The use of general primers in the polymerase chain reaction permits the detection of a broad spectrum of human papillomavirus genotypes.
A novel polymerase chain reaction (PCR) method was developed that permits the detection of 11 different human papillomavirus (HPV) genotypes using two general primer sets. By computer-assisted sequence analysis, two pairs of general primers were selected from the conserved L1 open reading frame and tested in the PCR on a set of cloned HPV genotypes. Experimental analysis showed that up to three mismatches between primers and target DNA did not influence the efficiency of the assay. The use of these primers in the PCR enabled the detection of HPV genotypes HPV-1a, -6, -8, -11, -13, -16, -18, -30, -31, -32 and -33, and was also successfully applied to well characterized cervical carcinoma cell lines and clinical samples. For the HPV types tested sub-picogram amounts of cloned DNA could be detected after general primer-mediated PCR and subsequent hybridization. The specificity of the amplification products was confirmed by blot hybridization procedures and RsaI restriction enzyme digestion. The results indicate that this PCR method can be a powerful tool for identifying novel HPV genotypes in dysplasias and squamous cell carcinomas suspected of having an HPV aetiology.
Snijders
P J
PJ
Department of Pathology, Free University Hospital, Amsterdam, The Netherlands.
van den Brule
A J
AJ
Schrijnemakers
H F
HF
Snow
G
G
Meijer
C J
CJ
Walboomers
J M
JM
D001483:Base Sequence; D015139:Blotting, Southern; D002294:Carcinoma, Squamous Cell / Q000382:microbiology; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D005260:Female; D005784:Gene Amplification; D005838:Genotype; D006367:HeLa Cells; D006801:Humans; D007830:Larynx / Q000382:microbiology; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D027383:Papillomaviridae / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms / Q000382:microbiology
D004279:DNA, Viral