CRITICAL: Thaw all frozen reagents thoroughly before use.
Preparation of synthetic RNA
1. Run PCR to amplify 316 bp SARS-CoV-2 N gene sequence in the pUCIDT plasmid. The 25-μL PCR system contains 1× SsoAdvanced™ Universal SYBR® Green PCR Supermix, 0.4 μM of T7-PCR-F, 0.4 μM of PCR-R, and 1.0 μL of 1.3× 105 copies/μL SARS-CoV-2 N plasmid solution. The thermal cycling is 2.5 min at 98°C for initial denaturation, 35 cycles of 15 s at 95°C for denaturation and 30 s at 60°C for annealing and extension.
2. Pipette 20 μL of the PCR products for 2% agarose gel electrophoresis analysis (120 V and 45 min). Then cut the band at the size of about 336 bp, followed by gel extraction using PureLink™ Quick Gel Extraction Kit. Further confirm the sequence by shipping the purified PCR products to Genewiz® for Sanger sequencing.
3. Incubate the solution containing 8 μL of 5× T7 Transcription Buffer, 3 μL each of 100 mM rNTPs, 4 μL of the Enzyme Mix with T7 RNA polymerase, 16 μL of the gel-extracted PCR products at 37°C for 4 h. The reagents are from the RiboMAX™ Large Scale RNA Production Systems-T7 Kit.
4. Treat the transcription products using the DNase (from the TURBO DNA-free TM Kit) to degrading the DNA.
5. Purify the resulting RNA using the RNeasy@ MinElute TM Cleanup Kit.
6. Determined the purity and concentration of the collected nucleic acid using NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometry.
7. Keep the synthetic RNA at -80°C before use. TIP: Do not thaw-freeze the RNA too many times in case of degradation.
Extraction of RNA from SARS-CoV-2 samples
· This procedure strictly follows the handbook of QIAamp DSP Viral RNA Mini Kit. The handbook can be downloaded at this link, https://www.qiagen.com/us/resources/resourcedetail?id=46638e95-df58-4874-9015-732e75587524&lang=en
1. Prepare Component A by mixing 12.5 μL of 2× Reaction Buffer from the TwistAmp® Liquid Basic Kit, 2.5 μL of 10× Basic E-mix from the TwistAmp® Liquid Basic Kit, 1.25 μL of 280 mM MgOAc from the TwistAmp® Liquid Basic Kit, 0.08 μL of 100 μM FP, 0.08 μL of 100 μM RP, 0.2 μL of 100 mM dATP, 0.2 μL of 100 mM dGTP, 0.2 μL of 100 mM dTTP, 0.2 μL of 100 mM dCTP, and 0.8 μL of 10 U/μL AMV reverse transcriptase. TIP: Prepare the mixture enough for at least 8 reactions for easy and accurate pipetting.
2. Prepare Component B by mixing 2 μL of ssDNA-FQ and 1.25 μL of 20× Core Reaction Buffer from the TwistAmp® Liquid Basic Kit. TIP: Thoroughly vortex the Core Reaction Buffer before pipetting.
3. Pipette 2.5 μL of the synthetic RNA or the extracted nucleic acid solutions into the prepared Component A, followed by adding 3.25 of the prepared Component B. The assembled mixture is left at room temperature during the preparation of Component C. TIP: Thoroughly vortex the assembled mixture and invert the tube 3 times prior to slight centrifuging.
4. Prepare Component C by mixing 0.32 μL of 50 μM crRNA1, 0.32 μL of 50 μM crRNA2, 0.32 μL of 100 μM EnGen® Lba Cas12a (Cpf1), and 0.28 μL nuclease-free water. TIP: It is preferable to leave the mixture for about 10 min at room temperature for fully forming the crRNA-Cas12a complex. Prepare the mixture enough for at least 2 reactions for easy and accurate pipetting. For better performance, the Component C is recommended to be fresh prepared when testing.
5. Pipette 1.24 μL of the Component C into the assembled mixture in Step 3 to form the final reaction system. TIP: Thoroughly vortex the reaction system and invert the tube 3 times prior to slight centrifuging.
6. Incubate the final reaction system at 37°C for 20-40 min prior to fluorescence detection.
· Take out the tubes after the AIOD-CRISPR reaction and place them in the LED blue transilluminator or the Imaging System for visual fluorescence detection.
· Real-time fluorescence detection can be achieved by incubating the AIOD-CRISPR reaction systems in the CFX96 Touch™ Real-Time PCR Detection System.