A one-pot assay based on PAM-free recombinase polymerase amplification and CRISPR/Cas12a for rapid detection of SARS-CoV-2 N gene

Jun 11, 2025Talanta

Rapid detection of the SARS-CoV-2 N gene using a single-step DNA amplification and CRISPR-based test

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Abstract

The assay can detect SARS-CoV-2 N gene DNA within 30 minutes with a sensitivity of 100 aM.

A one-pot assay combines recombinase polymerase amplification and CRISPR/Cas12a for SARS-CoV-2 detection.
The method avoids PAM site constraints by using two individual crRNAs targeting different regions of the N gene.
Activation of Cas12a by the amplified N gene DNA leads to significant fluorescence signal generation.
The assay demonstrates high sensitivity and excellent specificity for SARS-CoV-2 detection.
Practical application was confirmed through analysis of spiked samples.

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SARS-CoV-2 is a pathogenic virus, which exhibits high contagiousness. Therefore, a rapid and sensitive SARS-CoV-2 detection strategy is imperative. Herein, a one-pot assay by the combination of protospacer adjacent motif (PAM)-free recombinase polymerase amplification with CRISPR/Cas12a for detecting SARS-CoV-2 N gene was reported. To avoid the constraint of the PAM site for double-stranded DNA (dsDNA) in CRISPR/Cas12a system, we designed two individual crRNAs to hybridize with two different regions of the target sequence. The presence of N gene DNA was able to initiate the amplification of RPA, exposing the recognition site of crRNA and activating the Cas12a. Whereafter, the Cas12a activation resulted in the digestion of nontarget DNA reporters to induce significant fluorescence signal. The assay completed the detection of N gene DNA within 30 min. And a high sensitivity of 100 aM was obtained because of RPA amplification and Cas12a trans cleavage activity. Meanwhile, the proposed assay showed excellent specificity due to the site-specific recognition ability of CRISPR/Cas12a. More importantly, analysis of spiked samples verified the excellent practical application of the proposed method. Thus, the assay earned promising potential in molecular diagnostics.

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A one-pot assay based on PAM-free recombinase polymerase amplification and CRISPR/Cas12a for rapid detection of SARS-CoV-2 N gene

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