Rapid direct nucleic acid amplification test without RNA extraction for SARS-CoV-2 using a portable PCR thermocycler

Soon Keong Wee; Suppiah Paramalingam Sivalingam; Eric Peng Huat Yap

All samples used for the spike-in experiments were freshly prepared. Briefly, 8.5 µL of sputum and nasal exudates sample mix with Sputasol were aliquoted. 0.5 µL (20 U) of RNaseOUT™ Recombinant Ribonuclease Inhibitor (Life Technologies, USA) was added to each aliquot and inverted to mix. Meantime, the RNA template was ten-fold serially diluted in DNase/RNase-free water to achieve 8 orders of magnitude. Next, 1 µL of diluted RNA template was spiked into the sputum and nasal exudate mix forming a total volume of 10 µL. Subsequently, 2 μL of RNA spiked-matrices containing 42.5% sputum and nasal exudate respectively were directly added into the 20 µL reaction volume of inhibitor-resistant PCR reaction mix containing a PCR enhancer and inhibitor-resistant enzymes (Direct One-Step S/P RT-qPCR TaqProbe Kit, VitaNavi Technology LLC, USA), forward and reverse primers, FAM-based probe, and RNaseOUT™ Recombinant Ribonuclease Inhibitor (Life Technologies, USA) as described above. RNA template in DNase/RNasefree water was used as positive control. A sputum only and nasal exudate only sample containing Sputasol were also added as a blank non-template control (NTC) for the respective matrices. The reaction mixture was incubated at 50°C for 15 minutes, denatured at 95°C for 1 minute, followed by 45 cycles of denaturation at 95°C for 10 seconds and annealing at 55°C for 45 seconds on a standard benchtop real-time thermocycler (StepOnePlus Real-Time PCR System, Applied Biosystems, USA).