Quantification and genotyping of hepatitis B virus in a single reaction by real-time PCR and melting curve analysis.
OBJECTIVE
Both viral titer and genotype of hepatitis B virus (HBV) play critical roles in determining clinical outcome and response to antiviral treatment in hepatitis B patients. In this study, a method was developed to determine both parameters in a single-tube reaction.


METHODS
The method contains two consecutive steps, the first step used real-time PCR for quantification and second step used melting curve analysis for genotyping. For accurate quantification, the PCR primers and hybridization probes were selected from highly conserved regions to ensure the equivalent amplification and hybridization of all genotypes of HBVs. Within the sensor probe there exists signature single nucleotide polymorphisms (SNPs), which could effectively differentiate different HBV genotypes by showing different melting temperatures.


RESULTS
The quantification results showed great consistency with the commercial assays in linear range from 10(2) to 10(11) copies/ml. By comparison with the traditional restriction fragment length polymorphism (RFLP) methods, 99% of samples were accurately genotyped by current assay, and with a higher detection rate. In addition, this method can detect mixed HBV infections.


CONCLUSIONS
Currently, this methodology can be applied to areas prevalent with HBV genotypes B and C, providing an efficient alternative for clinical diagnosis and large-scaled longitudinal studies.
Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan, Republic of China.
Shiou-Hwei
SH
Yeh
Ching-Yi
CY
Tsai
Jia-Horng
JH
Kao
Chun-Jen
CJ
Liu
Ti-Jung
TJ
Kuo
Ming-Wei
MW
Lin
Wen-Ling
WL
Huang
Shu-Fen
SF
Lu
Jane
J
Jih
Ding-Shinn
DS
Chen
Pei-Jer
PJ
Chen
D003199:Computer Systems; D017931:DNA Primers; D005838:Genotype; D006509:Hepatitis B / Q000821:virology; D006515:Hepatitis B virus / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D015335:Molecular Probes; D016133:Polymerase Chain Reaction; D019562:Viral Load; D014773:Virology / Q000379:methods*
D017931:DNA Primers; D015335:Molecular Probes