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Journal of Clinical Microbiology, January 2003, p. 144-148, Vol. 41, No. 1
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.1.144-148.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Development and Evaluation of a PCR-Enzyme-Linked Immunosorbent Assay for Diagnosis of Human Brucellosis

Pilar Morata,1 María I. Queipo-Ortuño,1 Jose M. Reguera,2 Miguel A. García-Ordoñez,2 Ana Cárdenas,3 and Juan D. Colmenero2*

Department of Biochemistry and Molecular Biology, Faculty of Medicine,1 Infectious Diseases Unit, Internal Medicine Department,2 Microbiology Service, Carlos Haya University Hospital, Málaga, Spain3

Received 17 June 2002/ Returned for modification 1 September 2002/ Accepted 13 October 2002

In order to overcome some of the limitations of conventional microbiological techniques in the diagnosis of human brucellosis, a simple PCR-enzyme-linked immunosorbent assay (PCR-ELISA) was developed. After amplification of a 223-bp sequence of a gene that codes for the synthesis of an immunogenetic membrane protein specific for the Brucella genus (BCSP31), the digoxigenin-labeled amplified product was hybridized with a biotinylated capture probe which was complementary to the inner part of the amplicon. The hybrid was captured on streptavidin-coated microtiter plates and detected by using an antidigoxigenin Fab-peroxidase conjugate. The detection limit of the PCR-ELISA in a background of 3.5 µg of human genomic DNA was 10 fg (two bacterial cells). The PCR-ELISA showed an analytical sensitivity higher than that of ethidium bromide staining and equal to that obtained by conventional PCR followed by dot blot hybridization. In 59 peripheral blood samples from 57 consecutive patients with active brucellosis and 113 control samples, the PCR-ELISA was found to be 94.9% sensitive and 96.5% specific, whereas the sensitivity of the blood culture was only 70.1%. Since the assay can be performed in 1 day, is very reproducible, is easily standardized, and avoids the risk of infection in laboratory workers, this PCR-ELISA seems to be a practical and reliable tool for the diagnosis of human brucellosis.

* Corresponding author. Mailing address: Unidad de Enfermedades Infecciosas, Departamento de Medicina Interna, Hospital Universitario Carlos Haya, C/Avda. Carlos Haya s/n., 29010 Málaga, Spain. Phone: 34 951 030169. Fax: 34 951 030165. E-mail: colmene{at}interbook.net.

Journal of Clinical Microbiology, January 2003, p. 144-148, Vol. 41, No. 1
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.1.144-148.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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