Detection of HPV-16 in cell lines and cervical lavage specimens by a polymerase chain reaction-enzyme immunoassay assay.
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 log10 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 32P-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.
Coutlée
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F
Department of Microbiology and Infectious Diseases, Hôpital Notre-Dame, Montréal, Canada.
Bobo
L
L
Abbass
H
H
Dalabetta
G
G
Hook
N E
NE
Shah
K
K
Viscidi
R P
RP
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002584:Cervix Uteri / Q000382:microbiology*; D004279:DNA, Viral / Q000032:analysis*; D005260:Female; D006367:HeLa Cells; D006801:Humans; D007124:Immunoenzyme Techniques; D008969:Molecular Sequence Data; D027383:Papillomaviridae / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D007507:Therapeutic Irrigation; D014412:Tumor Virus Infections / Q000175:diagnosis
D004279:DNA, Viral