title: Introduction of a fast and sensitive fluorescent in situ hybridization method for single-copy detection of human papillomavirus (HPV) genome.
abstract: At present, in situ hybridization (ISH) is the only method for detection of specific genes in morphologically intact cells or tissue. We have developed a highly sensitive and quantitative fluorescence-based in situ hybridization (FISH) technique that can detect as few as one to five copies of the integrated human papillomavirus (HPV) type 16 genome in cervical cell lines, using digoxygenin tail-labeled oligonucleotides (Method 1). The entire procedure can be carried out in 4.5 hr through the elimination of some of the steps routinely used in other ISH protocols. We also compared the sensitivity of this new FISH method (Method 1) to four other FISH techniques: digoxigenin-labeled DNA probe (Method 2); fluorescein-15-d-ATP-labeled oligonucleotides (Method 3); fluorescein 15-d-ATP labeled DNA probe (Method 4); and biotin-DNA-labeled probe (Method 5), for their ability to detect HPV DNA in the HPV-positive human cervical cell lines CaSki (500 copies) and SiHa (1-5 copies), but not in C33-A and HT-3, which do not contain any copies of HPV. Our results indicate that Method 1 is more sensitive than the other methods employed. Method 1 was the only method that could reliably detect an HPV-16 genome in all SiHa cells. Our data suggest that the Method 1 FISH technique is highly sensitive and may therefore be of general use for detection and quantitation of a variety of viral genomes (including HIV), oncogenes, and drug-resistant genes, in a variety of morphologically intact cells and tissues.
authors.affiliation: Laboratories for Cell Biology, University of North Carolina, Chapel Hill 27599.
authors.forename: M
authors.initials: M
authors.lastname: Siadat-Pajouh
authors.forename: A H
authors.initials: AH
authors.lastname: Ayscue
authors.forename: A
authors.initials: A
authors.lastname: Periasamy
authors.forename: B
authors.initials: B
authors.lastname: Herman
mesh_terms: D001483:Base Sequence; D002584:Cervix Uteri / Q000737:chemistry / Q000473:pathology; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D005260:Female; D016679:Genome, Viral; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008969:Molecular Sequence Data; D027383:Papillomaviridae / Q000235:genetics*; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms / Q000737:chemistry / Q000473:pathology
chemical_list: D015342:DNA Probes; D004279:DNA, Viral