Detection of hepatitis C virus sequences in liver tissue by the polymerase chain reaction.
Although sensitive assays for serum antibodies to hepatitis C virus (HCV/anti-HCV) have been developed recently, the relation of anti-HCV to HCV infection of the liver has not been clarified. Therefore, we determined the presence of HCV RNA by the reverse transcription-polymerase chain reaction (PCR) in liver biopsy specimens of 21 patients with chronic liver disease and 5 control patients. RNA was extracted from frozen liver tissues by the guanidinium method, HCV cDNA was synthesized by reverse transcription, and core region and NS3 region sequences were amplified by PCR. The sensitivity and specificity of the reaction was significantly enhanced by double PCR with nested primers followed by Southern blotting with an HCV specific oligomer probe. NS3 region sequences were detected in the liver specimens of 12 out of 15 anti-HCV positive patients. Core region sequences were detected in 9 patients, all of whom were also positive for NS3 region sequences. HCV sequences were not detected in 11 anti-HCV negative patients. In all cases, the integrity of the extracted RNA was demonstrated by successful amplification of albumin mRNA as internal control. Our findings demonstrate the feasibility of the reverse transcription-double PCR method followed by Southern blotting for the detection of HCV sequences in liver tissues. In this system, the detection rate of NS3 region sequences is higher than that of core region sequences. There is a statistically significant correlation between high titer anti-HCV antibodies in serum and NS3 region sequences in liver tissue. However, not all anti-HCV positive patients had HCV positive hepatitis. The reverse transcription-polymerase chain reaction for HCV sequences on liver tissue extracts may reveal valuable information on the diagnosis of HCV infection and the pathogenesis of chronic hepatitis C.
Shieh
Y S
YS
Department of Pathology, Tulane University School of Medicine, New Orleans, Louisiana.
Shim
K S
KS
Lampertico
P
P
Balart
L A
LA
Jeffers
L J
LJ
Thung
S N
SN
Regenstein
F
F
Reddy
K R
KR
Farr
G
G
Schiff
E R
ER
D001483:Base Sequence; D002908:Chronic Disease; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D006508:Hepatitis Antibodies / Q000097:blood; D006526:Hepatitis C / Q000276:immunology; D006801:Humans; D008099:Liver / Q000382:microbiology*; D008107:Liver Diseases / Q000276:immunology / Q000382:microbiology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000032:analysis*
D006508:Hepatitis Antibodies; D012367:RNA, Viral