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Journal of Clinical Microbiology, June 2004, p. 2581-2586, Vol. 42, No. 6
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.6.2581-2586.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Multiplex PCR for Distinguishing the Most Common Phase-1 Flagellar Antigens of Salmonella spp.

Silvia Herrera-León,1* John R. McQuiston,2 Miguel A. Usera,1 Patricia I. Fields,2 Javier Garaizar,3 and M. Aurora Echeita1

Laboratorio Nacional de Referencia de Salmonella y Shigella, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid,1 Departamento de Inmunología, Microbiología y Parasitología, Facultad de Farmacia, Universidad del País Vasco, 01006 Vitoria-Gasteiz, Spain,3 Foodborne and Diarrheal Diseases Branch, Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 303332

Received 16 December 2003/ Returned for modification 3 March 2004/ Accepted 18 March 2004

Most Salmonella serotypes alternatively express either phase-1 or phase-2 flagellar antigens, encoded by the fliC and fljB genes, respectively. Flagellar phase reversal for the identification of both flagellar antigens is not necessary at the genetic level. Variable internal regions of the fliC genes encoding the H:i, H:r, H:l,v, H:e,h, H:z10, H:b, and H:d antigens have been sequenced; and the specific sites for each antigen in selected Salmonella serotypes have been determined. These results, together with flagellar G-complex variable internal sequences obtained by the Foodborne and Diarrheal Diseases Branch at the Centers for Disease Control and Prevention in Atlanta, Ga., have been used to design a multiplex PCR to identify the G-complex antigens as well as the H:i, H:r, H:l,v, H:e,h, Hz10, H:b, and H:d first-phase antigens. These antigens are part of the most common Salmonella serotypes possessing first-phase flagellar antigens. Salmonella enterica serotype Enteritidis is identified by adding a specific primer pair published previously (P. G. Agron, R. L. Walker, H. Kinde, S. J. Sawyer, D. C. Hayes, J. Wollard, and G. L. Andersen, Appl. Environ. Microbiol. 67:4984-4991, 2001). This multiplex PCR includes 13 primers. A total of 161 Salmonella strains associated with 72 different serotypes were tested. Each strain generated one first-phase-specific antigen fragment ranging from 100 to 500 bp; Salmonella serotype Enteritidis, however, generated two amplicons of 500 bp that corresponded to the G complex and a 333-bp serotype-specific amplicon, respectively. Twenty-three strains representing 19 serotypes with flagellar genes different from those targeted in this work did not generate any fragments. The method is quick, specific, and reproducible and is independent of the phase expressed by the bacteria when they are tested.

* Corresponding author. Mailing address: Laboratorio Nacional de Referencia de Salmonella y Shigella, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo, km.2, Madrid 28220, Spain. Phone: 34915097901. Fax: 3491 5097966. E-mail: sherrera{at}isciii.es.

Journal of Clinical Microbiology, June 2004, p. 2581-2586, Vol. 42, No. 6
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.6.2581-2586.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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