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

Quantitative RT-PCR was initially performed in monoplex single-tube reaction mixture using two different separate mastermixes. For standard RT-qPCR, PCR master mix containing Invitrogen SuperScript™ III Platinum™ One-Step RT-qPCR Kit Ribonuclease Inhibitor (Life Technologies, USA). 2 μL of ten-fold diluted RNA template in duplicates was added in a total volume of 20 µL. DNase/RNase-free water was used as the non-template control (NTC). All reactions were completed in a 96-well plate format (MicroAmp™ Fast Optical 96-Well Reaction Plate with Barcode, 0.1 mL). The RT-qPCR assays were performed under the following conditions: reverse transcription at 50°C for 15 minutes and initial denaturation at 95°C for 1 minute, 45 cycles of denaturation at 95°C for 10 seconds and annealing at 55°C for 45 seconds using a standard benchtop real-time thermocycler (StepOnePlus Real-Time PCR System, Applied Biosystems, USA). A specimen was considered positive if the amplification curve crossed the threshold line within 40 quantification cycle (Cq < 40).