Evaluation of immunoassays for the detection and typing of PCR amplified human papillomavirus DNA.
OBJECTIVE
To evaluate different hybridisation techniques to detect and type human papillomavirus (HPV) DNAs amplified by consensus primer polymerase chain reaction (PCR) in biopsy and cytological specimens.


METHODS
A hybrid capture-immunoassay in microtitre wells was performed to detect HPV sequences amplified by PCR and typed by specific oligoprobes. Consensus primers were used to amplify a sequence within the L1 open reading frame, and direct digoxigenin labelling of amplified products was performed during the amplification reaction. The amplified product was separately hybridised with six biotinylated type specific probes (HPV6, 11, 16, 18, 31, and 33); hybrids were then captured into streptavidin coated microtitre wells and detected by a spectrophotometer as an ELISA using antidigoxigenin Fab fragment labelled with peroxidase and a colorimetric substrate. The results were compared with the dot-blot immunoassay used to detect and type PCR amplified HPV DNA sequences. Consensus primers were used to generate the same unlabelled PCR product; digoxigenin labelled type specific probes for HPV6, 11, 16, 18, 31, and 33 were used and hybrids visualised by colorimetric immunoenzymatic reaction. Thirty nine biopsy specimens and 31 cytological samples were tested by the PCR-ELISA and by standard PCR followed by dot-blot hybridisation.


RESULTS
The PCR-ELISA proved to be more sensitive than standard PCR with dot-blot hybridisation typing. All samples positive for HPV-DNA in standard PCR with dot-blot hybridisation method were confirmed positive by the PCR-ELISA assay; however, seven samples were positive only by PCR-ELISA.


CONCLUSIONS
The PCR-ELISA assay, which can be performed in one day, is easily standardised and therefore seems to be a practical, sensitive, and reliable diagnostic tool for the detection and typing of HPV genomes in biopsy and in cytological specimens in the routine diagnostic laboratory.
Venturoli
S
S
Department of Clinical and Experimental Medicine, University of Bologna, Italy.
Zerbini
M
M
La Placa
M
M
D'Antuono
A
A
Negosanti
M
M
Gentilomi
G
G
Gallinella
G
G
Manaresi
E
E
Musiani
M
M
D001706:Biopsy; D004279:DNA, Viral / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D005069:Evaluation Studies as Topic; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D008297:Male; D009693:Nucleic Acid Hybridization / Q000379:methods; D015345:Oligonucleotide Probes; D027383:Papillomaviridae / Q000145:classification / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000175:diagnosis* / Q000821:virology; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014412:Tumor Virus Infections / Q000175:diagnosis* / Q000821:virology
D004279:DNA, Viral; D015345:Oligonucleotide Probes