Nucleic acid preparation. SARS-CoV-2 target sequences were designed using the available genomic sequence from NCBI as of 30 January 2021. These sequences were then aligned with bat SARS-like CoV and MERS to verify cross-reactivity (Table A1). LAMP primers were based on previously published sequences. Considering currently used target sites by the CDC and WHO, gRNAs were designed for the E gene, S gene, and the ORF1ab as well as a sample control gRNA that that was designed for previously published RNase P POP7 RT-LAMP primers. These were either based on open-source sequences (gRNA for E and N and RNase P) that had already been published or designed by root (Orf1ab gRNA). For the in vitro testing a synthetic ssRNA control was used. Oligonucleotide DNA sequences were synthesized by IDT with a yield of 25 nmole using standard desalting and then diluted to 100 µM of final concentration in nuclease-free water (IDT). The reporter molecule was an oligonucleotide functionalized with FAM (5´-end) and with BHQ (3’-end) using HPLC purification with a yield of 1µmole based on the sequence used by DETECTR. gRNAs were ordered from IDT with a yield of 2nmol and standard desalting all with 5’ and 3’ Alt-R end-blocking modifications and standard desalting with a stock solution of 50 µM.
5X Primer mixture with guanidine. LAMP primer mixtures (1X final, 10X stock diluted with guanidine to 5X) were assembled using a final concentration of 0.2 µM of F3/B3, 0.4µM of LF/LB, and 1.6µM of FIP/BIP in nuclease-free water. The stock solution of each primer (N/E/ORF1ab) 10X was mixed in equimolar proportions with guanidine (200mM in 5X primers mix and 40mM final concentration) to a concentration of 5X and this was the solution that was used for the simultaneous detection of the 3 different genes (Table A2).
RT-LAMP fluorescent assays. For the detection of SARS-CoV-2 RNA, several precautions were used to prevent sample contamination, such as using nuclease decontamination solution or RNase Away, filtered tips, and nuclease-free certified reagents in a dedicated clean bench as well as a laminar flow chamber with 20 min of UV decontamination when opening the plates was needed. For the RT-LAMP Vienna biocentre 12,13 reactions 11µL of tube 2A- LAMP MIX was mixed with 2.5 µL of tube 3A-SARS-CoV-2 As1 primer and with 2.5 µL of tube 4A-HNB dye and where referred, 0.4 µL of tube 7A- FLUO DYE (SYTO-9). Of this mixture, 16 µL were dispensed per well and 4µL of extracted RNA was added, vortexed, spined down, and incubated for 35 min at 63ºC. For the RT-LAMP mixture by SARS-CoV-2 Rapid Colorimetric LAMP Detection Assay Protocol (NEB #E2019) 12.5 µL of WarmStart Colorimetric LAMP 2X Master Mix with UDG, 2.5 µL of SARS-CoV-2 LAMP Primer Mix (N/E), 2.5 µL Guanidine Hydrochloride (40 mM of final concentration), 7.5 µL of nuclease-free-water and 2.0 µL of either sample nucleic acid (Extracted RNA), SARS-CoV-2 Positive Control (N gene) to a total reaction volume of 25 µL. When using WarmStart® LAMP Kit (DNA & RNA), for a final volume of 20.4 µL the reaction was assembled with 10 µL of WarmStart® Colorimetric LAMP 2X Master Mix (NEB #M1804), 4 µL of an equimolar 5X Primer mix (N/E/Orf1ab) with 200mM of Guanidine HCL, 0.7 mM of dUTP, 0.7mM of MgSO4, 0.02 U/µL of UDG, 0.32 U/µL of Bst 2.0 WarmStart® DNA Polymerase (#M0538M), 0.4 µL of 50X LAMP dye and 0.92 µL of nuclease-free-water, to this mixture 4 µL of extracted RNA samples was added in different concentrations. These reactions were incubated at either 65ºC or 63ºC where specified for 30 min. For the real-time fluorescent detection of the LAMP result, a 384-well plate was used in QuantStudio™ 5 Real-Time PCR Instrument using 30 cycles of 1 min at the specified temperature using the standard FAM emission and excitation filters. Melt curve analysis was performed from 35ºC to 95ºC with a heating ramp of 0.5 ºC/s.
CRISPR assay. For the CRISPR-based detection system, we used Lba Cas12a from New England Biolabs (EnGen® Lba Cas12a (Cpf1) #M0653T). Firstly, Cas12-gRNA complexes were generated by pre-incubating LbaCas12a (50nM, final concentration) with the gRNA for each gene (50 nM final concentration for all – 1/3 proportion) for 20 min at 37ºC in 1X NEBuffer 2.1 for a final volume of 20 µL. After the incubation, the fluorescent reporter molecule was added at a final concentration of 4 µM and placed on ice. After the desired target amplification by RT-LAMP, 2 µL of each reaction is added to 20 µL of the Cas12-gRNA mixture and incubated at 37ºC for 20 min. The real-time fluorescence acquisition was done using the Varioskan™ LUX multimode microplate reader using 20 cycles of 1 min.
In silico analysis. All SARS-CoV-2 sequences were aligned using Clustal omega using available genomes from GenBank (NCBI) as of January 2021. The specific target sites by LbaCas12a were compared with human coronavirus genomes (NC_045512.2), bat SARS-like CoV (NC_014470.1), and with MERS (NC_019843.3) to assess cross-reactivity. The LAMP primers were analysed via PrimerExplorer v.5 (https://primerexplorer.jp/e/) or based on previously published primers. All designed gRNAs were 100% specific (Figure A1, Figure A2, Figure A3) with Expect (E) values of 5x10− 6 in the plus/plus strand, the E gene gRNA enables the detection of SARS-CoV-2 (100%) bat coronavirus (95.24%) and MERS (93.75%). All RT-LAMP primers have been previously validated as highly specific with minimal cross reactivity as confirmed by the in-silico analysis.
Human clinical sample collection and preparation. Clinical nasopharyngeal swab samples were kindly provided by Instituto Ricardo Jorge (LNRVG) either from patients infected with SARS-CoV-2 (or other respiratory viruses) or from RT-qPCR negative individuals. Sample RNA was extracted according to the manufacturer's protocol. All experiments were performed in accordance with relevant guidelines and regulations to work with human samples. Protocols were approved by Faculdade de Farmácia da Universidade de Lisboa and ethical committee CEISH 03/2021.
Real-time RT-qPCR assay. The determination of positive or negative samples was confirmed by RT-PCR using “Novel Coronavirus (2019-nCoV) RT-qPCR Detection Kit (Fosun 2019-nCoV qPCR), this kit sets the maximum cycle threshold at 36 for a positive signal. This assay was performed on the Biorad Real-Time PCR system instrument.
Analytical validation (LoD). LoD is the lowest detectable concentration at which around 95% of all true positive replicates test positive. The FDA recommends testing a dilution series of three replicates per concentration with inactivated controls and then confirm the final concentration with 20 replicates. The analytical validation of the detection kit was performed by using synthetic RNA controls provided by Twist Biosciences. From this stock, ten-fold serial dilutions were made until the detection limit was reached (2/3 true positives were detected). This limit was then tested in 20 replicates achieving a total of 19/20 for the initial sample solution of 3 copies/µL.
Clinical validation. The analysis was carried out on 75 anonymized nasopharyngeal swabs with negative (n = 50) and positive (n = 25) results by RT-qPCR provided by INSA. The samples were freshly extracted on the 23rd of August 2021 and remained stored in the laboratory until they were delivered to our team on the 24th of August 2021. Once received at -20°C, the 75 samples were analysed for the E, Orf1ab, N viral genes as well as internal control such as Rnase P, to determine sensitivity and specificity values, as well as repeatability and limit of detection (analytical sensitivity). All experimental protocols were approved by Instituto Ricardo Jorge, Laboratório Nacional de Referência para o Vírus da Gripe (LNRVG). All methods were carried out in accordance with relevant guidelines and regulations (Figure A4).
Processing of saliva samples. Saliva samples were retrieved from healthy, covid negative donors and kept at -20ºC. Each saliva sample was heat inactivated by incubating at 65ºC for 30 min. Two different buffers were tested, the first one is composed of 100 mM TCEP and 1 mM EDTA, pH = 8 and the second one is composed of 2.5 mM TCEP, 1 mM EDTA and 0.1% Tween-20. A 2x solution of each of the buffers was mixed in a 1:1 proportion with saliva samples that were previously spiked with known amounts of in-activated virus and/or total swab controls. These were heat treated for 5 min at 95ºC and 22.5 µL of each was used to check for the presence of nucleases by incubating with the RnaseAlert substrate nuclease detection system (IDT), this takes advantage of a fluorescent RNA substrate in each re-action that will produce a signal if there are any Rnases in solution. In the positive control (PC) well, Rnase A is added. These solutions were incubated for 1h at 37ºC in a 384-well plate. For the detection tests, these samples were spiked with SARS-CoV-2 RNA controls from Twist Biosciences and treated using the developed protocol (1:1 in 2X Buffer with a final concentration of 100mM TCEP and 1mM EDTA pH = 8 for 5 min at 95ºC). The processed samples were then spiked with different initial concentrations. Informed consent was obtained from all subjects and/or their legal guardian(s).
Statistical analysis. Data were analysed using GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com. Statistical significance was determined by unpaired two-tailed t-test and all data were shown as mean ± S.D of 3 replicates. Asterisks indicate ** P < 0.01; *** P < 0.001; **** P < 0.0001 and “ns” is non-significant. The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.