[Improved method for detection and subtyping of HCV.RNA by nested polymerase chain reaction].
Amplification of hepatitis C virus RNA (HCV.RNA) by the reverse transcription nested polymerase chain reaction (RT-nested PCR) has been successfully applied to the detection for HCV.RNA in blood and its subtyping. Primers in the 5'-noncoding region (5'-NC) for detecting HCV.RNA and type-specific primers in the core region for subtyping HCV were used in this study. When 20 cycles of amplification with outer primers and another 20 cycles with inner primers were employed, good results were obtained in both specificity and sensitivity of nested PCR, especially by a single tube method. Of 755 subjects suspected of being infected with HCV, HCV.RNA was detectable in 488 (80.3%) of 608 subjects who were positive for HCV antibody and in 70 (47.6%) of 147 subjects who were negative; the total detection ratio was 73.9%. The distributions of HCV subtypes, designated as I, II, III, IV and mixed type (II+III, II+IV and III+IV), were 0, 85.0, 6.1, 4.8 and 4.0% respectively. Additionally, sensitivity in subtyping HCV.RNA was more improved by a single tube method. Using two primer sets (A:#32/#48 and B: #33/#48) prepared within the 5'-NC region, it was shown that the positivity of HCV.RNA was increased. Of 315 subjects, 232 (73.2%) were positive for HCV.RNA with both of A and B primer sets, and 20 or 22 were positive with either A or B primer set, thus indicating that a combination of these two primer sets increased the detection ratio of HCV.RNA approximately to 80%.(ABSTRACT TRUNCATED AT 250 WORDS)
Yamashita
K
K
Clinical Laboratory, Hyogo College of Medicine, Nishinomiya.
Ohtsuka
N
N
Kagawa
S
S
Matsuoka
A
A
D001483:Base Sequence; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D006526:Hepatitis C / Q000175:diagnosis; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000097:blood*; D012680:Sensitivity and Specificity; D012703:Serotyping
D012367:RNA, Viral