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United States Department of Agriculture

Agricultural Research Service

Title: SOME PCR PRIMER PAIRS TARGETING SALMONELLA ALSO AMPLIFY GUT ASSOCIATED BACTERIA

Authors
item Ziemer, Cherie
item Steadham, Shari

Submitted to: American Society for Microbiology Branch Meeting
Publication Type: Abstract Only
Publication Acceptance Date: September 30, 2002
Publication Date: October 12, 2002
Citation: Ziemer, C.J., Steadham, S.R. 2002. Some pcr primer pairs targeting salmonella also amplify gut associated bacteria. North Central Branch of the American Society for Microbiology. p. 12.

Technical Abstract: Nine pairs of PCR primers targeting Salmonella were evaluated for their specificity, prior to application to fecal samples. Each PCR primer set 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.

Last Modified: 4/16/2014
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