Study of stringency conditions for human papillomavirus DNA detection on cell lines, frozen and paraffin-embedded tissue sections by in situ hybridization with biotinylated probes.
In situ hybridization was mainly used for typing human papillomavirus (HPV) in paraffin-embedded or frozen sections under stringent conditions (SC). We tested 5 different conditions of stringency with biotinylated HPV 1, 2, 16 and 18 probes on 3 cell lines (Sihà and CaSki with HPV16, HeLa with HPV18) by varying the concentration of formamide in the hybridization mixture and washings in order to determine the stringency conditions to be used to assess the presence of HPV and its typing: A-low stringency, hybridization at 35 degrees C below the melting temperature of DNA (Tm-35 degrees C) and washings without formamide; B-low stringency, hybridization and washings at Tm-35 degrees C; C-medium stringency, hybridization at Tm-35 degrees C and washings at Tm-12 degrees C; D-high stringency, hybridization at Tm-12 degrees C and washing without formamide; E-very high stringency, hybridization and washings at -12 degrees C. This study showed that HPV typing required a high stringency. On the contrary, under non stringent conditions (NSC), each cell line was positive with the heterologous probes. When 3 to 5 stringency conditions were assayed on 4 frozen samples, similar results were obtained. Typing required high stringency conditions whereas NSC allowed HPV detection. Furthermore, this study demonstrated the specificity of the reaction in lesions positive with more than one type. Stringent (Tm-12 degrees C) and non stringent (Tm-35 degrees C) conditions of hybridization were further applied to 57 biopsy sections (17 frozen and 40 paraffin-embedded specimens) from typical wart lesions and lesions suspected of HPV.(ABSTRACT TRUNCATED AT 250 WORDS)
Guerin-Reverchon
I
I
INSERM U 209, CNRS DO 601, Clinique Dermatologique, Lyon, France.
Chardonnet
Y
Y
Chignol
M C
MC
Thivolet
J
J
D001706:Biopsy; D002460:Cell Line; D015342:DNA Probes; D015344:DNA Probes, HPV; D004279:DNA, Viral / Q000032:analysis*; D005629:Frozen Sections; D006801:Humans; D009693:Nucleic Acid Hybridization; D027383:Papillomaviridae / Q000302:isolation & purification*; D014412:Tumor Virus Infections / Q000382:microbiology* / Q000473:pathology
D015342:DNA Probes; D015344:DNA Probes, HPV; D004279:DNA, Viral