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.
Department of Clinical and Experimental Medicine, Division of Microbiology, University of Bologna, 40138 Bologna, Italy.
Simone
S
Ambretti
Mara
M
Mirasoli
Simona
S
Venturoli
Marialuisa
M
Zerbini
Mario
M
Baraldini
Monica
M
Musiani
Aldo
A
Roda
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