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  • Title: High-throughput polymerase chain reaction chemiluminescent enzyme immunoassay for typing and quantifying human papillomavirus DNAs.
    Author: Ambretti S, Mirasoli M, Venturoli S, Zerbini M, Baraldini M, Musiani M, Roda A.
    Journal: Anal Biochem; 2004 Sep 15; 332(2):349-57. PubMed ID: 15325304.
    Abstract:
    A miniaturized polymerase chain reaction (PCR) chemiluminescent enzyme immunoassay (CLEIA) based on a 384-well microtiter plate for detection and typing of oncogenic high- and low-risk human papillomavirus (HPV) in genital lesions is described. The assay relies on PCR consensus amplification, hybridization of the digoxigenin-labeled product by means of type-specific biotin-labeled oligoprobes immobilized on the streptavidin-coated wells of a 384-well microtiter plate, and quantification by means of a horseradish peroxidase-labeled antidigoxigenin antibody and chemiluminescence detection. The method provides semiquantitative information on the viral load, with a limit of detection of 10-50 DNA copies for HPV 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 54, 58, and 59 and high reproducibility (intraassay CV 7.5%, interassay CV 9.5%). Results obtained on 60 clinical samples were concordant with those obtained with a conventional PCR-enzyme-linked immunosorbent assay colorimetric assay. The 384 PCR-CLEIA method, which is amenable to automation, represents a fast and high-throughput method for detecting and typing HPV DNAs in screening programs and evaluating the viral load.
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