Author
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Ziemer, Cherie |
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Steadham, Shari |
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Submitted to: Letters in Applied Microbiology
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 9/16/2003 Publication Date: 12/1/2003 Citation: Ziemer, C.J., Steadham, S.R. 2003. Evaluation of the specificity of salmonella pcr primers using various intestional bacteria species.. Letters in Applied Microbiology. p. 463-469. Interpretive Summary: Polymerase chain reaction (PCR) techniques allow for more sensitive and specific detection of bacteria by amplifying a portion of the DNA many times. This decreases the time needed to get results. Application of PCR primers targeting Salmonella would improve our ability to rapidly assess its presence in fecal and manure samples. Both fecal and manure samples carry complex microbial communities, thus primers must be evaluated for their specificity to Salmonella in the presence of fecal-associated bacteria. Nine sets of PCR primers targeting Salmonella DNA were evaluated for their specificity. Each PCR primer pair was designed to amplify a different gene, or region of Salmonella DNA. 52 Salmonella strains were used to determine the extent of detection. Strains from 5 related genera (Enterobacter, Citrobacter, Proteus, Klebsiella and Pseudomonas) and 45 fecal-associated organisms were used to evaluate specificity. All primer pairs amplified Salmonella DNA and did not amplify any from the related genera. Only three primer pairs did not amplify DNA from the fecal-associated bacteria. The other 6 primer sets gave false positive results for a number of fecal-associated bacteria. These 3 primer pairs were determined to be suitable for PCR amplification of Salmonella in fecal samples. Application of these PCR primers will allow scientists to rapidly determine the presence of Salmonella in fecal and manure samples in order to accurately assess potential impact on the environment and/or food products. Technical Abstract: Nine sets of PCR primers targeting Salmonella were evaluated for their specificity, prior to application to fecal samples. Each PCR primer pair was designed to amplify a different gene. Gene targets included: 16S rDNA, a Salmonella pathogenicity island I virulence gene (hilA), Salmonella enterotoxin gene (stn), invA gene, Fur-regulated gene (iroB), histidine transport operon gene, the junction between SipB and SipC virulence genes, a Salmonella specific repetitive DNA fragment, and a multiplex targeting invA gene and spvC gene of the virulence plasmid. 52 Salmonella strains were used to determine the extent detection; 38 from Salmonella enterica subgroup I, and 2 from each of subgroups II, IIIa, IIIb, IV, VI, VII and 2 from S. bongori. Strains from 5 related genera (Enterobacter, Citrobacter, Proteus, Klebsiella and Pseudomonas) and 45 gut-associated organisms were used to evaluate specificity. All primers amplified target DNA from all or most of the Salmonella strains, only negative for strains from one or two subgroups other than I. There was no amplification of DNA from related genera organisms with most primer sets. Three primer pairs (invA, iroB, and the multiplex) generated non-specific amplification products with 1 or more Salmonella related bacteria. Three primer sets did not amplify any DNA from gut-associated bacteria (16S rDNA, stn, and histidine transport operon gene). The primers targeting SipB and SipC junction amplified fragments smaller than target size, hilA and the repetitive DNA fragment primers were positive for a few gut associated bacteria. The other three primer pairs amplified DNA from many gut-associated organisms. Only 3 primer pairs were determined to be suitable for PCR amplification of Salmonella in fecal samples - 16S rDNA, stn, and histidine transport operon gene. Further analysis will determine the detection limits of these primers as well as evaluate DNA extraction methods. |
