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Journal of Clinical Microbiology, April 2004, p. 1734-1738, Vol. 42, No. 4
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.4.1734-1738.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Development of a Multiplex PCR Technique for Detection and Epidemiological Typing of Salmonella in Human Clinical Samples

Juan Alvarez,1 Mertxe Sota,2 Ana Belén Vivanco,1 Ildefonso Perales,3 Ramón Cisterna,1,2 Aitor Rementeria,1 and Javier Garaizar1*

Department of Immunology, Microbiology, and Parasitology, University of the Basque Country, Vitoria-Gasteiz,1 Clinical Microbiology Service, Basurto Hospital,2 Public Health Laboratory, Basque Government Health Department, Bilbao, Spain3

Received 29 September 2003/ Returned for modification 14 November 2003/ Accepted 9 January 2004

We have developed a multiplex PCR assay for Salmonella detection and epidemiological typing. Six sets of primers were designed to detect the major Salmonella serotypes and phage types in Spain. An internal amplification control was designed in order to detect PCR inhibition. The different amplification profiles obtained allowed us to detect Salmonella bacteria and to distinguish the clinically prevalent Salmonella enterica serotypes Enteritidis, Typhimurium and subspecies I serotype 4,5,12:i:–. Using this method, we could detect a specific band for DT104 and U302 phage types in Salmonella serotype Typhimurium. Salmonella enterica serotype Hadar and other C2 serogroup strains showed two specific band profiles. In the validation stage, the assay was reproducible for all serotypes studied, apart from some C2 serogroup strains. When the technique was applied to clinical stool specimens, the prevalent serotypes Enteritidis and Typhimurium were detected with a sensitivity of 93%, specificity of 100%, and efficiency of 98%. Also, a low PCR inhibition rate (8%) was obtained. The overall agreement of the multiplex PCR with conventional culture-based techniques was 95% for Salmonella typing using Cohen's kappa index.

* Corresponding author. Mailing address: Department of Immunology, Microbiology, and Parasitology, F. Pharmacy, University of the Basque Country, Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Spain. Phone: 34 945 013912. Fax: 34 945 013014. E-mail: oipgacaj{at}vc.ehu.es.

Journal of Clinical Microbiology, April 2004, p. 1734-1738, Vol. 42, No. 4
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.4.1734-1738.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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