实时荧光定量pcr在临床医学中的应用课件.ppt

In particular, the mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU–VNTR)method has been considered as a good alternative method to RFLP 二．药物疗效观察??? 对病原体的药物治疗中，免疫学指标的变化通常比较滞后出现，且受个体差异影响较大，从而对临床判断难以及时地提供直接证据；而荧光定量PCR检测可直接反映病原体滴度是否受药物治疗发生变化，从而为药物疗效观察提供直接的依据，以供治疗方案参考；同时药物治疗中可能引起的基因变异等情况更依赖核酸检测提供的直接证据。 三．病情判断和预后评价??? 评估受侵器官或组织的炎症发生程度以及是否发生病变；长时间机体病毒高水平的患者尤其是慢性病患者往往预后不好。因此对病原体的定量PCR检测对病情和预后评估均有参考意义。四．加快疾病的诊断??? 许多病原体到目前为止仍旧依靠传统的细菌培养作为诊断手段，操作繁琐费时。以结核杆菌为例，传统培养法需要1~2个月，而荧光PCR可将检测时间缩短至半天，能更及时地为临床提供诊断依据。五．疾病的发病监控??? 许多病原体尤其是病毒病原体可以在感染人体后整合入细胞基因组内而成为整合型，而一旦机体的免疫力下降等又重新进入活动期并引致发病，临床上希望通过检测病原体基因的数量变化并结合临床表现找出其活动以及是否引起发病的规律。荧光定量PCR可以为此提供直接证据。六．遗传疾病的诊断??? 荧光PCR可以实现对点突变、缺失突变、插入突变、多基因突变等的检测。对产前诊断具有其它方法不可比拟的优势，有利于优生优育，提高人口素质。七．母婴传播的监控 High Resolution Melting analysis relies on a new generation of dsDNA binding dyes. LightCycler? 480 High Resolution Melting (HRM) Dye is a member of this new dye family. This unique dye can detect the presence of heteroduplexes formed during PCR (e.g., if the sample is heterozygous for a particular mutation). This feature is not shared with other dyes traditionally used in real-time PCR (e.g., SYBR Green I or ethidium bromide). LightCycler? 480 High Resolution Melting Dye is not toxic to amplification enzymes. Thus, high concentrations of the dye do not affect the PCR. These high concentrations completely saturate the dsDNA in the sample. dsDNA remains dye-saturated during the subsequent melting experiment. Under these conditions, even small changes in the melting curve result in subtle, but reproducible changes in fluorescence. Novel Signal-Improved HRM DyeProvided in High-Resolution Melting Master This picture shows High-Resolution Melting Curve data generated with the HRM dye in comparison to SYBR Green I. The HRM dye binds to double-stranded DNA in a saturating fashion. It gives higher signals and very sharp and distinct melting signals that, can resolve very subtle differences between e.g., wildtype and