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Applied and Environmental Microbiology, June 2003, p. 3456-3461, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3456-3461.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Evaluation of DNA Extraction Methods for Use in Combination with SYBR Green I Real-Time PCR To Detect Salmonella enterica Serotype Enteritidis in Poultry

Dario De Medici,^1* Luciana Croci,¹ Elisabetta Delibato,¹ Simona Di Pasquale,¹ Emma Filetici,² and Laura Toti¹

Laboratorio Alimenti,¹ Laboratorio di Batteriologia e Micologia Medica, Istituto Superiore di Sanità, 00161 Rome, Italy²

Received 17 October 2002/ Accepted 5 March 2003

The objective of this study was to develop a rapid, reproducible, and robust method for detecting Salmonella enterica serotype Enteritidis in poultry samples. First, for the extraction and purification of DNA from the preenrichment culture, four methods (boiling, alkaline lysis, Nucleospin, and Dynabeads DNA Direct System I) were compared. The most effective method was then combined with a real-time PCR method based on the double-stranded DNA binding dye SYBR Green I used with the ABI Prism 7700 system. The specificity of the reaction was determined by the melting temperature (T_m) of the amplicon obtained. The experiments were conducted both on samples of chicken experimentally contaminated with serotype Enteritidis and on commercially available poultry samples, which were also used for comparisons with the standard cultural method (i.e., ISO 6579/2001). The results of comparisons among the four DNA extraction methods showed significant differences except for the results from the boiling and Nucleospin methods (the two methods that produced the lowest threshold cycles). Boiling was selected as the preferred extraction method because it is the simplest and most rapid. This method was then combined with SYBR Green I real-time PCR, using primers SEFA-1 and SEFA-2. The specificity of the reaction was confirmed by the T_m, which was consistently specific for the amplicon obtained; the mean peak T_m obtained with curves specific for serotype Enteritidis was 82.56 ± 0.22°C. The standard curve constructed using the mean threshold cycle and various concentrations of serotype Enteritidis (ranging from 10³ to 10⁸ CFU/ml) showed good linearity (R² = 0.9767) and a sensitivity limit of less than 10³ CFU/ml. The results of this study demonstrate that the SYBR Green I real-time PCR constitutes an effective and easy-to-perform method for detecting serotype Enteritidis in poultry samples.

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* Corresponding author. Mailing address: Laboratorio di Alimenti, Viale Regina Elena 299, 00161 Rome, Italy. Phone: 39 06 49902477. Fax: 39 06 49902045. E-mail: dario.demedici{at}iss.it.

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Applied and Environmental Microbiology, June 2003, p. 3456-3461, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3456-3461.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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