Usefulness of PCR in situ hybridization as a technique for morphological detection of human papillomavirus in uterine cervical neoplasia.
This study was designed in order to devise fitting conditions in polymerase chain reaction (PCR)-in situ hybridization (ISH) for observing human papillomavirus (HPV) infection morphologically in uterine cervical neoplasias and to compare the detection rates of HPV by PCR-ISH and solution phase PCR (S-PCR) as well as fluorescence ISH (FISH). Tissues were obtained from 23 patients with cervical intraepithelial neoplasia 3, who visited our hospital between 1994 and 1997. To detect HPV-16, a HPVpF forward primer and a HPVp 16 reverse primer were used. Compared with the traditional methods, the PCR-ISH technique performed in this study was contrived as follows. To prevent detachment, the specimens were attached to silane-coated slides at 90 degrees C and successively left at room temperature for 36 hours. In proteopepsis, pepsin was used. PCR products were fixed with 4% paraformaldehyde. PCR-ISH, S-PCR, and FISH showed HPV-16 positivity in 52.2%, 56.5% and 21.7%, respectively. The positive rate of HPV-16 detected by PCR-ISH as well as S-PCR was significantly higher than that by FISH (p<0.01, respectively). There was no significant difference between the positive rates of HPV-16 detected by PCR-ISH and S-PCR. HPV-16 was detected by S-PCR in all 12 specimens in which HPV-16 expression was judged as positive using PCR-ISH. Similarly, HPV-16 was found by PCR-ISH in all five specimens in which HPV-16 expression was regarded as positive using FISH. While the FISH technique detected HPV-16 signals only in the superficial and middle layers of squamous cells, the PCR-ISH technique demonstrated them in all the layers including the parabasal and basal layers. The PCR-ISH technique contrived in this study has a high sensitivity to HPV-16 equal to that of S-PCR. The difference in detection rate and distribution of HPV DNA between PCR-ISH and FISH might suggest that HPV does not infect the superficial layer but rather the parabasal layer.
Oguchi
T
T
Department of Obstetrics and Gynecology, Hirosaki University School of Medicine, Japan.
Sato
S
S
Xiao
Y H
YH
Yokoyama
Y
Y
Saito
Y
Y
D000328:Adult; D000368:Aged; D004279:DNA, Viral / Q000302:isolation & purification*; D005260:Female; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000592:standards*; D008875:Middle Aged; D027383:Papillomaviridae / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000592:standards*; D011237:Predictive Value of Tests; D012680:Sensitivity and Specificity; D002583:Uterine Cervical Neoplasms / Q000175:diagnosis / Q000821:virology*; D002578:Uterine Cervical Dysplasia / Q000175:diagnosis / Q000821:virology*
D004279:DNA, Viral