Morata, PilarQueipo-Ortuño, María I.Reguera, Jose M.García-Ordoñez, Miguel A.Cárdenas, AnaColmenero, Juan D2016-10-062016-10-062003-01Morata P, Queipo-Ortuño MI, Reguera JM, García-Ordoñez MA, Cárdenas A, Colmenero JD. Development and evaluation of a PCR-enzyme-linked immunosorbent assay for diagnosis of human brucellosis. J Clin Microbiol. 2003; 41(1):144-80095-1137http://hdl.handle.net/10668/2450Evaluation Studies; Journal Article; Research Support, Non-U.S. Gov't;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.enBrucelosisEnzimoinmunoanálisis por adsorciónHumanosReacción en cadena de la polimerasaJuego de reactivos para diagnósticoReproducibilidad de los resultadosSensibilidad y especificidadMedical Subject Headings::Diseases::Bacterial Infections and Mycoses::Bacterial Infections::Gram-Negative Bacterial Infections::BrucellosisMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Immunologic Techniques::Immunohistochemistry::Immunoenzyme Techniques::Enzyme-Linked Immunosorbent AssayMedical Subject Headings::Organisms::Eukaryota::Animals::Chordata::Vertebrates::Mammals::Primates::Haplorhini::Catarrhini::Hominidae::HumansMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Genetic Techniques::Nucleic Acid Amplification Techniques::Polymerase Chain ReactionMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Equipment and Supplies::Reagent Kits, DiagnosticMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Evaluation Studies as Topic::Reproducibility of ResultsMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Epidemiologic Methods::Statistics as Topic::Sensitivity and SpecificityMedical Subject Headings::Organisms::Bacteria::Gram-Negative Bacteria::Gram-Negative Aerobic Bacteria::Gram-Negative Aerobic Rods and Cocci::Brucellaceae::Brucellaresearch article12517839open access1098-660XPMC149602