Quantitative reverse transcription strand displacement amplification: Quantitation of nucleic acids using an isothermal amplification technique
Quantitative reverse transcription strand displacement amplification: Quantitation of nucleic acids using an isothermal amplification technique
复制标题
DOI：
10.1006/abio.1998.2641
复制
发表时间：
1998-06-01
影响因子：
2.9
通讯作者：
Walker, GT
中科院分区：
文献类型：
--
作者：
Nycz, CM;Dean, CH;Walker, GT
核酸扩增技术的最新进展已经允许对临床样本中的病毒核酸水平进行定量。最普遍的检测是对艾滋病毒载量的检测。链置换扩增（SDA）是一种利用限制性内切酶和具有链置换特性的DNA聚合酶的等温DNA扩增系统。SDA适用于HIV gag序列的定量RNA扩增（QRT-SDA），包括AMV逆转录酶、定量对照序列和HIV和对照序列的P-32标记检测寡核苷酸。我还通过包括来自T4噬菌体基因32的单链结合蛋白（T4 gp 32）以增强链置换复制来提高扩增效率。在该技术的初步分析演示中，RT-SDA在从携带HIV gag序列的质粒产生的500- 500，000个转录物的范围内定量为两倍。QRT-SDA可能是临床环境中病毒载量检测的一种方便的替代方法。(C)北京：科学出版社.
Recent advances in nucleic acid amplification techniques have allowed for quantitation of viral nucleic acid levels in clinical specimens. The most prevalent testing is carried out for HIV viral load. Strand displacement amplification (SDA) is an isothermal DNA amplification system utilizing a restriction enzyme and a DNA polymerase with strand displacement properties. SDA was adapted for quantitative RNA amplification (QRT-SDA) of an HIV gag sequence by including AMV reverse transcriptase, a quantitative control sequence, and P-32-labeled detector oligonucleotides for the HIV and the control sequences. me have also improved the amplification efficiency by including the single-strand binding protein from gene 32 of T4 bacteriophage (T4gp32) to enhance strand displacement replication. In a preliminary analytical demonstration of the technique, RT-SDA was quantitative to within twofold over a range of 500-500,000 transcripts that were generated from a plasmid bearing an HIV gag sequence. QRT-SDA potentially represents a convenient alternative for viral load testing in a clinical setting. (C) 1998 Academic Press.