FEMMAN chip preparation
Surface chemical modification on pGOLD chip
The pGOLD chips, provided by Nirmidas Biotech, were immersed in Phosphate Buffer Saline (PBS, Lablead) containing 200 mg/mL bovine serum albumin (BSA, Sigma Aldrich) for 2 h, resulting in a dense layer of BSA on pGOLD substrate. After rinsing with PBS buffer and water, the slides were then immersed into 1 mM Sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate (Sulfo-SMCC, HWRK Chem) in water for 1 h, resulting in a layer of maleimide groups ready to react with thiol functionalized DNA probe33.
To regulate the distance between detection fluorophore and plasmonic gold film, we constructed a series of surface chemical modification on pGOLD substrate with 1 mM cysteamine (Sigma) in water, 1 mM mercapto-poly(ethylene glycol)-amine (HS-PEG-NH2, Mw = 1,000, Peng Sheng Biological) in water, 1 mM HS-PEG-NH2 (Mw = 5,000, Peng Sheng Biological) in water, or 200 mg/mL BSA in PBS buffer, following with 1 mM sulfo-SMCC. The comparison results of background signal and fluorescence intensity for naked pGOLD substrate, different surface chemical modification and glass substrate were listed in Extended Data Fig. 1c, d.
Comparison of plasmonic gold film, evaporated gold film and glass substrate
The glass slides used in comparison were purchased from Fisher Scientific. Evaporated gold film on glass slides were deposited via e-beam evaporation with Innotec ES26C E-Beam Evaporator. The pGOLD chips and evaporated gold slides were immersed in 200 mg/mL BSA solution in PBS buffer following with 1 mM Sulfo-SMCC before DNA probe immobilization. Glass slides were immersed into 1 mM (3-Aminopropyl)triethoxysilane (APTES, Sigma) in ethanol overnight34. After rinsing and drying, the amine modified glass slides were treated with 1 mM Sulfo-SMCC in water to form a layer of maleimide groups before DNA probe immobilization.
DNA microarray arraying
The maleimide activated slides described above were loaded into Nano-Plotter™ 2.0 (GeSiM mbH) where 2 μM thiolated DNA probe in TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, Coolabor) with 25 mM Tris(2-carboxyethyl)phosphine (TCEP, Meryer)35, 0.01% Tween 20 (Solarbio) and 0.1% Glycerol (Aladdin) was arrayed using Nano Tip J (GeSiM mbH) at 25 °C, resulting in microarray feature with diameters of ~ 400 μm. Typically, each slide was segmented into 16 blocks with a 8 × 2 fashion and each block contained triplicate spots of different probes. After arraying, the slides were stored in humid chamber for 2 h to complete the reaction, dried in a desiccator and then blocked with 0.01% mercapto-hexanol (Macklin) to quench free maleimide groups. The established FEMMAN chips could be stored at 4 °C. The FEMMAN assay affords throughput of 56 samples in one set (Extended Data Fig. 7, 14 blocks in a chip and 4 chips in a frame).
Dynamic range and sensitivity of FEMMAN assay
Synthetic DNA targets
Biotin labeled synthetic DNA targets were ordered from Sangon Biotech and resuspended in DNase/RNase-free water. The resuspended DNA solution was serially diluted from 2 nM to 2 fM in binding buffer and used as inputs to FEMMAN chip.
DNA microarray processing
100 µL of each biotinylated DNA solution (2 nM to 2fM) in binding buffer (TE buffer with 1 M NaCl) was applied to each set of DNA microarray in FEMMAN chip, incubated at room temperature for 1 h to overnight. The chip was washed with TE buffer for 3 times, followed by blocking with 200 mg/mL BSA in PBS buffer and incubated with IRDye800 (Licor Biosciences) or Cy5 (GLPBIO) labeled streptavidin in PBS buffer with 200 mg/mL BSA, the incubation lasted for 30 min at room temperature in dark. Chips were washed for 3 times with PBS-Tween 20 (0.05%) buffer, followed by brief immersion in water and subsequent drying with centrifuge.
For binding buffer optimization, we screened TE buffer with 0.1 M NaCl, TE buffer with 0.5 M NaCl, TE buffer with 1M NaCl, TE buffer with 0.1 M MgCl2, TE buffer with 0.5 M MgCl2, TE buffer with 1 M NaCl and 0.1 M MgCl2, TE buffer with 0.5 M KH2PO4, TE buffer with 0.5 M K2HPO4, 2 × saline sodium citrate (SSC) buffer, 5 × SSC buffer, 6 × SSC buffer, 6 × SSC buffer with 0.1 M NaCl, 6 × SSC buffer with 1 M NaCl, 6 × SSC buffer with 0.1 M MgCl2, 6 × SSC buffer with 0.5 M KH2PO4, 6 × SSC buffer with 0.5 M K2HPO4 (Extended Data Fig. 1e).
Microarray scanning and wide field imaging
The commercial MidaScan-IR™ dual-channel confocal scanner (Nirmidas Biotech) was used to scan the IRDye800 or Cy5 labeled microarrays on different substrates with the 785 nm channel for IRDye800 and 670 nm channel for Cy5 with the gain set to 40 as defined by the system. Microarray fluorescence images were analyzed by built-in software of the scanner, and the spot features were automatically identified by the software. The fluorescence intensity of each spot was background subtracted, and the average of median pixel intensity values for each probe arrayed in triplicates was used to represent signal intensity.
Single-fluorophore imaging was performed on Cy5-labed DNA at varying concentrations on both plasmonic substrates and quartz slides. Imaging was done using a 658 nm laser diode excitation source with an 80 μm spot focused with a 100 × lens (Olympus). The excitation light was filtered through a 750 nm short pass filter (Thorlabs) as well as a 655 nm/40 nm bandpass filter (Semrock). The resulting NIR images were collected using an 800 nm short pass filter as well as a 715 nm/40 nm bandpass filter (Semrock) on a 1344 × 1024 pixel silicon CCD camera (Hamamatsu). A binning of 4 and an exposure time of 400 ms was used to obtain videos of varying target DNA concentrations on both the plasmonic and gold substrates. Initial frames were selected to demonstrate the starting Cy5 fluorescent intensity and fluorophore density.
FEMMAN-RPA assay for SARS-CoV-2 detection
Synthetic viral RNA targets
Reference material for SARS-CoV-2 wild type Genomic RNA was purchased from GeneWell Biotechnology Co., Ltd, reference material for SARS-CoV-2 Delta Variant (B.1.617.2) Genomic RNA was purchased from National Institute of Metrology.
Lentivirus Preparation and Processing
Lentiviruses containing RNA fragments for Spike protein of SARS-COV-2 wild type, B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta), C.37 (Lambda), and B.1.621 (Mu) variants were constructed by GeneCopoeia™. Lentivirus containing RNA fragments of Spike protein of B.1.1.529 (Omicron) was constructed by General Biol. Pseudotyped lentiviral particles were titrated using qRT-PCR to determine the physical copy numbers of viral genomic RNA. The lentiviral particles were generated according to standardized protocol using highly purified plasmids and EndoFectin-Lenti™ and TiterBoost™ reagents. The lentiviral transfer vector pEZ-Lv201 was co-transfected into 293Ta cells with Lenti-Pac™ HIV packaging mix. The lentivirus particles were purified and stored at -80 °C in aliquots (purified particles). The construction of lentiviral expression was validated by full-length sequencing, restriction enzyme digestion and PCR-size validation using gene-specific and vector-specific primers. For FEMMAN-RPA assay, the lentiviruses were spiked into the nasopharyngeal swab sample preservation solution (Biocomma®, YMJ15C) and serially diluted, the titer of lentiviruses was determined by ddPCR. 1,000 copies of each lentivirus were spiked into the sample preservation solution respectively for subsequent viral RNA extraction with QIAamp Viral RNA minikit (Qiagen), serially diluted lentiviruses were thermal lysed at 95 °C for 2 min before being used as input for RPA.
Clinical samples
For clinical nasopharyngeal swab samples, RNA was extracted from 140 μL input material using QIAamp Viral RNA minikit (Qiagen) according to the manufacturer’s instructions. Samples were eluted in 80 μL of nuclease free water and stored at -80 °C until use. For thermal lysis, the clinical sample was heated at 95 °C for 2 min and used immediately.
Reverse Transcription Recombinase Polymerase Amplification (RT-RPA)
The design principle of Primers for RPA was described in Supplementary Discussion 5. The effect factors include the amplicon size (between 80 and 90 nt), primer melting temperatures (between 54 °C and 67 °C), and primer size (between 30 and 35 nt)23. Forward primers were ordered as 5’ terminal biotin modified DNA, and reverse primers were ordered as 5’ terminal phosphate modified DNA (Sangon Biotech).
RT-RPA reactions were performed as instructed using TwistAmp® Basic (TwistDx) or RPA Basic Amplification Kit (ZC Bioscience), the total volume of amplification system was 10 μL for each reaction. The primers used in RPA were 600 nM for Y144 site, 400 nM for K417 site, and 200 nM for L452 site. Reactions were run with 1 µL (extracted RNA) or 2.5 µL (thermal lysed sample) of input template, 0.5 µL RiboLock RNase Inhibitor (Thermo) and 0.5 µL PrimeScript Reverse Transcriptase (PrimeScript RT, Takara) for 30 min at 40 °C. For specimens with low copy number, the volume of RPA system was increased to 40 μL or 50 μL for each reaction, and other reagents were augmented correspondingly. For the blank sample, only buffer was used as input for amplification.
Amplicon digestion by Lambda Exonuclease
The amplified sample was digested into single stranded DNA (ssDNA) by Lambda Exonuclease (Lambda Exo, New England Biolabs Inc.)36. For each 10 μL of RPA system, the amplified sample was diluted to 50 µL with 1 µL Lambda Exo in 1 × reaction buffer, then incubated for 30 min at 37 °C. For RPA system with volume more than 10 μL, the upper limit for the digestion system was 100 μL with 2 µL Lambda Exo. The digested sample was diluted to 1 × TE buffer and 1 M NaCl. The mixture was denatured at 95 °C for 5 min, followed by quelling at 4 °C before being loaded onto FEMMAN chip.
Processing and imaging of FEMMAN-RPA assay
FEMMAN assay was performed as described previously. In brief, up to 150 µL of digested amplicon was applied to each set of DNA microarray in FEMMAN chip and incubated for 1 h to overnight. For the blank sample, amplification and digestion were also performed using DNase & RNase free water instead of template before being loaded to FEMMAN chip. After washing and blocking, the chip was incubated with IRDye800 labeled streptavidin in dark. All imaging was conducted using MidaScan-IR™ dual-channel confocal scanner with the 785 nm channel. The fluorescence intensity of each spot was analyzed by built-in software of the scanner with automatic spot identification.
Quantitative PCR (qPCR) analysis with TaqMan probes
To compare FEMMAN quantification with other established methods, we performed qPCR with diluted thermal lysed lentivirus and clinical samples. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using a commercial kit targeting the ORF1b and N gene of SARS-CoV-2 (ShenZhen ZiJian Biotech Co., Ltd.). TaqMan probes of N501N or N501Y, and corresponding primer set (Supplementary Table 5) were designed according to Centers for Disease Control and Prevention of U.S. (CDC)37 and synthesized by Sangon Biotech. Assays were performed using the HiScript II U+ One Step qRT-PCR Probe Kit (Vazyme) on a QuantStudio 5 (Thermo Fisher).
Digital droplet PCR (ddPCR) quantification
The concentration of viral RNA in reference material and thermal lysed lentivirus used in Figure 1, 3 was measured by ddPCR. The reference material for SARS-CoV-2 wild type genomic RNA was provided as 5.24 × 105 copies/µL, the reference material for SARS-CoV-2 Delta Variant (B.1.617.2) genomic RNA was provided as (2.45±0.38) × 103 copies/µL, the lentivirus was provided as 4.88 × 105 copies/µL (Wild type), 5.38 × 105 copies/µL (Alpha), 1.13 × 106 copies/µL (Beta), 1.02 × 106 copies/µL (Gamma), 1.20 × 106 copies/µL (Delta), 7.04 × 105 copies/µL (Lambda), 6.59 × 106 copies/µL (Mu), and 2.26 × 103 copies/µL (Omicron).
Sanger sequencing preparation
Extracted viral RNA was prepared for Sanger sequencing. Following extraction, double-stranded complementary DNA (cDNA) was created and amplified using Spike protein primers (Supplementary Table 5) and PrimeScript™ One Step RT-PCR Kit (Takara). Sequencing was performed by Sangon Biotech.
Data analysis
General data analysis
The background subtracted fluorescence intensity for each spot was counted by the analysis software of scanner and the mean of the triplicate spots was taken for further analysis. All spots were divided into three groups (G142-Y145, K417, and L452) according to the corresponding primer pairs. Imaging data was analyzed with built-in software of the scanner. To calculate background subtracted fluorescence data, the fluorescence signal around the spot was subtracted as background to allow for comparisons between different assays. The averaged background subtracted fluorescence intensity of each probe would be normalized to positive control spot in each block.
Statistical analysis
The signal proportion of each wild type or mutation site was calculated as follows:
![](data:image/png;base64,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)
Where
refers to the mean value of fluorescence intensity for probe
,
refers to the number of probes for the same target site (G142-Y145, K417, or L452), and
refers to the probes in the same target site (Fig. 4e). Take Y144Y as example:
![](data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAlUAAAA2CAYAAADu+WxvAAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAADsMAAA7DAcdvqGQAABNdSURBVHhe7Z3dqxXVG8fH3335UlfiRVQXSUVgpwwrwaAMLyIpMhQkMExTuqjwpZeL6IVOkUKgvWAQkmloGOJbCl1USlZGQeJF2UVEV1ZWf8D5zWed+W7XWc7sPbPP9rSP+/uB2TOz1pr18qw1az17rWdmpozkZMYYY4wxZlz8r9gbY4wxxphxYKXKGGOMMaYHWKkyxhhjjOkBVqqMMcYYY3qAlSpjjDHGmB5gpcoYY4wxpgdYqTLGGGOM6QFWqowxxhhjeoCVKmOMMcaYHmClyhhjJoAvv/wymzJlSthee+21wvU8Bw8eDH633HJL9sMPPxSuWXbvvfe2rks34hSxuzHmv8FKlTHGTAB33HFH9swzz4Tjzz77LOzFH3/8kT3xxBPh+IUXXshuuummcAw7duwI+507d2Z8VYztiy++CG7EKQ4cOBD28jPGTDxWqowxZoI4efJktmTJkuLsPM8//3w2NDQUjufOnRv24vTp02F/9913hz2gTCm8+PHHH7Pp06ePUbSMMROLlSpjjJkgPv300+z+++8Pe8ES3i+//JLNmTMnKEpXXHFF4TPK8ePHgyKWun/zzTfF0SjMfj388MPFmTHmv8BKlTHGTADYTC1cuDCbNWtW4TK67PfII49kw8PD2e7du7N77rmn8DkP7h999NEYmymui+EcRW3+/PmFizHmv8BKlTHGTADHjh3LFi9enM2ePTucM0P13nvvZStXrgyK1rfffpvdfvvtwU+gLOGOvZRsqVavXn3BrNWJEyfC/tZbbw17Y8x/g5UqY4yZAJhtuu2221oK0alTp8Is1Lp161pKUWpPlbpjL7V169ZwHIPCxtLh1VdfHc71xKCeMkzPUdY4rlouxI9rUrhuxowZpU8dyi09N2aQsFJljDEXGWymQE/1sQy4atWqbPPmzeFcSlE6A7V///5S9xQUtnjpUIrXihUrwp4ZMma4UOBQjDZt2hTcz507F/YxLFN+9913xdlY1qxZk11zzTXF2Sg8lUgeZSCP/RduNpg3g4iVKmOMucjs2bMnPJknpk2bFl6vgOKBkoNSlIL7rl27OipUzAidOXMmmzp1auGShRkrlKijR4+GOP7++++WokV8L7/8cjZv3rxwHoPyt3379mzt2rWFy3meffbZ4J7mh9muv/76K1xLmOXLl9tg3gwsVqqMMeYigtKzfv36YBul5TcUHRQbWLZsWVCKYn/AHWUFA/TYPeXOO+8Me9LQjBgsXbo0e/zxx8MxM1R1IPyWLVuKs9FlQGD2CqVNs0///PNP9s4774RjwC6MmbKHHnooW7RoUeFqzOAxZQTrR2OMMZcUvJX9gQceyH7++efCZSwoey+99FJ2+PDh1rkUtBiGCOyr4tdAAE8sSllDwSK9MnsvYwaJvpup4p8RRo4YQxpjjOkOlCBsnarAUJ4lRsEsFAqUnjLE7kv/uVG85Ic7/vHs1969e8e8Bd6YQaWRUqWnOuKNtfP4xhwv3KjYGpS9r6Up/HNSPq+99trC9bzixpZOq3MN397qVCamycuejokpC4OyqLQ1Vc8/RLl1inOiQAbYRxhjJh9acsRWi+MU+hoM5Vly5DgNc+jQodJlR/pHZr7iGS3CEJb4jBl48n8etTlw4AB/W0bOnDkTzvN/KyPTp08fWb16dTjvFfk/oZG33367OBsfw8PDIc/kPQa3NN8K20ksO3fuDGHIZxVVYZAZ7rniWLiMQrj8X+XI2bNnC5eLD3msKgN57FUdGGOMMYNAo5kqvi3F47R6FwrTxbxsLjaO7AVff/11dv311xdn40OPFGNYKfhnxZMx8fo/szI33HBDx4+R8k/tjTfeaH0YtYx2YZAZU/IYoAr+JTIzxhR7pyd9egXpkcebb765cBlL3jayxx57rDgzxhhjTCcaKVV8Wyr+GCgDMwqQBmaeaGF5i6UjQFHhPF7SQuFgyVDLXenyIf4oHLx1mOsUJgXFiCU9/NlXKXYoKeT5k08+CefkkXKkBpU8idPpqRXy+eijj2bbtm0b8/hyTJ0w+JMPoLx8pqKJQoVcKTd70pM846n6WM7UAYakqgeUuCuvvDJM/b/yyishjPyIg3PVoSAdljO1fIk/aVRBGoTrtJUtTRhjjDGTkdpKFYMq6+b6jAJKDC+CA2aDOP/888+zffv2hcGaAZ/Hazds2NBSugizYMGCMDCfPXs2bITVi+jgq6++CjM5fL5hx44dYeYotQtAAUAxOnLkSIgDeA9MFXzAlDhQLJidId5uIJ8oRO0MMuuE4WvzKI4oMDyd8/HHH9dWqFDGbrzxxvDkDeXn6/Y8Ar1w4cIw0wapnH/66ads48aNrXpgtoxHuIE9s1J6AgibNuJKbdqQOR9wpb4Iz4wl5awiNnpttxHOGGOMuSTIB7ZayJ4q3pYsWTLy/fffFyFGkS1R6g6Ex24oBpue2K5HYWLbIuLDFgmIPx/Qgz+2Xdj94C87rzIISxhd1w7ZPKVQfvImsL8i38Qn9zphBGWsklMdsAerKg9pxTIFbN9iuzLJsQzc47CScZyW7M8uFsTtzZs3b9689ctWh9qjIobVVYNwDIN9qkAIMpUaPxMnA7RIB38pOVI+UBZUQK4lvU6KCcoA4VEkOlGlVJEvpZtuUmDqhBEoVamhfBMoe5UhOenFZUU+uMVKEXVUlj7KaRqWvKd1yrVpmWIkx06blGVjjDFmslNbqZIC04mqwV4DezyIpgpT2eCfDt74x0pYHVDS0nirqFKqUjQL1Y6qMFLyYuWxCVJ8ypRJ5T+Wc9XMVZmSiVs6m0jYWObkH7cqpc4YY4wZRGrZVGHPhO3NVVddVbiUgy0P4fgSewo2RvlAnB0/fjycE+d9990XnsKT/RH2VPDbb7+FPTZQb731VrAfEvmA3/rYJ3Ze2CVhv9WON998M+zr2C3pKUHK0g6+pVX1pmJRFQZ7Mbj88svDvik8HIAsy+y2Zs6cGfZ6sR+yyRXd8MQmhuX6tAQ2XaRPOWWkDtjFISfCyvAd+ypkTnwKT5wPPvhg8DfGGGNMTqFctYVg2uIZkBRmOZjBqAJ/ZrKIh30648SsVByG2ZV0NoZz+ZMW17SbgWLZUnlvN7NEuvgrLHGXpQ+EVbiq2buqMJo109YNxFe1xAoqs/KvNHEXKiuyjGfMZD9F/JIrccgGrI7MjTHGmEHE3/4zxhhjjOkBjd5TZcxEo/dmVb3TSu/sSt93pmvKNkF8couXQCcD5LdMHlXEZdWybszBgweDX/r+MdLRdekWpx+Ha5KvyQRla0K3bbeuzGN30xxk2fS+j+umDO4f/NJ7TNeUbYL8yK1pvkwfEearjOljtPSYLhezFM1yJH7pcqSWPONXbZQ9OMCSKHFMtuVMytFuKb6MeFk4hrJrST19eAI/3FnOFnoYIkbh0jq6lEjLXIdu2m5dmauNN20HZhTklt4LnVDdlMmdOqZO0wd94FLvj8x5PFNl+h4efsg7n+LsPLyJHiN6Hl5IH0Lgk0q465NKMG/evAs+y3Py5MkQR92Xr05mKOuS6IsIghfIIiuYO3du2IvTp0+HPS+sFfrUUozCldXTINNN260rc9p4PgD7BboTCHVTdg/xAM+7774bHuBJX5wM7o8GBytVpq9hSpzvS9IZ8RZ9wfT6ypUrs3PnzpV2YoRN3/jO4MPniGL4SgBv3B8EVFb2AvkyIMyZM6d0gOdpXQaR1J2368cQjgGl3ZcEBo1u225dmRNn2Se8zMWDuuFeoa3rSXbgE17vv/9++OKEvjoS4/5ocLBSZfqaQ4cOZXfdddeY13lg88O/Ql7pEH86SWCfgvuqVataNgrptwwBOyJg4LvUoazMmMyaNatwGZUTMybDw8PZ7t27Swd43PnEk+TIFtv/iKrrB5lu2i7Ukbna+Pz58wsXMxFQN8ww8b1ZwWtqpk2b1nodTzrb6/5owCiWAY3pS4aGhsa8FgKwQ8CeQW6p/UHqju1CbJ8isF/AlmgyQHk6bandTgxl5XUZsglBfoRnkxtyi0nduabsFSIKVybjyQplpUydttSuJqabtltX5ro+ttEx7aGtI7NOWxXUDfZOwP1EXeJGH8IeN+o8Ja3rS6E/MtVYqTJ9CwOGOjENcigGdD5Q1YlVuacQRnGBOlUNlOk5+WGAq1Je6GTL/NQZKx6Vha3svA4anOtCZ613rpEOcpSMqgb4KveUNBx541yySM8JxzHvQitD8iijSsaC6+rUPWkTtolSUpWnMrptu3Vlnl7PNWxqE+l5p7aLO7JNIR9x2xWEp02xkQ/i75SHTvVelgfFoU0oPckY1M7SvFZBuLIyV4EipLwrr5xTZ4Ac4v5EVNV1Stn1yAw3yknZpMDJr6k8KQNxEFfqR/q4S65cH9O0DRGec8VZdV0VndIj7tiP+lRabGn5hMKV1VUM/SXhyv7UtMPLf6Zv4c3xshmRMe7GjRuzJ598MhwfOXKkdMmJpZNOS1HYEaX2D3mHE+yKlFbeWQU3zlm22bNnT/bnn38GvxQej2cJoIw1a9YEGwxBfPmNmuU3dTifPXt2SDe/icN5r6GsIHunvLMJSxGbN28O58eOHSu1p9q/f3+pe0oabuvWrWG/YsWKsF+8eHEo77p168JSyKZNm4I7NkUp5PXDDz8szsbSTsYiV1xqLUPu2rUr1ElsONxLum27dWWetvHxtF2WnfSVipS07YKWME+cONH6YgRybJeHTvVelQfaaj5OtTZBerQpZEBdwnPPPRfuKaXfa/jahOydWAJkSY+yLFq0KJSvyp6q2/4Ili1bFh52wC9XosKDCbSNbuWJjKg34qSNYvcnnnrqqVBnyHnfvn3Zq6++2pJtN23o999/D/cr8eWKZ7Z+/frCpzPd9re5ktVqK4cPHy5cx0L7oJxldRVDf0l8TZfYrVSZvgXDTzoRwfEHH3wQOhVuOjqaFDoJOoypU6cWLuVww0L8qSAGQT7fQwfHTbt8+fLWwMgNhlKAkWoKHQrplfkRz9q1ay8YJJ9++unQ2dI58vTdtm3bLpqRN2WN5UhnpMGH9MlHCu50qJ0G97JwGvCOHj0a/PhckxQtwmGcy6BUBga/L774YnF2nnYyJg1sXLBVef3118d0lrQTvVuIMOQXaCd1lK9u6abt1pV5WRvvtu0Sfvv27aGNplS13RQZ0LfLQ7t6b5eHdjDgouQga8BwvNNA2S2qG/UXl112WahTtWv1Jynj6Y+4FsVny5Yt4Z5ChpJ1t/JECeZa4kNZ5g+dQGlcsGBBOKZv2LBhQ/brr7+G827aEHEgM2THHzcU7Lp029+2gzY5Y8aMsPGnJrZ9I071IWqzwAMGjW3cco3OmL6DaV2aJ1vZdL78Uv8q95h4mpip8BjSxU1LZSn4s4m8wxyzJBD7syygY6aiOWcJSODGpun8JnBdVfli4rLG+RbEU+Zf5Z4Sh8s7zcJ1NN180Km0s8Kfa2OYjleZiI9jtnYyVjrx0ma8PEJdEgb/OBzXV+WtCuKuA3FLJipPjPxS/7oyj69HNoJrmrRdIE3kRD44ln+ntotfWf3i3i4PSiemKg9AGiprvFxDuTnnOsJwrnjqUpaXKginfKQQj/xS/9idcGXE18f9EctOcZnLKCuD5CC/WJ6CuqROY9J6I+30Ws5Tt3bpqWzEHadHOJU53ggfk6ZXpy8gnrRtEmZoaKiVT46Fzomba7hWxPVRFytVxkTQ2dCZVRHfxMBx2jHQYUBZxxFfS+fQdGAXxJ12QP0EnXO7Dom8S04ilRUb4drJOJahBlZBXSo8nWbcqasTbQLx9DNN2y6ylXziDTq1XUB+6UDcKQ9pvbfLA6iO2DPYKa1YKSA9zuOBsg5lbbCfIG8qYyynmKbyBO6XtN9J71cpq8QX06QNxShcU9L0OE7Tisuv9oLcYuWIY/lR9jhO+hDFxbHaGHG0a8tVePnPmIi9e/e2XYbDboDlLMEUdX4fhS2/UcOmtXz28stv/CzvWEJ4YEqcKeh4+r0JxH2xbEd6AfYm+SBXnF3IqVOnWktxQrJi0zllbCdj7ElYMmEJgqVDXmHAVD5QT/m/7XAd9mMsQQiW3/7999+wxFIX5atfadp2ka3kStukjaqMVW0X2eoTLGVLWp3ykNZ7uzxQN9RfGaRz3XXXheOlS5cGG8Gmy7mkrXbUj7BEpzY7c+bMsFyXD/ThXDSRJ7AcByxxUZdq/9yvkp+WzAlDfDFN2hDthCU3YLm0G5r0t6SHyUEZLEvTXikvdmUsm6qfoA9BrsTJ8qdevssLW3kdCq/MaEQekTEmJ79B6Q3ClpJ3GC2/sjD8s8s7lOBO2Bj9C4yvUVj2lxqxrFJZgPyqwvAPEfdUNmUy1mwU7rpO/8JjuWupSPDPlWtit8nMeNouIB/ciScmbbvIlnNkxz6e8WiXB5CftrTe0zzEaVNf1DWQJm7xLALhqP9Libj8zMKlM3G4x1sneTJTU3YN90DsRpppO+imDZF/3a9pW+lEp/TK+gLip53gRnqxPDQbRR7JH+HIH+i6uI0Bx7g1yTdM4SdPzBhjjDHGjAMv/xljjDHG9AArVcYYY4wxPcBKlTHGGGNMD7BSZYwxxhjTA6xUGWOMMcb0ACtVxhhjjDE9wEqVMcYYY8y4ybL/Aw4/tNRqDyiOAAAAAElFTkSuQmCC)
Heat map was generated from the proportion of the mean fluorescence intensity of each probe in group (Fig. 4f). In the heat map of Fig. 3e, mutation indexes were normalized by column.
SARS-CoV-2 detection of clinical samples
Background subtraction and normalization were performed as in general data analysis. For each sample, the signal of each target site (G142-Y145, K417, or L452) was calculated as follows:
![](data:image/png;base64,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)
Where
refers to G142-Y145, K417, or L452 target site,
refers to the mean value of fluorescence intensity for target
,
refers to the number of probes in target
, and
refers to the probes in target site
.
For each target site, the threshold for SARS-CoV-2 viral RNA detection in FEMMAN-RPA assay was defined at 3 × s.d. above the
value (
,
and
) of blank (dotted box in Fig. 4b).
Methods-only References
33 Dovgan, I., Kolodych, S., Koniev, O. & Wagner, A. 2-(Maleimidomethyl)-1,3-Dioxanes (MD): a Serum-Stable Self-hydrolysable Hydrophilic Alternative to Classical Maleimide Conjugation. Sci. Rep. 6, 30835, doi:10.1038/srep30835 (2016).
34 Wei Wang, M. W. V. Morphology and amine accessibility of (3-aminopropyl) triethoxysilane films on glass surfaces. 30 2, 13, doi:10.1002/sca.20097 (2008).
35 Deb-Choudhury, S., Plowman, J. E. & Harland, D. P. Isolation and Analysis of Keratins and Keratin-Associated Proteins from Hair and Wool. Methods Enzymol. 568, 279-301, doi:10.1016/bs.mie.2015.07.018 (2016).
36 Deng, J. et al. Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. Nat. Biotechnol. 27, 353-360, doi:10.1038/nbt.1530 (2009).
37 Matic, N. et al. Rapid Detection of SARS-CoV-2 Variants of Concern, Including B.1.1.28/P.1, British Columbia, Canada. Emerg. Infect. Dis. 27, 1673-1676, doi:10.3201/eid2706.210532 (2021).