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 s...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.展开更多
目的建立TaqMan-MGB探针检测CYP2C19基因*2、*3和*17三个多态性位点的方法。方法针对CYP2C19基因*2、*3和*17位点设计合成相应引物及双标记探针。选取2021年3月至2022年6月贵州省人民医院心内科门诊已知基因型的剩余全血样本42例,覆盖...目的建立TaqMan-MGB探针检测CYP2C19基因*2、*3和*17三个多态性位点的方法。方法针对CYP2C19基因*2、*3和*17位点设计合成相应引物及双标记探针。选取2021年3月至2022年6月贵州省人民医院心内科门诊已知基因型的剩余全血样本42例,覆盖每个位点的每个基因型各10例,提取DNA进行聚合酶链反应(PCR)检测,产物进行电泳和Sanger法测序分析。加样1、10、100、800 ng DNA,进行PCR检测以评价灵敏度。每个位点的每个基因型5例,每周1次对其检测,连续3周,以评价重复性。结果设计的引物扩增出目的基因片段,电泳显示明亮的单一的DNA条带;90个基因型的PCR结果与测序结果完全一致(P>0.05);不同加样量PCR法均可准确判读基因型,所有基因型在1 ng水平的Ct值均<34。同一基因型3次重复检测的结果一致。结论本研究成功建立了TaqMan-MGB探针检测CYP2C19基因*2、*3和*17三个多态性位点的方法,具有快速、准确、灵敏、简便的特点。展开更多
文摘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.
文摘目的建立TaqMan-MGB探针检测CYP2C19基因*2、*3和*17三个多态性位点的方法。方法针对CYP2C19基因*2、*3和*17位点设计合成相应引物及双标记探针。选取2021年3月至2022年6月贵州省人民医院心内科门诊已知基因型的剩余全血样本42例,覆盖每个位点的每个基因型各10例,提取DNA进行聚合酶链反应(PCR)检测,产物进行电泳和Sanger法测序分析。加样1、10、100、800 ng DNA,进行PCR检测以评价灵敏度。每个位点的每个基因型5例,每周1次对其检测,连续3周,以评价重复性。结果设计的引物扩增出目的基因片段,电泳显示明亮的单一的DNA条带;90个基因型的PCR结果与测序结果完全一致(P>0.05);不同加样量PCR法均可准确判读基因型,所有基因型在1 ng水平的Ct值均<34。同一基因型3次重复检测的结果一致。结论本研究成功建立了TaqMan-MGB探针检测CYP2C19基因*2、*3和*17三个多态性位点的方法,具有快速、准确、灵敏、简便的特点。
