title: Development and evaluation of a PCR-enzyme-linked immunosorbent assay for diagnosis of human brucellosis.
abstract: 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 micro g 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Medicine, Carlos Haya University Hospital, Málaga, Spain.
authors.forename: Pilar
authors.initials: P
authors.lastname: Morata
authors.forename: María I
authors.initials: MI
authors.lastname: Queipo-Ortuño
authors.forename: Jose M
authors.initials: JM
authors.lastname: Reguera
authors.forename: Miguel A
authors.initials: MA
authors.lastname: García-Ordoñez
authors.forename: Ana
authors.initials: A
authors.lastname: Cárdenas
authors.forename: Juan D
authors.initials: JD
authors.lastname: Colmenero
mesh_terms: D002002:Brucella / Q000235:genetics / Q000302:isolation & purification*; D002006:Brucellosis / Q000175:diagnosis* / Q000382:microbiology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008828:Microbiological Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D011933:Reagent Kits, Diagnostic