Genotyping of hepatitis C virus by melting curve analysis with SYBR Green I.
BACKGROUND
Recent studies have focused on whether different hepatitis C virus (HCV) genotypes are associated with different profiles of pathogenicity, infectivity, and response to antiviral therapy. We needed to develop a convenient screening test for HCV genotypes 1 and 2.


METHODS
We tested 55 patients with known chronic HCV infection. Viral RNA was extracted from serum samples using an automatic viral RNA purification system, and HCV genotypes were determined by reverse transcriptase-polymerase chain reaction using LightCycler melting curve analysis with SYBR Green I.


RESULTS
HCV RNA was detected in all samples and each genotype was determined. The mean (standard deviation) melting temperatures for subtypes 1b (n = 32), 2a (n = 15) and 2b (n = 8) were 93.14 degrees C (0.51 degrees C), 91.08 degrees C (0.49 degrees C) and 91.77 degrees C (0.27 degrees C), respectively. Genotypes 1 and 2 were differentiated within 3 h by this method.


CONCLUSIONS
Our melting curve analysis is a rapid and convenient screening test for differential identification of HCV genotypes 1 and 2.
Fujigaki
Hidetsugu
H
Department of Clinical Laboratory Medicine, Gifu University School of Medicine, Gifu 500-8705, Japan. g2104021@gueda.cc.gifu-u.ac.jp
Takemura
Masao
M
Takahashi
Kanako
K
Yamada
Yasuhiro
Y
Fujii
Hidehiko
H
Wada
Hisayasu
H
Saito
Kuniaki
K
Ohnishi
Hiroo
H
Seishima
Mitsuru
M
D052160:Benzothiazoles; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics*; D003959:Diamines; D016679:Genome, Viral; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics* / Q000472:pathogenicity; D006526:Hepatitis C / Q000097:blood / Q000235:genetics*; D006801:Humans; D009691:Nucleic Acid Denaturation / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000235:genetics / Q000379:methods; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral / Q000097:blood / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014774:Virulence / Q000235:genetics
D052160:Benzothiazoles; D017931:DNA Primers; D018076:DNA, Complementary; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C098022:SYBR Green I