Abstract
Objective : We visualized single-copy HPV-DNA using in situ PCR, comparing staining results of in situ PCR to CSA-ISH.
Study Design : Study materials were cell lines HeLa, SiHa, and HL60, and liquid cervical cytological specimens from 5 women diagnosed with LSIL. We initially studied optimum conditions for pretreatment and in situ PCR, comparing staining results between in situ PCR and CSA-ISH for different fixed times.
Results : Pretreatment with 0.01% trypsin at between 2 and 2 min 50s gave optimum results. Optimum conditions for in situ PCR with HPV-18 primers were initial DNA denaturation for 1 min followed by 25 amplification cycles consisting of denaturation for 30s at 90°C, primer annealing for 30s at 59°C, and extension for 30s at 72°C. HeLa cells showed diffuse nuclear positivity for HPV-18 under these conditions. Compared to both types of staining, which differ with fixed times, in situ PCR staining was unaffected by fixed time. SiHa cells showed specific nuclear positivity for HPV-16 in in situ PCR and CSA-ISH.
Conclusion : Using this improved in situ PCR, we visualized single-copy HPV-DNA in cells from smear preparations fixed in ethanol.
