Development of Novel Biological Indicators to Evaluate the Efficacy of Microwave Processing
ProQuest, 2008 - 166 pages
In the second study, a molecular method was developed for the detection and discrimination of viable and non-viable spores of Clostridium sporogenes. In the first phases of the research, a method to extract spore-associated DNA was developed, which included the combined steps of decoating and lysozyme digestion. The process resulted in recovery of high yields of quality DNA. A Sybr green-based quantitative real-time (qPCR) assay targeting the GerAB gene was also designed. When combined with the DNA extraction steps, the assay was log linear over a range of from 102-10 9 spores/mL, with a lower limit of detection of approximately 10 2 spores/mL. It was confirmed that exposure of spores to 121°C for as little as 2 min resulted in near complete degradation of the DNA and loss of PCR amplifiability, suggesting that under stringent heat treatment, the PCR-based method would be able to distinguish viable spores from those which had been killed. Under less stringent processing conditions, the DNA from non-viable spores remained detectable. In this case, the decoated spores were treated a 12.5 mug/ml concentration of propidium monoazide (PMA), a DNA intercalating agent, which selectively enters inactivated bacterial cells, binds to the DNA and makes it unavailable for amplification. Unfortunately, the PMA was not able to selectively inhibit the amplification of DNA derived from dead spores. This was evidenced by the fact that CT values obtained for live and thermally treated spores were nearly identical, regardless of PMA treatment status. Further options for the selective detection of DNA derived from viable spores are under investigation.
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Feasibility of utilizing bioindicators for testing microbial inactivation in sweetpotato
100 spores/ml spores/ml acid alginate beads amplification Appl Bacillus stearothermophilus Bacillus subtilis bacterial cells bacterial spores Bacteriol Bell and Kyriakides biological indicators botulism buffer capillary tube cell wall Chipley Clostridium botulinum Clostridium sporogenes commercial sterilization cortex CT values dead bacterial detection dielectric DNA extraction Driks endospores enumeration Environ Figure forespore Geobacillus germination heat resistance Holdsworth Hulland immobilized spores inactivated spores incubation lethality lysozyme method Microbiol microorganisms microwave heating microwave processing Montville Montville and Matthews multiphase aseptic processing multiphase food product NACMCF Nocker nutrients peptidoglycan phase contrast microscopy PMA treatment pouches qPCR Quesnel real-time PCR Russell salsa con queso Serp Shapiro simulated particles sodium alginate sodium citrate spore coat extraction spore crop spore populations spore suspension sporogenes spores sporulation stearothermophilus D-values stearothermophilus spores sterile dH2O subtilis and G subtilis D-values surrogate sweetpotato puree target Technol thermal inactivation thermal processing toxin vegetative cells viable spores