Detection of human papillomavirus DNA and mRNA using synthetic, type-specific oligonucleotide probes.
Type-specific 30'mer-36'mer oligonucleotide probes complementary to mRNA transcribed from the E6 and E7 open reading frames of HPV 6b/11, 16, 18 and 33 were designed using the published nucleotide sequences. As oligonucleotides are easily and relatively cheaply synthesized in large amounts and are free of vector DNA, they were assessed for potential use in routine clinical detection and typing of HPV. Multiple Southern and dot blots of cloned HPV 6b, 11, 16, 18, 31 and 33 DNA, and of DNA extracted from cell lines carrying integrated HPV 16 and 18 genomes were prepared. In addition, Northern and dot blots of RNA extracted from the HPV-containing cell lines HeLa, CaSki and SiHa, were also prepared. All filters were first probed with the oligonucleotide and then with the corresponding full-genomic HPV DNA probe and their relative sensitivities and specificities compared: both probe types were labelled with 32P. The oligonucleotide probes were all as specific as the full-genomic probes for Southern, DNA and RNA dot blot hybridisations. The HPV 16 and 18 oligonucleotide probes detected HPV transcripts of the appropriate sizes in the cell line RNA. For DNA detection, oligonucleotide probes were up to 10 times less sensitive than the full-genomic probes, but for RNA detection, they were more sensitive. The sensitivity for both HPV DNA and RNA detection could be improved by using two type-specific oligonucleotide probes in combination, without reducing the specificity. The ease of preparation and handling of oligonucleotide probes, together with their lack of contaminating vector DNA, suggests that they may have some advantages over full-genomic probes for the clinical detection and typing of HPV.
Microbiology Department, Royal Women's Hospital, Carlton, Australia.
B E
BE
Faulkner-Jones
S N
SN
Tabrizi
A J
AJ
Borg
P J
PJ
Roche
J
J
Haralambidis
J P
JP
Coghlan
S M
SM
Garland
D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D015344:DNA Probes, HPV; D004279:DNA, Viral / Q000302:isolation & purification*; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry; D016366:Open Reading Frames / Q000235:genetics; D027383:Papillomaviridae / Q000235:genetics*; D012333:RNA, Messenger / Q000302:isolation & purification*; D012367:RNA, Viral / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014412:Tumor Virus Infections / Q000175:diagnosis*
D015344:DNA Probes, HPV; D004279:DNA, Viral; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D012367:RNA, Viral