A CRISPR/Cas9 eraser strategy for contamination-free PCR end-point detection.
Biotechnol Bioeng
; 118(5): 2053-2066, 2021 05.
Article
in English
| MEDLINE | ID: covidwho-1092501
ABSTRACT
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.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Polymerase Chain Reaction
/
Reverse Transcriptase Polymerase Chain Reaction
/
CRISPR-Cas Systems
Type of study:
Diagnostic study
/
Prognostic study
Limits:
Animals
/
Humans
Language:
English
Journal:
Biotechnol Bioeng
Year:
2021
Document Type:
Article
Affiliation country:
Bit.27718
Similar
MEDLINE
...
LILACS
LIS