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Detection of HPV‐16 in cell lines and cervical lavage specimens by a polymerase chain reaction‐enzyme immunoassay assay
Author(s) -
Coutlée François,
Bobo Linda,
Abbass Hawwari,
Dalabetta Gina,
Hook Ned E.,
Shah Keerti,
Viscidi Raphael P.
Publication year - 1992
Publication title -
journal of medical virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 121
eISSN - 1096-9071
pISSN - 0146-6615
DOI - 10.1002/jmv.1890370105
Subject(s) - microbiology and biotechnology , immunoassay , polymerase chain reaction , dna , virology , biotinylation , biology , rna , nucleic acid thermodynamics , antibody , gene , biochemistry , genetics
A gene amplification method that combines the polymerase chain reaction with detection of amplified DNA in a solution hybridization/enzyme immunoassay (PCR‐EIA) was developed for HPV‐16 DNA. Samples were amplified with primers for the E7‐E1 region of HPV‐16. Amplified DNA products were identified and quantitated by hybridization in solution with a biotinylated RNA probe. Labeled DNA/RNA hybrids were measured semiquantitatively in an enzyme immunoassay using solid phase anti‐biotin antibody and liquid phase B‐d‐galactosidase labeled monoclonal antibody against DNA‐RNA hybrids. Enzyme bound to the solid phase was quantitated with a fluorogenic substrate. The assay was linear over 2 log 10 dilutions of SiHa cells and the detection limit was three copies of HPV‐16 genome. The sensitivity of PCR‐EIA for detection of PCR amplified products compared favorably with slot and Southern blots using a 32 P‐labeled RNA probe. The assay was used to assess HPV‐16 infection of uterine cervix in women attending a clinic for sexually transmitted diseases. Twenty‐one of the 81 specimens (25.9%), obtained by cervicovaginal lavage, were positive for HPV‐16 by PCR‐EIA. The assay provides a convenient means to objectively measure HPV DNA amplified with PCR. © 1992 Wiley‐Liss, Inc.