36B-12 |
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S. E. HANNA, Dept. of Food Science and Technology, The Ohio State Univ., 2015 Fyffe Rd., Columbus, OH 43210 and H. Wang, Dept. of Food Science & Technology, Ohio State Univ., 2015 Fyffe Ct., 219 Parker Food Science Bldg., Columbus, OH 43210-1007. Changes in gene expression can provide key insights into how cells respond to different stimuli, but quantification of gene expression can be a challenging task. One way to achieve this is through relative quantification of gene expression, in which the expression of a gene of interest is compared to that of an internal standard gene, the expression of which remains unchanged during experimental conditions. Our objectives were to determine the suitability of the rplD gene, encoding the L4 ribosomal protein, to serve as an internal standard for gene expression study in Listeria monocytogenes, and to investigate the influence of stress conditions on L. monocytogenes virulence factor expression using real-time RT-PCR. Primers and TaqMan probe were developed for rplD. The L. monocytogenes strain Scott A, grown in tryptic soy broth (TSB), was exposed to various stress conditions. RNA was extracted and quantified. Equal amounts of total RNA were used in real-time RT-PCR to examine the consistency of rplD expression under various conditions. Following this validation, rplD was multiplexed with existing primers and probe for inlA, which codes for the virulence factor internalin A. Our results show that rplD is a suitable internal standard for L. monocytogenes exposed to several stress conditions, with no statistically significant changes in expression compared to the controls (p<0.05). Using multiplexed real-time RT-PCR, the expression of inlA was found to be significantly increased in strain Scott A when exposed to increased acidity. These results demonstrated that semi-quantitative, rapid evaluation of gene expression in L. monocytogenes could be achieved by real-time RT-PCR using rplD gene as an internal standard. In addition, the data suggested that the expression of internalin A is upregulated in acidic environments. Therefore this L. monocytogenes strain can become more virulent after being exposed to acids such as in the stomach or in acid foods.
Session 36B, Biotechnology: General
2005 IFT Annual Meeting, July 15-20 - New Orleans, Louisiana |