Detection of hepatitis B virus DNA by real-time PCR using TaqMan-MGB probe technology.
OBJECTIVE
To develop a real-time PCR for detecting hepatitis B virus (HBV) DNA based on TaqMan technology using a new MGB probe.


METHODS
Plasmid containing the sequence of X gene (1414-1744 nt) was constructed as HBV-DNA standard for quantitative analysis. A TaqMan-MGB probe between primers for amplification was designed to detect PCR products. The interested sequence contained in the plasmid and in clinical specimens was quantitatively measured.


RESULTS
The detection limit of the assay for HBV DNA was 1 genome equivalent per reaction. A linear standard curve was obtained between 10(0) and 10(9) DNA copies/reaction (r>0.990). None of the negative control samples showed false-positive reactions in duplicate. HBV DNA was detected in 100% (50/50) of HBV patients with HbeAg, and in 72.0% (36/50) with HBsAg, HBeAb and HBcAb. The coefficient of variation for both intra- and inter-experimental variability demonstrated high reproducibility and accuracy.


CONCLUSIONS
Real-time PCR based on TaqMan-MGB probe technology is an excellent method for detection of HBV DNA.
Zhao
Jin-Rong
JR
Institute of Genetic Diagnosis, Fourth Military Medical University, 17 Changle West Road, Xi'an 710032, Shaanxi Province, China. zhaojrr@fmmu.edu.cn
Bai
Yu-Jie
YJ
Zhang
Qing-Hua
QH
Wan
Yan
Y
Li
Ding
D
Yan
Xiao-Jun
XJ
D001483:Base Sequence; D015342:DNA Probes / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis; D006515:Hepatitis B virus / Q000235:genetics* / Q000302:isolation & purification*; D019694:Hepatitis B, Chronic / Q000175:diagnosis*; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011994:Recombinant Proteins / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019914:Taq Polymerase
D015342:DNA Probes; D004279:DNA, Viral; D011994:Recombinant Proteins; D019914:Taq Polymerase