编辑: 没心没肺DR | 2018-07-07 |
the specific product, non-specific products and primer dimers are detected equally well.? The LightCycler Instrument allows melting curve analysis Once the melting point of the product has been determined the LightCycler Instrument'
s flexible programming allows the user to acquire fluorescence above the melting temperature of the primer dimers, but below the melting temperature of the product.? 缺点:存在非特异性产物,可以与非特异性产物,甚至引物二聚体结合发出荧光. 可以使用熔点曲线分析进行鉴别,明显提高诊断的特异性.(F1通道)
2、TaqMan水解探针技术 杂交探针技术对PCR产物定量又称荧光共振能量转移,FRET需要一对引物和一对探针,探针A的3`端为荧光染料供体;
探针B的5`端作为荧光染料受体.2个探针首尾相连,仅差一个碱基.检测在退火时进行. Advantages of the LightCycler SystemWatch amplification as it occurs: real-time, on-line fluorescence allows you to see the results of every cycle as soon as PCR proceeds Save time: extremely rapid PCR,
20 minutes for
30 cycles Verify amplicon specificity: melting curve analysis provides differentiation between amplification products Detect mutations: changes in the melting curve analysis can be used to identify mutations Minimize contamination: amplification and detection are performed in the same closed tube 荧光定量PCR技术的优点1)定量准确,实时监控2) 敏感性和特异性高3)简单快速,30个循环20几分钟就能够完成.4)污染可能性小5)不需要常规的产物后期复杂处理,直接观测结果6)可以进行点突变分析,根据熔点曲线的不同直接分析点突变的情况. Figure 3. Mutation detection by melting curve analysis. Top panel: melting curves of amplified gene fragments from wild-type and mutant individuals. Bottom panel: negative first derivatives of the melting curves showing the unique melting peak of each genotype. The gene fragment amplified from the heterozygous individual exhibits a melting curve with both the wild-type and mutant peaks (red plot). HBV的定量检测 标本:病人血清200μl检测:HBV病毒核酸载量检测样本:阳性对照、阴性对照、空白对照 标准品、病人HBV DNA检测步骤:HBV DNA提取;
荧光定量 PCR;
结果分析定量PCR方法:TaqMan水解探针技术 标准品扩增曲线 标准曲线 报告结果: HBV DNA拷贝数/ml,如6.20*106拷贝数/ml或6.20 E +06?参考范围:最小检测值,5*102拷贝数/ml(5E+02) ........