Oligonucleotides and crRNAs preparation. Primers with a T7 promoter for RPA amplification are compatible with a CRISPR/Cas12a or CRISPR/Cas13a detection system. The primers for RdRp gene amplification were forward primer with T7 promoter 5’-GAAATTAATACGACTCACTATAGGGctaatgagtgtgctcaagtattgagtgaaat-3’ and reverse primer 5’-caaatgttaaaaacactattagcataagcagt-3’. The primers for N gene amplification were forward primer with T7 promoter 5’-GAAATTAATACGACTCACTATAGGGcagcagtaggggaacttctcctgctagaatgg-3’ and reverse primer 5’-tggcctttaccagacattttgctctcaagctg-3’. The crRNA for RdRp gene (5’-GGGAAUUUCUACUGUUGUAGAUacauauagugaaccgccacaca-3’) and the crRNA for N gene (5’-GGGAAUUUCUACUGUUGUAGAUcugcugcuugacagauuga-3’) were from synthetic oligo fragments. Each oligo fragment contained a T7 promoter which was used as the template for in vitro transcription at 37°C for 16 h using HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB). RNA was purified using Agencourt RNAClean XP (Beckman Coulter). RNA was quantified by Nanodrop and diluted in nuclease-free water to working concentrations. The crRNAs and ssDNA reporter (5’-6-FAM-TTATTATT-BHQ1-3’ and 5’-6-FAM-TTATTATT-Biotin-3’) were used for DETECTR detection. The crRNAs (5’-GATTTAGACTACCCCAAAAACGAAGGGGACTAAAACgcagcagcaaagcaagagcagcatcac-3’) targeting N gene and ssRNA reporter (5’-6-FAM-UUUUUC-BHQ1-3’) were used for SHERLOCK (Specific High-Sensitivity Enzymatic Reporter UnLOCKing) detection. All these oligonucleotides were synthesized by General Biosystems, Ltd. (Anhui).
Artificial sample preparation. COVID-19 RNA reference material for limit of detection (LoD) determination was purchased from the Chinese Academy of Metrology. ORF1ab gene segment (14911-15910, GenBank No.NC_045512) was 1.1×106 copies/μl and N gene was 8.38×105 copies/μl. The RNA standard of the RdRp gene was diluted with TE buffer to 6 concentrations of 100 copies/μl, 50 copies/μl, 25 copies/μl, 12.5 copies/μl, 10 copies/μl, 5 copies/μl, and extra 0 copies/μl (blank control). The RNA standard of the N gene was diluted with TE buffer to 7 concentrations of 80 copies/μl, 40 copies/μl, 20 copies/μl, 10 copies/μl, 5 copies/μl, 2.5 copies/μl, 1.25 copies/μl, and extra 0 copies/μl (blank control).
Pseudoviruses sample preparation. Pseudoviruses from seven coronaviruses including SARS-Cov-2, SARS-CoV, MERS-CoV, hCoV-229E, hCoV-NL63, hCoV-OC43 and hCoV-HKU1 were purchased from Cobioer Biotechnology Company in Nanjing. A pseudovirus of Influenza A (H1N1) was purchased from Genewell Gene Technology Company in Shenzhen. The eight pseudoviruses were diluted from the original concentration to the 200 copies/μl with TE buffer containing 1% TritonX-100. We then used the TE buffer containing 1% TritonX-100 to serially dilute 200 copies/μl of SARS-Cov-2 pseudovirus, 200 copies/μl, 20 copies/μl, 10 copies/μl, 2 copies/μl, 1 copies/μl, 0.2 copies/μl, 0.1 copies/μl and extra 0 copies/μl (blank control). The diluted pseudoviruses were then incubated at 90°C for 10 minutes to rupture the virus and release nucleic acid.
Human clinical sample collection and preparation. Pharyngeal swab samples were collected from 14 fever patients in Shenzhen Second Peoples’ Hospital by the Clinic Diagnosis Laboratory and the nucleic acids of each sample were extracted with the pre-packaged nucleic acid extraction kit (Da’An Gene., Ltd.), according to the manufacturer’s instructions, resulting in 55 μl extracts for each sample.
An additional 40 pharyngeal swab samples from 40 fever patients in Shenzhen Second Peoples’ Hospital were also collected and extracted. rRT-PCR assay employing 5 μl of extract from each sample, were all negative. The remaining 50 μl extract of each sample was combined and 5 μl of the mixture was once again analyzed by rRT-PCR and confirmed to be SARS-CoV-2 negative. The RNA mix was named N50B.
Patients with fever underwent nucleic acid testing for two consecutive days. Patients who tested positive for two consecutive nucleic acid tests were transferred to designated hospitals for treatment. We continued to test patients who had negative tests and no ground glass shadow on chest X-ray to find the cause and determine treatment. The samples used in this study were nucleic acids extracted from patients’ pharyngeal swabs on day one. These patients underwent two rRT-PCR tests and chest X-rays and were evaluated as negative.
OR-DETECTR assays. The OR-DETECTR platform combines RPA and CRISPR/Cas12a detection. One RPA pellet per tube was resuspended with 29.5 μl rehydration buffer in the TwistAmp basic RPA kit. RT-RPA reactions containing 5 µl of sample extract, 14.75 μl resuspended RPA solution, 1.2 μl of each primer (10 mΜ), 0.75 μl RNA reverse transcriptase (100 U/ml), 0.85 μl nuclease-free water and 1.25 μl MgAc (280 mM), with the total volume of 25 μl. The multiple RT-RPA reaction contains four primers, each of which is 0.6 μl (10 mΜ). After being gently mixed and centrifuged, the mixture was placed at the bottom of a centrifuge tube. The CRISPR/Cas12a reaction mix consisted of 2 μl NEB Buffer 3.1 (10×), 2 μl LbCas12a (100 ng/μl), 2 μl crRNA (200 nM), 2 μl ssDNA reporter, and 12 μl nuclease-free water. This 20 μl CRISPR/Cas12a mix was added to the lid of the centrifuge tube before sealing. The tube was placed in a thermocycler or water bath at 42°C and incubated for 30 min. The CRISPR/Cas12a mixture in the cap was combined with the RPA reaction product with a short spin. If ssDNA reporter was 5’-6-FAM-TTATTATT-BHQ1-3’, the centrifuge tube was then placed in the PE microplate reader at 42°C, with the fluorescence signal collected every min for 20 min total. If ssDNA reporter was 5’-6-FAM-TTATTATT-Biotin-3’, the centrifuge tube was then placed in a thermocycler or water bath at 42°C. The reaction product (3 μl) was diluted in 400 μl water, after which 80 μl was added to the sample hole of the test card (GenDx Suzhou).
OR-SHERLOCK assay. The OR-SHERLOCK platform is a one-tube detection platform based on RT-RPA and SHERLOCK, termed OR-SHERLOCK. The platform combines RT-RPA and CRISPR/Cas13a detection. The RPA amplification step is the same as for the OR-DETECTR. The CRISPR/Cas13a reaction mix consisted of 2 μl Buffer (400 mM Tris, pH 7.4), 2 μl LwCas13a (20 ng/μl), 1 μl crRNA (10 nM), 2 μl ssRNA reporter (4 μM), 0.5 μl RNAse inhibitor (40 U/μl), 0.1 μl T7 Polymerase (100 U/μl), 0.8 μl rNTP (100 mM each), 1 μl MgCl2 (120 mM), and 10.6 μl nuclease-free water. The centrifuge tube was incubated in a PCR machine or water bath at 42°C for 30 min. The detection process of OR-SHERLOCK was the same as OR-DETECTR.
rRT-PCR assays. Real-time RT-PCR reaction kit was supplied by Biogerm, which uses the primers recommended by the China CDC. Reactions were conducted in a 25 μl volume following the kit instructions. Reaction cycle parameters were set as reverse transcription at 50°C for 10 min, denaturation at 95°C for 5 min, followed by 40 cycles of amplification, 95°C for 10 s and 55°C for 40 s.
Statistical Analysis. Statistical analyses were performed using GraphPad Prism. P<0.001 was considered to indicate a statistically significant difference.