Fig 1.
Workflow of Cas12a-based detection for SARS-CoV-2.
Extracted RNA or lysed samples can be used as an input for RPA-Cas12a-based detection. (a) The results can be achieved within 50 minutes using a fluorescent reader or with visual detection methods. (b) The minimum equipment needed to run the assay following RNA extraction or rapid sample lysis includes UV light imagers, heat blocks (42°C and 37°C), pipettes and tips, Eppendorf tubes, reagents, and lateral flow strips. Cas, CRISPR associated proteins; RPA, recombinase polymerase amplification; RT, room temperature; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; UV, ultraviolet.
Fig 2.
Primers for ORF1ab (a–d) and N (e–h) were screened using a single forward or reverse primer against the corresponding reverse or forward primers, selecting the best performing primer and then using it to perform second round screening to determine the primer pairs with the best performance. Fluorescent signal was obtained at 10 minutes for the Cas12a reaction. The data are presented as the means ± SD (n = 3; b, d, f, h). Numerical source data underlying this figure can be found in S1 Data. Cas, CRISPR associated proteins; RFU, relative fluorescence unit; SD, standard deviation.
Fig 3.
Sensitivity and specificity analysis.
(a, b) Serial dilutions of in vitro–transcribed ORF1ab (a) and N (b) for LoD determination. After amplification at 42°C for 20 minutes, 10 μL of each reaction was transferred to 40 μL of the Cas12a mixture for cleavage assays. Bar graphs represent fluorescent signal obtained at 10 minutes for the Cas12a reaction. The data are presented as the means ± SD (n = 15). Unpaired 2-tailed t test was used to analyze the difference from NTC. ***P < 0.001. (c) The locations of primers and crRNAs of ORF1ab (upper) and N (lower) from 4 SARS-CoV-2 strains and 4 other human coronaviruses. Sequences of different types of coronavirus were obtained from GenBank and aligned by the MEGA program. Positions with complete alignment to the primer or crRNA are labeled in yellow. (d, e) Specificity assessment of the RT-RPA assay for ORF1ab gene (d) and N gene (e) of SARS-CoV-2. Only the RNA from SARS-CoV-2 produced signals, whereas RNAs from other pathogens and the negative control did not produce any detected signals. Four human coronaviruses (SARS, HKU1, MERS, and OC43) and 11 other respiratory pathogens including avian influenza A viruses (H7N9 and H5N1), human influenza A viruses (H1N1 [swine flu] and H3N2 subtypes), influenza B viruses (Yamagata and Victoria lineages), adenovirus (ADV3, 7), human parainfluenza virus (PIV2), Staphylococcus aureus, and Streptococcus pneumonia were evaluated. Numerical source data underlying this figure can be found in S1 Data. LoD, Limit of Detection; MERS, Middle East Respiratory Syndrome; NTC, no-template control; RPA, recombinase polymerase amplification; RT, room temperature; SARS, Severe Acute Respiratory Syndrome; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; SD, standard deviation.
Fig 4.
Validation of the system with pseudovirus and clinical samples.
(a–d) The LoD was determined using the lysed pseudovirus for ORF1ab (a, b) and N (c, d). Fluorescent signal was obtained at 10 minutes for the Cas12a reaction (b, d). Fifteen replicates were conducted for each test. The data are presented as the means ± SD. Unpaired 2-tailed t test was used to analyze the difference from NTC. ***P < 0.001. (e, g) The signals of ORF1ab (e) and N (g) were detected with a gel imaging system after 20 minutes of Cas12a cleavage at the indicated viral load of pseudovirus, and the signals were visible with the naked eye; rnx, per reaction. (f, h) Lateral flow detection of ORF1ab (f) and N (h) genes at the indicated viral load. T, test line; C, control line; rnx, per reaction. (i) RPA-only detection of ORF1ab and N gene. RT-RPA reaction was performed at 42°C for 12 minutes, after which the product was diluted 1:5 in HybriDetect Assay Buffer, and the strips were inserted and incubated for 2 minutes at RT. Then, the strips were removed and imaged using a smartphone camera. T, test line; C, control line. (j) Real-time (left panel) and end point (right panel) fluorescence detection using primers specific to ORF1ab and N gene of SARS-CoV-2 in clinical samples. Eleven PCR–positive and PCR–negative samples each with 2 μl of the RNA input were used for the evaluation. 1–11, PCR–positive samples; 12–22, PCR–negative samples. Box-and-whisker plots represent fluorescent values at 20 minutes for the Cas12a reaction. The lower and upper borders of the box represent first and third quartile of the values, respectively. The line within the box shows the median, and the whiskers falling outside the box are plotted as the minimum and maximum of the data. Unpaired 2-tailed t test was used to analyze the difference from negatives (n = 11); ***P < 0.001. (k, l) Visual detection for ORF1ab (k) and N (l) genes of SARS-CoV-2 in clinical samples. Eleven PCR–positive and PCR–negative samples were used for the evaluation. The signals were obtained with a gel imaging machine at 20 minutes for the Cas12a reaction. (m, n) Eleven PCR–positive and PCR–negative clinical samples were detected for ORF1ab (m) and N (n) genes of SARS-CoV-2 using lateral flow strips. RT-RPA reaction was performed at 42°C for 20 minutes and followed by 20 minutes of Cas12a cleavage, and then the product was diluted and overflowed on a strip for 2 minutes. T, test line; C, control line. Numerical source data underlying this figure can be found in S1 Data. Cas, CRISPR associated proteins; LoD, Limit of Detection; NTC, no-template control; PCR, polymerase chain reaction; RPA, recombinase polymerase amplification; RT, room temperature; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; SD, standard deviation.