A second-generation method of genotyping hepatitis C virus by the polymerase chain reaction with sense and antisense primers deduced from the core gene.
A second-generation method of genotyping hepatitis C virus (HCV) was developed by the polymerase chain reaction (PCR) with sense as well as antisense primers deduced from the core gene. HCV RNA specimens extracted from sera were reverse-transcribed and amplified with universal primers in the first round of PCR to obtain fragments of 433 base pairs representing nucleotides 319-751. In the second round of PCR, portions of PCR products were amplified separately with sense and antisense primers specific for each of the five common genotypes prevailing across the world, i.e., I/1a, II/1b, III/2a, IV/2b and V/3a. The specificity of the method was verified by a panel of 177 HCV isolates of various genotypes in the genetic groups 1-9. It allowed clear differentiation of genotype I/1a from II/1b which was not always accomplished by the previous method. When 501 sera from blood donors and hepatitis patients with HCV viremia from various countries were genotyped by the second-generation method, 478 (95.4%) were classified into the five genotypes. HCV RNA samples from 23 (4.6%) sera were not classifiable into any of the five common genotypes and, by sequence analysis, 22 were found to be of four genotypes in group 4 and one of genotype 1c in Simmond's classification.
Okamoto
H
H
Immunology Division, Jichi Medical School, Tochigi-Ken, Japan.
Kobata
S
S
Tokita
H
H
Inoue
T
T
Woodfield
G D
GD
Holland
P V
PV
Al-Knawy
B A
BA
Uzunalimoglu
O
O
Miyakawa
Y
Y
Mayumi
M
M
D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis*; D005838:Genotype; D016174:Hepacivirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D006526:Hepatitis C / Q000097:blood / Q000821:virology*; D006801:Humans; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017421:Sequence Analysis; D014758:Viral Core Proteins / Q000145:classification / Q000235:genetics*
D017931:DNA Primers; D004279:DNA, Viral; D016376:Oligonucleotides, Antisense; D014758:Viral Core Proteins; C069130:nucleocapsid protein, Hepatitis C virus