Detection of low copy numbers of HPV DNA by fluorescent in situ hybridization combined with confocal microscopy as an alternative to in situ polymerase chain reaction. 1998

G Lizard, and M C Chignol, and C Souchier, and P Roignot, and Y Chardonnet, and D Schmitt
INSERM U498, CHU/Hôpital du Bocage, Laboratoire de Biochimie Médicale, Dijon, France.

In genital lesions infected by human papillomavirus (HPV), histological criteria and HPV DNA typing are of prognostic value. Therefore, non-radioactive methods such as in situ hybridization are used extensively since they preserve the histological organization of the tissue, and allow the detection and characterization of HPV DNA. However, the sensitivity of these methods is often limited to detection of low copy numbers of HPV DNA in isolated cells or in tissue sections, and therefore alternative techniques have been explored. In the present study, 1-2 copies of HPV DNA were visualized in SiHa cells either by in situ amplification of nucleic acid sequences with the polymerase chain reaction (PCR) or by fluorescent in situ hybridization (FISH) associated with observation by laser scanning confocal microscopy (LSCM). The latter procedure was evaluated for use on histological tissue sections to identify low copy numbers of HPV DNA. Genital lesions which were negative by enzymatic in situ hybridization and FISH but histologically suspected of HPV infection were investigated, and intense signals were obtained both with in situ PCR and with the combined use of FISH and LSCM. Therefore, the combination of FISH with LSCM examination may be as valuable as in situ PCR to detect viral genes present in small amounts in isolated cells and in tissue sections.

UI MeSH Term Description Entries
D003218 Condylomata Acuminata Sexually transmitted form of anogenital warty growth caused by the human papillomaviruses. Genital Warts,Venereal Warts,Warts, Genital,Warts, Venereal,Genital Wart,Venereal Wart,Wart, Genital,Wart, Venereal
D004279 DNA, Viral Deoxyribonucleic acid that makes up the genetic material of viruses. Viral DNA
D005835 Genitalia The external and internal organs involved in the functions of REPRODUCTION. Accessory Sex Organs,Genital Organs,Sex Organs, Accessory,Genital System,Genitals,Reproductive Organs,Reproductive System,Accessory Sex Organ,Genital,Genital Organ,Genital Systems,Organ, Accessory Sex,Organ, Genital,Organ, Reproductive,Organs, Accessory Sex,Organs, Genital,Organs, Reproductive,Reproductive Organ,Reproductive Systems,Sex Organ, Accessory,System, Genital,System, Reproductive,Systems, Genital,Systems, Reproductive
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D014407 Tumor Cells, Cultured Cells grown in vitro from neoplastic tissue. If they can be established as a TUMOR CELL LINE, they can be propagated in cell culture indefinitely. Cultured Tumor Cells,Neoplastic Cells, Cultured,Cultured Neoplastic Cells,Cell, Cultured Neoplastic,Cell, Cultured Tumor,Cells, Cultured Neoplastic,Cells, Cultured Tumor,Cultured Neoplastic Cell,Cultured Tumor Cell,Neoplastic Cell, Cultured,Tumor Cell, Cultured
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D017404 In Situ Hybridization, Fluorescence A type of IN SITU HYBRIDIZATION in which target sequences are stained with fluorescent dye so their location and size can be determined using fluorescence microscopy. This staining is sufficiently distinct that the hybridization signal can be seen both in metaphase spreads and in interphase nuclei. FISH Technique,Fluorescent in Situ Hybridization,Hybridization in Situ, Fluorescence,FISH Technic,Hybridization in Situ, Fluorescent,In Situ Hybridization, Fluorescent,FISH Technics,FISH Techniques,Technic, FISH,Technics, FISH,Technique, FISH,Techniques, FISH
D018613 Microscopy, Confocal A light microscopic technique in which only a small spot is illuminated and observed at a time. An image is constructed through point-by-point scanning of the field in this manner. Light sources may be conventional or laser, and fluorescence or transmitted observations are possible. Confocal Microscopy,Confocal Microscopy, Scanning Laser,Laser Microscopy,Laser Scanning Confocal Microscopy,Laser Scanning Microscopy,Microscopy, Confocal, Laser Scanning,Confocal Laser Scanning Microscopy,Confocal Microscopies,Laser Microscopies,Laser Scanning Microscopies,Microscopies, Confocal,Microscopies, Laser,Microscopies, Laser Scanning,Microscopy, Laser,Microscopy, Laser Scanning,Scanning Microscopies, Laser,Scanning Microscopy, Laser
D018628 Gene Dosage The number of copies of a given gene present in the cell of an organism. An increase in gene dosage (by GENE DUPLICATION for example) can result in higher levels of gene product formation. GENE DOSAGE COMPENSATION mechanisms result in adjustments to the level GENE EXPRESSION when there are changes or differences in gene dosage. Gene Copy Number,Copy Number, Gene,Copy Numbers, Gene,Dosage, Gene,Dosages, Gene,Gene Copy Numbers,Gene Dosages,Number, Gene Copy,Numbers, Gene Copy
D027383 Papillomaviridae A family of small, non-enveloped DNA viruses infecting birds and most mammals, especially humans. They are grouped into multiple genera, but the viruses are highly host-species specific and tissue-restricted. They are commonly divided into hundreds of papillomavirus "types", each with specific gene function and gene control regions, despite sequence homology. Human papillomaviruses are found in the genera ALPHAPAPILLOMAVIRUS; BETAPAPILLOMAVIRUS; GAMMAPAPILLOMAVIRUS; and MUPAPILLOMAVIRUS.

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