High sensitivity detection of HPV-16 in SiHa and CaSki cells utilizing FISH enhanced by TSA.
Detection of integrated human papillomavirus type 16 (HPV-16) DNA in SiHa and CaSki cells was used as a model system to demonstrate sensitivity and resolution of a well defined target. Using 293- to 1987-base polymerase chain reaction (PCR)-synthesized probes to the E6 and E7 open reading frames of HPV-16, several fluorescent in situ hybridization (FISH) detection methods, enhanced with tyramide signal amplification (TSA), were compared. The synthetic probes were biotin labeled by a nick translation method and the hybridized probes were detected by various fluorescent TSA methods using cyanine 3 tyramide, biotinyl tyramide and a biotin TSA Plus reagent. High sensitivity detection in SiHa cells was demonstrated using a 619-base probe to detect two single copies of integrated HPV-16 DNA. In CaSki cells, which contain up to 600 copies of HPV-16 DNA, a 293-base probe was used for detection. The results of these comparisons show that with refinement of TSA methods and reagents, increasing levels of high sensitivity detection can be achieved and that these methods allow subnuclear localization as well.
Adler
K
K
NEN Life Science Products, Boston, MA 02118, USA.
Erickson
T
T
Bobrow
M
M
D002232:Carbocyanines / Q000737:chemistry; D004279:DNA, Viral / Q000032:analysis*; D019793:Fluorescein; D005456:Fluorescent Dyes; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D027383:Papillomaviridae / Q000235:genetics*; D050725:Papillomavirus E7 Proteins; D012097:Repressor Proteins; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D014439:Tyramine; D016662:Virus Integration
D002232:Carbocyanines; D004279:DNA, Viral; C068651:E6 protein, Human papillomavirus type 16; D005456:Fluorescent Dyes; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012097:Repressor Proteins; C089187:cyanine dye 3; C059731:oncogene protein E7, Human papillomavirus type 16; D019793:Fluorescein; D014439:Tyramine