重组酶聚合酶扩增
反式激活crRNA
核糖核酸
化学
分子生物学
清脆的
互补DNA
放大器
逆转录酶
核糖核酸酶P
核酸酶
核糖核酸酶H
DNA
Cas9
生物
环介导等温扩增
核酸
基因
聚合酶链反应
生物化学
作者
Wei Feng,Hanyong Peng,Jingyang Xu,Yanming Liu,Kanti Pabbaraju,Graham Tipples,Michael Joyce,Holly A. Saffran,D. Lorne Tyrrell,Shawn Babiuk,Hongquan Zhang,X. Chris Le
出处
期刊:Analytical Chemistry
[American Chemical Society]
日期:2021-09-10
卷期号:93 (37): 12808-12816
被引量:73
标识
DOI:10.1021/acs.analchem.1c03456
摘要
CRISPR-Cas systems integrated with nucleic acid amplification techniques improve both analytical specificity and sensitivity. We describe here issues and solutions for the successful integration of reverse transcription (RT), recombinase polymerase amplification (RPA), and CRISPR-Cas12a nuclease reactions into a single tube under an isothermal condition (40 °C). Specific detection of a few copies of a viral DNA sequence was achieved in less than 20 min. However, the sensitivity was orders of magnitude lower for the detection of viral RNA due to the slow initiation of RPA when the complementary DNA (cDNA) template remained hybridized to RNA. During the delay of RPA, the crRNA-Cas12a ribonucleoprotein (RNP) gradually lost its activity in the RPA solution, and nonspecific amplification reactions consumed the RPA reagents. We overcame these problems by taking advantage of the endoribonuclease function of RNase H to remove RNA from the RNA-cDNA hybrids and free the cDNA as template for the RPA reaction. As a consequence, we significantly enhanced the overall reaction rate of an integrated assay using RT-RPA and CRISPR-Cas12a for the detection of RNA. We showed successful detection of 200 or more copies of the S gene sequence of SARS-CoV-2 RNA within 5–30 min. We applied our one-tube assay to 46 upper respiratory swab samples for COVID-19 diagnosis, and the results from both fluorescence intensity measurements and end-point visualization were consistent with those of RT-qPCR analysis. The strategy and technique improve the sensitivity and speed of RT-RPA and CRISPR-Cas12a assays, potentially useful for both semi-quantitative and point-of-care analyses of RNA molecules.
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