Detection of hepatitis B virus DNA by real-time PCR using TaqMan-MGB probe technology |
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Authors: | Zhao Jin-Rong Bai Yu-Jie Zhang Qing-Hua Wan Yan Li Ding Yan Xiao-Jun |
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Affiliation: | Institute of Genetic Diagnosis, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China |
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Abstract: | AIM: 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 100 and 109 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. CONCLUSION: Real-time PCR based on TaqMan-MGB probe technology is an excellent method for detection of HBV DNA. |
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Keywords: | Hepatitis B Virus DNA TaqMan-MGB probe Real-time PCR |
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