Viral RNA and S-gene target failure (SGTF) detection by real-time PCR
Extracted RNA was transferred to 96-well PCR plate containing 20 µl of TaqPath 1-step Multiplex Master Mix No ROX (Applied Bioscience, Cat number: A28523). This was followed by a one-step RT-PCR (TaqPath COVID-19 assay kit; Thermo-Fisher). Thereafter, the plate was sealed with MicroAmp clear adhesive strip (Applied Bioscience, Cat number: 4306311). The plate was loaded onto a QuantStudio 5 Real-Time PCR System (Applied Bioscience, Cat number: AB-A28574) and the following amplification program was used: 25 °C for 2 min, X1 cycle 53 °C for 10 min, X1 cycle 95 °C for 2 min, X1 cycle 95 °C for 3 s, followed by 60 °C for 30 s, X40 cycles Ct threshold values were preset using the following values/parameters: MS2-15,000; by cycle 37; S gene- 20,000 by cycle 37; Orf1ab- 20,000 by cycle 37; Ngene- 20,000 by cycle 37. Samples that passed the Threshold is a Ct value >37 were re-tested or considered weak positive. Above threshold values of MS2, Orf1ab, and Ngene, but not S gene was considered S-gene target failure (SGTF). SGTF serves as a proxy for identifying B.1.1.7 cases (