1、HBV DNA定量检测PCR的基本原理的基本原理 The software displays the fluorescence signals in real-time immediately after each measurement.Signals from the samples are obtained as the machine positions the capillaries sequentially over the optical unit.During thermal cycling,fluorescence can be measured once per cycle
2、 for every capillary and the fluorescence values are displayed immediately on the screen and updated after every cycle(Fig.3).The generated data are stored for further analysis.荧光定量荧光定量PCRPCR在每一次循环在每一次循环后对进行实时监控，因此后对进行实时监控，因此能最准确反映出对数期。能最准确反映出对数期。确定准确的初始浓度。确定准确的初始浓度。软件系统可以进行参软件系统可以进行参数设定、保存数据和数设定、保存
3、数据和报告结果，并将结果报告结果，并将结果自动保存在计算机中。自动保存在计算机中。相同模板进行相同模板进行96次扩增的扩增曲线图。次扩增的扩增曲线图。CtCt值，值，C C代表代表CycleCycle，t t代表代表thresholdthreshold，CtCt值的含义值的含义是：每个反应管内的荧光信号到达设定的域值时所经历是：每个反应管内的荧光信号到达设定的域值时所经历的循环数。的循环数。CtCt值分析实际上就是低浓度的荧光值分析。值分析实际上就是低浓度的荧光值分析。由于是低浓度，影响因素小，所以具有良好的重现性。由于是低浓度，影响因素小，所以具有良好的重现性。研究表明，研究表明，每个模板的
4、每个模板的CtCt值与该模板的起始拷贝数值与该模板的起始拷贝数的对数存在线性关系，的对数存在线性关系，起始拷贝数越多，起始拷贝数越多，CtCt值越小。值越小。利用已知起始拷贝数的标准利用已知起始拷贝数的标准品可作出标准曲线，其中横品可作出标准曲线，其中横坐标代表起始拷贝数的对数，坐标代表起始拷贝数的对数，纵坐标代纵坐标代CtCt值。因此，只要值。因此，只要获得未知样品的获得未知样品的CtCt值，即可值，即可从标准曲线上计算出该样品从标准曲线上计算出该样品的起始拷贝数。的起始拷贝数。The biggest disadvantage of SYBR is that it binds to any
5、dsDNA;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 Instruments flexible programming allows the user to acquire fluorescence abo
6、ve 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，缺点：存在非特异性产物，可以与非特异性产物，甚至可以与非特异性产物，甚至引物二聚体结合发出荧光引物二聚体结合发出荧光。可以使用可以使用熔点曲线分析熔点曲线分析进进行鉴别，明显提高诊断的行鉴别，明显提高诊断的特异性。（特异性。（F1通道）通道）2 2、TaqManTaqMan水解探针技术水解探针技术杂交探针技术对杂交探针技术对PCR产物定量又称荧光共振能量转移，产物定量又称荧光共振
7、能量转移，FRET需要一对引物和一对探针，探针需要一对引物和一对探针，探针A的的3端为荧光染料供体；探端为荧光染料供体；探针针B的的5端作为荧光染料受体。端作为荧光染料受体。2个探针首尾相连，仅差一个个探针首尾相连，仅差一个碱基。检测在退火时进行。碱基。检测在退火时进行。Advantages of the LightCyclerAdvantages of the LightCycler SystemSystemWatch amplification as it occurs:Watch amplification as it occurs:real-time,on-line fluoresce
8、nce real-time,on-line fluorescence allows you to see the results of allows you to see the results of every cycle as soon as PCR every cycle as soon as PCR proceeds proceeds Save time:extremely rapid PCR,Save time:extremely rapid PCR,20 minutes for 30 cycles 20 minutes for 30 cycles Verify ampliconVe
9、rify amplicon specificity:specificity:melting curve analysis provides melting curve analysis provides d i f f e r e n t i a t i o n b e t w e e n d i f f e r e n t i a t i o n b e t w e e n amplification products amplification products Detect mutations:changes in the Detect mutations:changes in the
10、melting curve analysis can be melting curve analysis can be used to identify mutations used to identify mutations M i n i m i z e c o n t a m i n a t i o n:M i n i m i z e c o n t a m i n a t i o n:amplification and detection are amplification and detection are performed in the same closed tube perf
11、ormed in the same closed tube 荧光定量荧光定量PCRPCR技术的优点技术的优点1 1）定量准确，实时监控）定量准确，实时监控2 2）敏感性和特异性高敏感性和特异性高3 3）简单快速，）简单快速，3030个循环个循环2020几分几分钟就能够完成。钟就能够完成。4 4）污染可能性小）污染可能性小5 5）不需要常规的产物后期复杂）不需要常规的产物后期复杂处理，直接观测结果处理，直接观测结果6 6）可以进行点突变分析，根据）可以进行点突变分析，根据熔点曲线的不同直接分析点突熔点曲线的不同直接分析点突变的情况。变的情况。Figure 3.Mutation detection
12、 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).标准品扩增曲线标准品扩增曲线标准曲线标准曲线报告结果：报告结果：HBV DNA拷贝数拷贝数/ml，如如 6.20106拷贝数拷贝数/ml或或6.20 E+06 参考范围：最小检测值，参考范围：最小检测值，5102拷贝数拷贝数/ml（5E+02）
