期刊
BIOTECHNOLOGY AND BIOENGINEERING
卷 118, 期 5, 页码 2053-2066出版社
WILEY
DOI: 10.1002/bit.27718
关键词
contamination‐ free; CRISPR; Cas9; end‐ point detection; PCR; RT‐ PCR
资金
- Special Project of Science and Technology Development of Guangdong Province [2017B020207011]
- National Natural Science Foundation of China [21874049, 21904042, 81772246, 91959128]
- Opening Project of State Key Laboratory of Chemo/Biosensing and Chemometrics of Hunan University [2019004]
- Special Support Program of Guangdong Province [2016TQ03R749]
- Open Funds of the State Key Laboratory of Electroanalytical Chemistry [SKLEAC202001]
The study introduced a CRISPR/Cas9 eraser strategy to eliminate the risk of contamination amplification in PCR. By engineering primers and pre-incubating Cas9/sgRNA, selective cleavage of contaminated products was achieved to improve the accuracy of virus detection.
Polymerase chain reaction (PCR), a central technology for molecular diagnostics, is highly sensitive but susceptible to the risk of false positives caused by aerosol contamination, especially when an end-point detection mode is applied. Here, we proposed a solution by designing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 eraser strategy for eliminating potential contamination amplification. The CRISPR/Cas9 engineered eraser is firstly adopted into artpcr reverse-transcription PCR (RT-PCR) system to achieve contamination-free RNA detection. Subsequently, we extended this CRISPR/Cas9 eraser to the PCR system. We engineered conventional PCR primers to enable the amplified products to contain an implanted NGG (protospacer adjacent motif, PAM) site, which is used as a code for specific CRISPR/Cas9 recognition. Pre-incubation of Cas9/sgRNA with PCR mix leads to a selective cleavage of contamination amplicons, thus only the template DNA is amplified. The developed CRISPR/Cas9 eraser, adopted by both RT-PCR and PCR systems, showed high-fidelity detection of SARS-CoV-2 and African swine fever virus with a convenient strip test.
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