High-throughput polymerase chain reaction chemiluminescent enzyme immunoassay for typing and quantifying human papillomavirus DNAs.
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.
Ambretti
Simone
S
Department of Clinical and Experimental Medicine, Division of Microbiology, University of Bologna, 40138 Bologna, Italy.
Mirasoli
Mara
M
Venturoli
Simona
S
Zerbini
Marialuisa
M
Baraldini
Mario
M
Musiani
Monica
M
Roda
Aldo
A
D002138:Calibration; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics*; D007124:Immunoenzyme Techniques / Q000379:methods*; D008163:Luminescent Measurements / Q000379:methods*; D027383:Papillomaviridae / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004279:DNA, Viral