Simultaneous quantitation and genotyping of hepatitis B virus by real-time PCR and melting curve analysis.
Hepatitis B virus (HBV) genotype and HBV DNA levels have been implicated in clinical evaluation and prognosis of patients with chronic HBV infection. The aim of the present study was to develop a rapid and sensitive method for simultaneous HBV DNA quantitation and differentiation between HBV genotypes B and C in a single-step reaction by real-time PCR and melting curve analysis using SYBR Green I fluorescent dye. The genotypes obtained by this method were compared with those examined by PCR-RFLP and direct sequencing on 52 serum samples of patients with chronic HBV infection. Using the results obtained by direct sequencing and phylogenetic analysis as the reference, the accuracy of HBV genotyping by PCR-RFLP and melting curve analysis was 90.38 and 92.31%, respectively. The geometric mean of HBV DNA levels was 3.42 x 10(6), 2.10 x 10(6), 1.19 x 10(5) and 3.10 x 10(4) copies/microl in asymptomatic carriers, patients with chronic hepatitis, cirrhosis and hepatocellular carcinoma, respectively. It is concluded that this method has the advantages of rapidity, reproducibility and accuracy, which would be feasible and attractive for large-scale analysis, particularly in regions where HBV genotypes B and C are prevalent.
Center of Excellence in Viral Hepatitis Research Unit, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand.
Sunchai
S
Payungporn
Pisit
P
Tangkijvanich
Pojchanad
P
Jantaradsamee
Apiradee
A
Theamboonlers
Yong
Y
Poovorawan
D004279:DNA, Viral / Q000032:analysis / Q000302:isolation & purification; D005456:Fluorescent Dyes; D005838:Genotype; D006515:Hepatitis B virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D019694:Hepatitis B, Chronic / Q000821:virology*; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D044366:Transition Temperature
D004279:DNA, Viral; D005456:Fluorescent Dyes