Detection of hepatitis C virus genome in human serum by multi-targeted polymerase chain reaction.
A multi-targeted "hemi-nested" PCR (M-PCR) assay in which the primer pairs derived from the 5' non-coding (5'NC) and the nonstructural protein 3 (NS3) regions of HCV genome were concurrently used for amplification in order to compare the sensitivity and specificity of polymerase chain reaction (PCR) with different primer pairs in detecting hepatitis C virus (HCV) genome. Sera from patients with virus-associated liver diseases were examined for the presence of HCV RNA by the M-PCR method following reverse transcription to cDNA. The amplified products derived from both the 5'NC and the NS3 regions were detected in 28 (70%) of the 40 HCV RNA-positive samples. However, 12 samples (30%) were devoid of the signal of NS3-derived product. Sensitivity tests using serial dilutions of HCV RNA revealed that the 5'NC-derived band was still detectable in the 10(5)-fold diluted sample by the M-PCR method, yet the NS3-derived band could hardly be detected in the 10(4)-fold diluted sample. Thus, as previously demonstrated by a single-targeted "nested" PCR assay, the present study using the M-PCR assay has clearly shown that the 5'NC-derived primers are more sensitive and specific than the NS3-derived primers in detecting HCV RNA.
Okamoto
M
M
Department of Microbiology, Fukushima Medical College, Japan.
Baba
M
M
Kodama
E
E
Sekine
K
K
Takagi
T
T
Kasukawa
R
R
Shigeta
S
S
D001739:BK Virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D016026:Bone Marrow Transplantation; D002648:Child; D004279:DNA, Viral / Q000032:analysis / Q000652:urine; D005260:Female; D016679:Genome, Viral; D006801:Humans; D016030:Kidney Transplantation; D008969:Molecular Sequence Data; D019384:Nucleic Acid Synthesis Inhibitors; D030361:Papillomavirus Infections / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D012680:Sensitivity and Specificity; D019914:Taq Polymerase; D014412:Tumor Virus Infections / Q000382:microbiology*; D014556:Urine / Q000382:microbiology
D004279:DNA, Viral; D019384:Nucleic Acid Synthesis Inhibitors; D019914:Taq Polymerase