| 酸检测的新方法——重组酶聚合酶扩增(RPA) | | 点击： 作者：51protocol收集 来源： 时间： 2007-05-28 本站论坛 |
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酸检测的新方法——重组酶聚合酶扩增(RPA)
DNA扩增是大多数核酸检测方法中的一个基本步骤，但是现有的技术需要尖端的仪器，例如温度调节仪器，或者复杂的加样流程，因此在专业实验室以外进行核酸分析受到了限制。
在最新一期的《PLoS Biology》上， Cambridge的Olaf Piepenburg等报道的方法——重组酶聚合酶扩增(RPA)克服了上述难题：RPA不需要模板热变性这个步骤，可以在持续的较低温度下进行，同时，辅以一种新的探针检测方法。
RPA通过反向寡聚核苷酸引物定位到模板DNA以及在DNA聚合酶的延伸，获得了特定DNA片段的等温扩增（见图1A）。RPA的关键是动态反应环境的建立，即重组酶—引物复合体的形成和解聚达到一种动态的平衡（见图1B）。为了设计一个高敏感性的PRA检测系统，他们开发了一个新的探针检测方法（见图3A）。他们用的探针含有一个类似四氢呋喃的脱碱基位点（THF），在核酸的侧面非常靠近地用荧光基团和淬灭基团修饰。为了证明RPA和探针/核酸检测组合后的效果，他们设计了引物和探针用于检测一种常见的医院病原抗甲氧西林金黄色葡萄球菌(MRSA)（见图3B）。
大量研究研究和诊断应用可能从应用RPA中得到好处。最明显的可能是，这个技术为非实验室试验设计的发展提供了显著的突破，当结合手持工具或者无需仪器的DNA检测系统时，RPA将为多种病原检测以及用于其他母体田间试剂盒提供一个操作简便的系统。
相关图片
图1
此主题相关图片如下：
Figure 1.
Schematic of the RPA Process and the Recombinase/Primer Filament Formation
(A) Recombinase/primer filaments scan template DNA for homologous sequences (red/blue). Following strand exchange, the displaced strand is bound by gp32 (green), primers are extended by Bsu polymerase (blue). The net result of two opposing primer binding/extension events is the generation of one complete copy of the amplicon in addition to the original template. Repetition of the process results in exponential DNA amplification.
(B) In the presence of ATP, uvsX (gray) binds cooperatively to oligonucleotides (red, top). Upon ATP hydrolysis, the nucleoprotein complex disassembles (left) and uvsX can be replaced by gp32 (green, right). The presence of uvsY and Carbowax20M shifts the equilibrium in favour of recombinase loading.
图3
此主题相关图片如下：
Figure 3.
Schematic of the Probe-Based RPA Detection Method
(A) Signal generation by separation of a fluorophore and a quencher depends on cutting of the probe by double-strand specific Nfo.
(B) Arrangement of primers and probes relative to the targets used in Figures 4 and 5. See Protocol S1 for sequences of MRSA isoforms. A PCR fragment that fused an unrelated sequence to the target sites sccIII and orfX served as internal control.
英文摘要：
DNA amplification is essential to most nucleic acid testing strategies, but established techniques require sophisticated equipment or complex experimental procedures, and their uptake outside specialised laboratories has been limited. Our novel approach, recombinase polymerase amplification (RPA), couples isothermal recombinase-driven primer targeting of template material with strand-displacement DNA synthesis. It achieves exponential amplification with no need for pretreatment of sample DNA. Reactions are sensitive, specific, and rapid and operate at constant low temperature. We have also developed a probe-based detection system. Key aspects of the combined RPA amplification/detection process are illustrated by a test for the pathogen methicillin-resistant Staphylococcus aureus. The technology proves to be sensitive to fewer than ten copies of genomic DNA. Furthermore, products can be detected in a simple sandwich assay, thereby establishing an instrument-free DNA testing system. This unique combination of properties is a significant advance in the development of portable and widely accessible nucleic acid–based tests.
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