数字PCR技术在肿瘤分子病理转化研究中的应用.pptx

王俊果 Digital PCR Product Manager Genetic Analysis Division Life Technologies Solution 数字PCR技术在分子病理临床转化研究中的应用 374.219.022 What is Digital PCR?（什么是数字PCR） “Divide and Conquer” Digital PCR是一种基于单分子模版PCR扩增，进行核酸拷贝数精确定量的分析方法。 dPCR Working Principle（什么是数字PCR） dPCR Working Principle（什么是数字PCR） 微滴式数字PCR流程 将20ul含有样品的PCR预混液制备成20,000个微滴 进行emulsion PCR PCR扩增完成后，对每个样品中的微滴逐个检测，有荧光信号的微滴为阳性微滴；反之为阴性微滴 根据记录到的阳性微滴/总微滴数目计算给出靶分子的拷贝数 Applications for Digital PCR（主要应用） Gene Expression , miRNA Copy Number Variation Generation of references and standards NGS library quantification Absolute quantification Rare cancer target detection Allele detection Absolute quantification of pathogen (e.g. viral load) Detection of harmful strains of pathogens GMO detection and monitoring vol. 96 no. 16  Bert Vogelstein,  9236–9241, doi: 10.1073/pnas.96.16.9236 Development History of Digital PCR Development History of Digital PCR Pohl, G; Shih, I-M (2004). Principle and applications of digital PCR. Expert Rev Mol Diagn 4 (1): 41–47. Digital PCR Application in cfDNA detection JCO November 1, 2003 vol. 21 no. 21 3902-3908 N Engl J Med 2008; 359:366-377July 24, 2008DOI: 10.1056/NEJMoa0800668 Single-Molecule Detection of EGFR Mutations in Plasma Clin Cancer Res 2009;15(6) March 15, 2009 Annals of Oncology 25: 2304–2313, 2014 Digital PCR Application in cfDNA detection Use Digital PCR inQuantify T790M Mutation CANCER GENOMICS PROTEOMICS 1: 31-38 (2015) A hypothesis regarding acquired tyrosine kinase inhibitor (TKI) resistance using digital PCR Identification of EGFR T790M in a patient plasma sample /en-us/product/qx200-droplet-digital-pcr-system?pcp_loc=catprod Detection Of Rare Somatic Mutation Down To 0.01% /us/en/home/life-science/pcr/digital-pcr/quantstudio-3d-digital-pcr-system.html Assays meet requirements for cfDNA Dr. Atocha Romero from Hospital Clinico de Madrid Looked at PIK3CA H1047R mutation in cfDNA A) Sample with mutation (positive) B) Normal sample – no mutation present “…and we have