Contamination-free visual detection of SARS-CoV-2 with CRISPR/Cas12a: A promising method in the point-of-care detection

Abstract

The outbreaks of the infectious disease COVID-19 caused by SARS-CoV-2 seriously threatened the life of humans. A rapid, reliable and specific detection method was urgently needed. Herein, we reported a contamination-free visual detection method for SARS-CoV-2 with LAMP and CRISPR/Cas12a technology. CRISPR/Cas12a reagents were pre-added on the inner wall of the tube lid. After LAMP reaction, CRISPR/Cas12a reagents were flowed into the tube and mixed with amplicon solution by hand shaking, which can effectively avoid possible amplicon formed aerosol contamination caused by re-opening the lid after amplification. CRISPR/Cas12a can highly specific recognize target sequence and discriminately cleave single strand DNA probes (5'-6FAM 3'-BHQ1). With smart phone and portable 3D printing instrument, the produced fluorescence can be seen by naked eyes without any dedicated instruments, which is promising in the point-of-care detection. The whole amplification and detection process could be completed within 40 min with high sensitivity of 20 copies RNA of SARS-CoV-2. This reaction had high specificity and could avoid cross-reactivity with other common viruses such as influenza virus. For 7 positive and 3 negative respiratory swab samples provided by Zhejiang Provincial Center for Disease Control and Prevention, our detection results had 100% positive agreement and 100% negative agreement, which demonstrated the accuracy and application prospect of this method.

Keywords: CRISPR/Cas12a; RT-LAMP; SARS-CoV-2; Visual detection.

Fig. 1

In the whole gene sequence of the SARS-CoV-2, the ORF gene, E gene, N gene were chosen as the targets for detection.

Fig. 2

The RT-PCR amplification curves and corresponding Ct values for ORF gene of different concentrations of RNA templates using methods recommended by Chinese Center for Disease Control and Prevention.

Fig. 3

The real time RT-LAMP amplification curves and corresponding threshold time of different concentrations of RNA templates with a) E gene as the target. b) N gene as the target. c) ORF gene as the target.

Fig. 4

a) The diagram of overall experimental flow and principle of the contamination-free visual detection method. b) Visual detection of different concentrations of synthesized RNA templates of ORF gene. c) The photo of 3D printing instrument and smart phone used to observe fluorescent signal.

Fig. 5

The detection results of 7 positive and 3 negative real respiratory swab samples. For each picture, the left side was sample to be detected and the right side was no template control.