Use of Viability qPCR for Quantification of Salmonella Typhimurium and Listeria monocytogenes in Food Safety Challenge Studies
Open Access
- Author:
- Connolly, Charles
- Graduate Program:
- Food Science
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- February 23, 2021
- Committee Members:
- Catherine Nettles Cutter, Thesis Advisor/Co-Advisor
Jonathan Alexander Campbell, Committee Member
Edward G Dudley, Committee Member
Jasna Kovac, Committee Member
Robert F Roberts, Program Head/Chair - Keywords:
- Salmonella
Listeria
Challenge Studies
qPCR
Food Safety
Viability qPCR - Abstract:
- Validating food safety interventions can be expensive, time-intensive, and resource-intensive using culture-based plate and count methods (PAC). Viability Quantitative Polymerase Chain Reaction (qPCR) has the potential to increase speed, while also reducing costs and waste associated with quantifying pathogens in challenge studies. The purpose of this research was to develop an efficient viability qPCR protocol and compare its ability to quantify viable pathogens with that of PAC in two, small-scale challenge studies. Listeria monocytogenes and Salmonella enterica serovar Typhimurium were chosen as pathogens of interest due to their association with many foodborne outbreaks, as well as the inherent physiological differences between the two organisms. Development of the viability qPCR protocol began with the selection and validation of qPCR primers, probes, and reagents. Standard curves measuring the qPCR reaction efficiency for L. monocytogenes yielded efficiencies of 96.9±2.1% (mean, σ, n=9). S. Typhimurium yielded standard curves with qPCR reaction efficiencies of 96.1±1.5% (mean, σ, n=9). R2 values for all curves exceeded 0.99. These results were well within the acceptable range of 90% to 110% for efficiency and R2 values above the 0.99. Four DNA extraction kits were then selected and tested for highest yield of pathogen target sequence. Of the four DNA extraction kits tested, Kit Q, the Qiagen DNeasy PowerFood Microbial Kit, demonstrated the greatest ability to extract the target sequences for qPCR analysis of L. monocytogenes and S. Typhimurium at multiple dilution levels (p ≤ 0.05, n=8). To mitigate the PCR amplification of DNA originating from dead cells, the performance of multiple DNA intercalating agents was evaluated. Of the six DNA intercalation treatments tested, treatment P using the product PMAxx, demonstrated the greatest reduction of dead cell DNA amplification across a variety of conditions, without reducing live cell signal (p ≤ 0.05, n=9). The optimized viability qPCR protocol was compared to PAC in challenge studies utilizing two different intervention steps: 60°C heat treatment of experimentally inoculated ground beef and a 6% lauric arginate (LAE) dip of experimentally inoculated meat. In the heat challenge study, qPCR and PAC yielded similar starting cell estimates for both organisms; however, qPCR overestimated final counts by 4.94 log10 CFU/mL for L. monocytogenes and 3.31 log10 CFU/mL for S. Typhimurium (p ≤ 0.05, n=9). In the LAE experiments, starting estimates for qPCR and PAC were similar for S. Typhimurium; however, the qPCR underestimated the starting L. monocytogenes counts by 0.82 log10 CFU/mL (p ≤ 0.05, n=12). qPCR underestimated live cells in LAE-treated S. Typhimurium samples by 2.59 log10 CFU/mL (p ≤ 0.05, n=12). L. monocytogenes results trended similarly. These results highlight the potential use of viability qPCR for quantifying pathogens in challenge studies. However, more research is needed to address method limitations. While live cell qPCR estimates were accurate in 3 of the 4 conditions tested, intervention-treated live cells were both overestimated and underestimated, depending on the intervention used. If further development can overcome these challenges, qPCR holds promise for improving food safety and replacing costly, resource intensive, culture-based quantification methods for pathogens.