Comparison of Methods of Extracting Salmonella enterica Serovar Enteritidis DNA from Environmental Substrates and Quantification of Organisms by Using a General Internal Procedural Control

• DOI: 10.1128/aem.02266-05
• Publication Date: 2006-06-02T20:36:26Z
• Authors: M. M. Klerks; A. H. C. van Bruggen; C. Zijlstra; M. Donnikov
• Keywords: DNA, Bacterial; 0301 basic medicine; outbreak; Gene Amplification; polymerase-chain-reaction; escherichia-coli o157-h7; amplification; assay; Sensitivity and Specificity; soil; Manure; recovery; 03 medical and health sciences; Salmonella enteritidis; manure; typhimurium; Animals; real-time pcr; Soil Microbiology; Environmental Monitoring

ABSTRACT

This paper compares five commercially available DNA extraction methods with respect to DNA extraction efficiency of Salmonella enterica serovar Enteritidis from soil, manure, and compost and uses an Escherichia coli strain harboring a plasmid expressing green fluorescent protein as a general internal procedural control. Inclusion of this general internal procedural control permitted more accurate quantification of extraction and amplification of S. enterica serovar Enteritidis in these samples and reduced the possibility of false negatives. With this protocol it was found that the optimal extraction method differed for soil (Mobio soil DNA extraction kit), manure (Bio101 soil DNA extraction kit), and compost (Mobio fecal DNA extraction kit). With each method, as little as 1.2 × 10 3 to 1.8 × 10 3 CFU of added serovar Enteritidis per 100 mg of substrate could be detected by direct DNA extraction and subsequent S. enterica -specific TaqMan PCR. After bacterial enrichment, as little as 1 CFU/100 mg of original substrate was detected. Finally, the study presents a more accurate molecular analysis for quantification of serovar Enteritidis initially present in soil or manure using DNA extraction and TaqMan PCR.