RPA—PCR技术的革命.ppt

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,RPA,PCR,技术的革命,什么是,RPA,为什么说,RPA,是一场技术革命,应用及相关,1,什么是,RPA,？,自,PCR,技术诞生以来已经有三十年了。从经典,PCR,、实时定量,PCR,再到现在的数字,PCR,，这一技术在不断蜕变却从未淡出我们的视野。,RPA,（,Recombinase Polymerase Amplification,）,全称重组酶聚合酶扩增技术，被称为可以替代,PCR,的核酸检测技术。,2,RPA,技术主要依赖于三种酶：,能结合单链核酸,(,寡核苷酸引物,),的重组酶,单链,DNA,结合蛋白,(SSB),链置换,DNA,聚合酶。,这三种酶的混合物在常温下也有活性，最佳反应温度在,37C,左右。,3,RPA,的原理,4,5,Previouly established RPA Conditions,2006,6,7,8,9,10,引物设计,RPA,分析的关键在于扩增引物和探针的设计。,PCR,引物多半是不适用的，因为,RPA,引物比一般,PCR,引物长，通常需要达到,30-38,个碱基。引物过短会降低重组率，影响扩增速度和检测灵敏度。在设计,RPA,引物时，变性温度不再是影响扩增引物的关键因素。,RPA,的引物和探针设计不像传统,PCR,那样成熟，用户需要自己摸条件进行优化。,11,Speed,10 to 15 minute detection time,Sensitivity,Single molecule,Cost,Cheap access as little or no hardware is required,Simplicity,Stabilised reaction format,minimal sample preparation,Robustness,To sample contaminants and temperature fluctuations,Portability,Handheld instrumentation or disposable test format,为什么说,RPA,是一场技术革命,12,RPA,具体应用举例,Clinical,Food safety,Agricultural,Blood bank,screening,Environmental,Animal health,13,Recombinase Polymerase Amplification(RPA)of CaMV-35SPromoter and,nos Terminator for Rapid Detection of,Genetically Modified Crops,Chao Xu 1,Liang Li 1,2,Wujun Jin 1,2 and Yusong Wan 1,2,*,1 Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081,China;,E-Mails:xuchao1667(C.X.);liliang(L.L.);jinwujun(W.J.),2 Inspection and Testing Center for Environmental Risk Assessment of Genetic Modified Plant-Related,Microorganisms(Beijing),Ministry of Agriculture,Beijing 100081,China,14,1.Introduction,The International Service for the Acquisition of Agri-biotech Applications(ISAAA)estimates that millions of farmers cultivated genetically modified(GM)crops over more than 170 million hectares across 27 countries in 2013;the major GM crop species were canola,maize,cotton,and soybean 1.,Although the polymerase chain reaction(PCR)is one of the most widely used amplification methods for GMO screening detection 3,the need for delicate equipment and complicated procedures limit the use of PCR amplification in point-of-use and field settings.Rapid,specific,and highly effective methods for identifying the presence of GMOs in food and feed are important and necessary 4.,The most frequently used method for detecting GMO material is screening for the CaMV-35S promoter(P-35S)from the cauliflower mosaic virus(CaMV)and the 3 non-translated region of the nopaline synthase gene(T-,nos)from Agrobacterium mefaciens 11.In this work,we describe the,initial development of a real-time RPA assay to detect P-35S and T-,nos sequences for purposes of,GMO screening and etection.,15,16,2.2.Sensitivity of the RPA Assays,17,18,2.3.Application to Practical Sample Analysis,19,3.1.Materials,3.2.Extraction of Genomic DNA,3.3.Oligonucleotide Primers and Probes,RPA real time fluorescent assays include a forward primer,a reverse primer,and a probe.,3.4.RPA Assays,RPA reactions were performed in a total volume of 50 L using a TwistAmp Exo kit(TwistDX,Cambridge,UK),29.5,L of TwistAmp rehydration buffer,420 nM each RPA primer,120 nM RPA probe,14 mM magnesium acetate,and 1 L of genomic DNA.,mix freeze-dried reaction tube add Magnesium acetate and rehydrated material Twista tube scanner device(39 C for 1525 min),Fluorescence measurements were taken every 20 s,A probit regression,3.Experimental Section,20,4.Conclusions,In this research,we have developed a rapid real-time RPA technique for the detection of P-35S and T-,nos regulatory elements,which are widely employed in GM crops.This novel method can be easily,adapted to other target genes for GMO detection.,21,