title: Detection of transcripts of human papillomaviruses 16 and 18 in cancer-derived cell lines and cervical biopsies by enzyme immunoassay for DNA-RNA hybrids following solution hybridization.
abstract: mRNAs of human papillomaviruses (HPV) 16 and 18 were detected in cancer-derived cell lines and genital tract biopsy specimens by a novel hybridization assay. Biotinylated whole genomic HPV DNA probes were hybridized in solution to extracted total nucleic acids. Hybrids between the labeled probes and RNA transcripts were captured on a microplate coated with an antibiotin antibody. Bound hybrids were incubated with a beta-galactosidase-labeled monoclonal antibody to DNA-RNA hybrids and measured by the addition of a fluorogenic substrate. HPV 18 and HPV 16 mRNAs were detected in nucleic acids from 2.3 x 10(3) HeLa cells and 10(4) SiHa cells, respectively. The specificity of the assay for mRNA was demonstrated by the low reactivity of nucleic acids from SiHa cells after treatment with T1 RNase and by the selective reactivity of cellular nucleic acids which bound to an oligo(dT) column. With HPV 16 subgenomic probes, E6-E7 transcripts but not L1-L2 transcripts were detected in SiHa cells. Tests of 58 biopsy specimens from 31 patients showed that the detection of HPV 16 and HPV 18 transcripts in tissue specimens was feasible. Analysis of biopsy specimens with subgenomic probes revealed HPV 16 E6-E7 transcripts in all specimens that reacted with the whole genomic probe, while L1-L2 transcripts were found infrequently.
authors.affiliation: Department of Pediatrics, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205.
authors.forename: F
authors.initials: F
authors.lastname: Coutlée
authors.forename: K V
authors.initials: KV
authors.lastname: Shah
authors.forename: J S
authors.initials: JS
authors.lastname: Rader
authors.forename: J L
authors.initials: JL
authors.lastname: Currie
authors.forename: R P
authors.initials: RP
authors.lastname: Viscidi
mesh_terms: D002584:Cervix Uteri / Q000382:microbiology; D015344:DNA Probes, HPV; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D009693:Nucleic Acid Hybridization; D027383:Papillomaviridae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured / Q000382:microbiology
chemical_list: D015344:DNA Probes, HPV; D012333:RNA, Messenger; D012367:RNA, Viral