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Research Support, Non-U.S. Gov't
- PCR Method Based on the ogdH Gene for the Detection of Salmonella spp. from Chicken Meat Samples
- Un-Ho Jin , Sung-Hak Cho , Min-Gon Kim , Sang-Do Ha , Keun-Sung Kim , Kyu-Ho Lee , Kwang-Yup Kim , Duck Hwa Chung , Young-Choon Lee , Cheorl-Ho Kim
- J. Microbiol. 2004;42(3):216-222.
- DOI: https://doi.org/2086 [pii]
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Abstract
- In a previous paper, the ogdH gene that encodes 2-oxoglutarate dehydrogenase was isolated from Salmonella typhimurium. The catalytic N-terminal region in the enzyme was found to be very specific for the Salmonella species. Therefore, the aim of the present study was to detect S. typhimurium in food sources using primers designed for OGDH-1 and OGDH-2 which were based on the salmonella-specific region of the ogdH gene. A simple polymerase chain reaction (PCR) detection method was developed to detect low numbers of S. typhimurium in a chicken meat microbial consortium. Using the ogdH-specific primers under stringent amplification conditions and for gene probe analysis, fewer than 100 colony-forming units (CFUs) were detectable when pure cultures were employed. When the PCR assay was run on S. typhimurium-contaminated meat contents, only the positive meat samples containing as few as 200 CFUs reacted to the assay. The method employed for sample processing is simple and it was determined to provide a sensitive means of detecting trace amounts of S. typhimurium-specific sequences in the presence of mixed meat microbial populations. When compared with six representative intestinal gram-negative bacterial strains in foods, including Vibrio parahaemolyticus, V. vulnificus, Enterobacter cloacae, E. coli O157:H7, Pseudomonas aeruginosa, and Proteus sp., S. typhimurium had a unique and distinct PCR product (796 bp). In conclusion, the two OGDH primers were found to be rapid and sensitive detectors of Salmonella spp for the PCR method.
- The invariant region I sequence of the adenovirus serotype 2 DNA polymerase influences template specificity during DNA synthesis
- Houng , In Sil
- J. Microbiol. 1998;36(3):222-230.
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Abstract
- Mutants in highly conserved region I (YGDTDS) of the adenovirus serotyope 2 DNA polymerase (Ad Pol) have been shown previously to be defective in assays for initiation and elongation of adenovirus DNA replication in vitro. A selected subset of these mutants was characterized in a number of assays to determine in more detail the nature of the defect that they cause in Ad Pol. The single amino acid substitution in this sequence motif had no detectable effect on binding either to factors required for viral DNA replication or to Ad DNA origin. However, in the deletion mutant mimicking a similar sequence found in the Klenow fragment and in RNA polymerases, binding to Ad DNA origins was reduced. When the nucleotide and template specificity of partially purified mutant Ad Pol proteins was checked there were no significant differences between mutant and wild-type Ad Pols in RNA polymerase assays both on DNA templates and on RNA templates. In reverse transcription assays, both wild type and mutant Ad Pol were inactive, with the exception of a Gly to Met replacement mutant which mimicked the sequence found in reverse transcriptase; this mutant showed low but reproducible levels of reverse transcriptase activity when compared to wt Ad Pol. Taken together, these results suggest that region I may influence template specificity during DNA synthesis, although the single replacement is not sufficient to convert Ad Pol to a reverse transcriptase or an RNA polymerase. Region I probably acts independently of other regions of Ad Pol that are responsible for DNA binding, since mutations in this sequence did not significantly alter adenovirus DNA origin interactions in gel shift assays.
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