RPA——PCR技術(shù)的革命

1、RPAPCR技術(shù)的革命什么是RPA為什么說RPA是一場(chǎng)技術(shù)革命應(yīng)用及相關(guān)什么是RPA？ 自PCR技術(shù)誕生以來已經(jīng)有三十年了。從經(jīng)典PCR、實(shí)時(shí)定量PCR再到現(xiàn)在的數(shù)字PCR，這一技術(shù)在不斷蛻變卻從未淡出我們的視野。 RPA（Recombinase Polymerase Amplification） 全稱重組酶聚合酶擴(kuò)增技術(shù)，被稱為可以替代PCR的核酸檢測(cè)技術(shù)。 RPA技術(shù)主要依賴于三種酶： 能結(jié)合單鏈核酸(寡核苷酸引物)的重組酶 單鏈DNA結(jié)合蛋白(SSB) 鏈置換DNA聚合酶。 這三種酶的混合物在常溫下也有活性，最佳反應(yīng)溫度在37C左右。RPA的原理的原理Previouly establis

2、hed RPA Conditions , 2006 引物設(shè)計(jì)引物設(shè)計(jì) RPA分析的關(guān)鍵在于擴(kuò)增引物和探針的設(shè)計(jì)。PCR引物多半是不適用的，因?yàn)镽PA引物比一般PCR引物長(zhǎng)，通常需要達(dá)到30-38個(gè)堿基。引物過短會(huì)降低重組率，影響擴(kuò)增速度和檢測(cè)靈敏度。在設(shè)計(jì)RPA引物時(shí)，變性溫度不再是影響擴(kuò)增引物的關(guān)鍵因素。RPA的引物和探針設(shè)計(jì)不像傳統(tǒng)PCR那樣成熟，用戶需要自己摸條件進(jìn)行優(yōu)化。Speed10 to 15 minute detection timeSensitivitySingle moleculeCostCheap access as little or no hardware is req

3、uiredSimplicityStabilised reaction format, minimal sample preparationRobustnessTo sample contaminants and temperature fluctuationsPortability Handheld instrumentation or disposable test format 為什么說為什么說RPA是一場(chǎng)技術(shù)革命是一場(chǎng)技術(shù)革命 RPA具體應(yīng)用舉例ClinicalFood safetyAgriculturalBlood bankscreeningEnvironmentalAnimal he

4、althRecombinase Polymerase Amplification (RPA) of CaMV-35SPromoter and nos Terminator for Rapid Detection ofGenetically Modified CropsChao 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

5、: (C.X.); (L.L.); (W.J.)2 Inspection and Testing Center for Environmental Risk Assessment of Genetic Modified Plant-RelatedMicroorganisms (Beijing), Ministry of Agriculture, Beijing 100081, China1. IntroductionThe International Service for the Acquisition of Agri-biotech Applications (ISAAA) estimat

6、es 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 method

7、s 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 mos

8、t 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 re

9、al-time RPA assay to detect P-35S and T-nos sequences for purposes of GMO screening and etection.2.2. Sensitivity of the RPA Assays2.3. Application to Practical Sample Analysis3.1. Materials3.2. Extraction of Genomic DNA3.3. Oligonucleotide Primers and Probes RPA real time fluorescent assays include

10、 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.

11、 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 regression3. Experimental Section4. ConclusionsIn this research, we have developed a rapid real-time RPA technique for