated a class of capture reagents known as magnetic ionic liquids (MILs) which would be ideal for in-field applications as their use requires minimal electrical equipment and no cold-chain storage. The MIL formulations used had already been evaluated for capture of bacterial pathogens from liquid food matrices, but had not yet been tested with non-enveloped viruses. Therefore, we tested a number of parameters impacting MIL-based capture and recovery of both an intact human norovirus surrogate (bacteriophage MS2) and purified viral ssRNA from aqueous suspension. All MIL formulations were effective for both targets, though they varied some in capture and recovery efficiency, and none appeared to significantly damage the virus capsid. We also determined that maximizing MIL dispersion is critical for ensuring optimal performance, and is determined by both the complexity of the input suspension and the relative volume of MIL used. We also showed that MILs have some capacity to concentrate the target. Most interestingly, they were able to effectively capture and recover free RNA while also appearing to give some protection from degradation in suspension. This raised the possibility that they could play a role in nucleic acid extraction. The last sample preparation step needed for nucleic acid-based detection is genomic extraction. However, though many established methods exist for viral nucleic acid extraction, few are designed with a focus toward in-field applications. Therefore, we explored the use of MILs as a binding substrate similar to the magnetic silica beads in commercial kits, which would enable target separation, concentration, and genomic extraction to occur in a single tube with minimal target loss. We found that some MIL formulations were able to recover target RNA at levels comparable to magnetic silica beads when used with commercial RNA extraction reagents. We also developed our own lysis, wash, and elution buffers that showed comparable performance to the commercial reagents when used with both MILs and magnetic silica beads. Though there is still much to explore, this work constitutes a meaningful step toward development of a complete and portable sample preparation method for foodborne virus detection. When combined with a portable endpoint detection method, this could significantly reduce barriers to in-field pathogen detection, facilitating faster outbreak tracking and more routine testing for foodborne viruses. | |
| dc.description.degree | Doctor of Philosophy (Ph.D.) | |
| dc.description.sponsorship | Work for chapter 3 and 4 was partially funded through the Chemical Measurement and Imaging Program at the National Science Foundation (Grant number CHE-2203891). Other funding sources for collaborators include the Alice Hudson Professorship at Iowa State University and the Iowa Agriculture and Home Economics Experiment Station Project No. IOW04202, sponsored by the Hatch Act and State of Iowa Funds. This work was also supported by the USDA National Institute of Food and Agriculture, Agricultural and Food Research Initiative Competitive Program, grant numbers: 2023-67011-40392 FELLOWSHIP and 2022-67021-36408. | |
| dc.identifier.orcid | https://orcid.org/0000-0002-5936-2968 | |
| dc.identifier.uri | https://hdl.handle.net/20.500.14394/57428 | |
| dc.language.iso | en_US | |
| dc.restriction.embargo | No | |
| dc.rights | Attribution-NonCommercial-NoDerivatives 4.0 International | en |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.title | Novel Reagents and Approaches for Portable Sample Preparation and Detection of Foodborne Pathogens | |
| dc.type | Dissertation (1 Year Campus Access Only) | |
| dspace.entity.type | Publication |