Simultaneous quantification and genotyping of hepatitis B virus for genotypes A to G by real-time PCR and two-step melting curve analysis.
Both the viral titer and the genotype significantly determine clinical outcomes and responses to antiviral treatment in chronic hepatitis B virus (HBV) infection. A method was developed for large-scale A-to-G genotyping with simultaneous viral quantification. The assay was run on a LightCycler instrument using hybridization probes. The genotype was determined from the melting points of the probes in a two-step manner. Set 1 amplicons differentiated genotypes B, E, and F from A, C, D, and G and simultaneously quantified viremia by real-time PCR. Melting curve analysis using the set 2-1 amplicon or the set 2-2 amplicon reaction mixture was then used to differentiate these genotype groups into single genotypes. HBV DNA quantification was consistent with that of the Amplicor assay and linear in a range from 10(2) to 10(13) copies/ml. By comparison with the restriction fragment length polymorphism method, 92.3% of 441 samples were accurately genotyped by the current assay. The method should be useful for genotyping and quantification of HBV DNA in areas where all genotypes exist.
Liu
Wen-Chun
WC
Institute of Basic Medical Sciences, Department of Medicine, National Cheng Kung University, Tainan City 704, Taiwan, Republic of China, and Liver Unit, Hospital General Universitari Vall d'Hebron, Barcelona, Spain.
Mizokami
Masashi
M
Buti
Maria
M
Lindh
Magnus
M
Young
Kung-Chia
KC
Sun
Koun-Tem
KT
Chi
Yun-Chan
YC
Li
Hsi-Hsien
HH
Chang
Ting-Tsung
TT
D017931:DNA Primers; D004279:DNA, Viral / Q000737:chemistry* / Q000235:genetics; D005838:Genotype; D006509:Hepatitis B / Q000175:diagnosis / Q000821:virology*; D006515:Hepatitis B virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D044366:Transition Temperature; D019562:Viral Load
D017931:DNA Primers; D004279:DNA, Viral