Detection of hepatitis C virus RNA using ligation-dependent polymerase chain reaction in formalin-fixed, paraffin-embedded liver tissues.

摘要: Abstract Reverse transcription polymerase chain reaction (RT-PCR) has been used to detect hepatitis C virus (HCV) sequences in liver tissue. However, RT-PCR a variable detection sensitivity, especially on routinely processed formalin-fixed, paraffin-embedded (FFPE) specimens. RNA-RNA and RNA-protein cross-links formed during formalin fixation is the major limiting factor preventing reverse trans criptase from extending primers. To overcome this problem, we applied ligation-dependent PCR (LD-PCR) for of HCV RNA FFPE This method uses two capture probes isolation hemiprobes subsequent PCR. Despite cross-links, are able form hybrids with RNAs released The isolated through binding paramagnetic beads. then ligated by T4 DNA ligase full probe that serves as template Taq polymerase. A total 22 specimens, 21 hepatocellular carcinoma (HCC) 1 biliary cirrhosis secondary bile duct atresia were selected study, which 13 patients seropositive 9 seronegative. was detectable ID-PCR all HCV-seropositive HCCs 5 8 HCV-seronegative but not atresia. By contrast, detected only HCV-sero-positive HCCs. resolve discordance between LD-PCR results, performed frozen tissue discrepant confirmed positive results. In conclusion, more sensitive than tissues. high rate infection (86%) found HCC indicating previously underestimated role pathogenesis.