Development of Novel Biological Indicators to Evaluate the Efficacy of Microwave Processing

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ProQuest, 2008 - 166 pages
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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 121C 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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Contents

CHAPTER
1
Chapter 2
35
Chapter 3
60
Feasibility of utilizing bioindicators for testing microbial inactivation in sweetpotato
125
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