Browsing by Author "Trevor Phister, Committee Member"

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    Comparison of RNA and DNA-Based Amplification Methods for the Discrimination of Viable from Non-Viable Salmonella Typhimurium
    (2008-11-26) Bingham, James Edmund; MaryAnne Drake, Committee Member; Trevor Phister, Committee Member; Lee-Ann Jaykus, Committee Chair
    Although nucleic acid-based foodborne pathogen detection strategies offer promise, a consistent concern has been their poor correlation to bacterial cell viability, largely due to long-term persistence of DNA even after cell death. Evidence has suggested that RNA may be a better target than DNA for detection of viable cells. The purpose of this study was to compare a real-time RNA amplification method (nucleic acid sequence-based amplification or NASBA) to a similar real-time DNA-based PCR method with respect to the ability to detect viable bacterial cells. Salmonella enterica serovar Typhimurium was grown in both pure culture and a chicken carcass rinse and subjected to different time and temperature conditions (no heat, 2.5-5 min at 60C, 10-20 min at 60C, and 15 min at 121C) followed by isolation and purification of both DNA and RNA and cultural enumeration by direct plating. Isolated RNA and DNA were amplified by real-time NASBA or molecular Beacon, SYBR Green, and/or TaqMan® real-time PCR, respectively. Detection limits for each assay were determined using a modified MPN approach. The SYBR Green and the TaqMan®-based PCR assays had a detection limit of 102-103 CFU/ml for each replicate, with a similar log linear quantification range (approximately 2.0-9.0 log10 CFU/ml); in comparison, the real-time NASBA assay had a detection limit of 104 CFU/ml indicating a less sensitive assay. Exposure of S. Typhimurium cells to 60C for 2.5-5 min resulted in a 4 log10 reduction in viable count when compared to the control (unheated) samples, while an >8 log10 reduction in viable count was observed after exposure to heat for 10-20 min at 60C or 15 min at 121C, regardless of the matrix. Based on the MPN equivalents estimate, there was no statistically significant difference between detection of S. Typhimurium when comparing treatments for any one real-time PCR method in either matrix. There was a gradual reduction in real-time NASBA signal as the heat treatments increased in severity for each matrix. The RNA-based amplification assay was more indicative of bacterial cell viability than was a parallel DNA-based amplification assay, suggesting that RNA may be a more reliable target in this regard. While the real-time NASBA assay was a better indicator of cell viability, it appears that mRNA can still be detected up to one hour after cell death. Consequently, the detection of either DNA or RNA using nucleic acid amplification methods cannot be relied upon to completely indicate the presence of live target bacterial cells.
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    Genomic Analysis of Lactobacillus gasseri Strains using Suppressive Subtractive Hybridization .
    (2010-04-08) Schroeter, Joel; Todd Klaenhammer, Committee Chair; Robert Kelly, Committee Member; Trevor Phister, Committee Member