Quantitation of HIV-1 by real-time PCR with a unique fluorogenic probe
Quantitation of HIV-1 by real-time PCR with a unique fluorogenic probe
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DOI：
10.1016/s0166-0934(00)00288-3
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发表时间：
2001-04-01
影响因子：
3.1
通讯作者：
Bucy, RP
中科院分区：
文献类型：
--
作者：
Saha, BK;Tian, BH;Bucy, RP
对HIV - 1特异性RNA和DNA进行定量对于理解HIV - 1疾病的病理生理学至关重要。已经开发出一种通过实时聚合酶链反应（PCR）对HIV - 1 DNA/RNA进行定量的方法，该方法使用一种独特的荧光引物 - 探针加合物，称为“ scorpion”（蝎形引物探针）。该探针在分子内与相邻引物的延伸部分杂交，这一过程在动力学和热力学上比传统的双分子探针 - 靶标杂交更具优势。本文所提供的数据表明，“ scorpion”检测法极其可靠，与基于信标（beacon）的检测法具有相当的可比性。“ scorpion”检测法也与定量竞争PCR（QC - PCR）检测法相当，但所需时间和精力仅为其一小部分。此外，“ scorpion”检测法的动态范围比传统的终点PCR检测法高几个数量级。在大量外源添加的基因组DNA存在的情况下，可检测到少至10个拷贝的病毒DNA。有限稀释分析表明，该检测法能够检测到单个病毒模板拷贝。因此，“ scorpion”检测法为通过实时PCR对DNA/RNA模板进行定量提供了一种特异性强且灵敏的方法。（C）2001年，爱思唯尔科学出版社（Elsevier Science B.V.）版权所有。
Quantitation of HIV-1 specific RNA and DNA is pivotal to understanding the pathophysiology of HIV-1 diseases. A method has: been developed for quantitation of HIV-1 DNA/RNA by real-time PCR using a unique fluorogenic primer-probe adduct known as scorpion. The probe hybridises to the extension of the adjoining primer intramolecularly, a process kinetically and thermodynamically more favourable than the conventional bimolecular probe-target hybridisation. Data presented in this paper indicate that the scorpion assay is extremely robust and is quite comparable to beacon-based assays. The scorpion assay is also comparable to quantitative competitive PCR (QC-PCR) assays but requires only a fraction of time and effort. Additionally, the dynamic range of the scorpion assay is several log-fold higher than the conventional end point PCR assays, As few as ten copies of VDNA can be detected in the presence of a large excess of exogenously added genomic DNA. Limiting dilution analysis indicates that the assay is capable of detecting a single copy of the viral template. Thus, the scorpion assay presents a specific and sensitive approach for quantitation of DNA/RNA templates by real-time PCR. (C) 2001 Elsevier Science B.V. All rights reserved.