COVID-19 diagnosis by RT-PCR and viral load quantification

The detection of viral SARS-CoV-2 was carried out using the ORF1ab probe (TaqMan™ 2019nCoV Assay Kit v1, Thermo Fisher Scientific, Cat. No. A47532) and one-step strategy. Each reaction contained 5 μl of TaqMan™ Fast Virus 1-Step Master Mix 4X, 1 μl of ORF1ab assay 20X, 1 μl of RNase P assay 20X, 11 μl of nuclease-free water, and 2 μl of the extracted RNA sample. The RT-PCR reaction was performed on the Agilent AriaMx Real-Time PCR System (Agilent Technologies, Part. No. G8830A). Serial 1/10 dilutions generated a standard curve with the positive control TaqMan 2019-nCoV Control Kit v1 (10⁴ copies/μL) (Thermo Fisher Scientific, Cat. No. A47533) by RT-PCR. Antilogarithm of the following equation of the line (y = −3.07^*X + 40.2)/2 was used to calculate the viral load. To obtain the number of viral copies per μl, this equation was divided by two according to the volume of RNA (2 μl) used in the RT-PCR reaction. The Cq got in the NPSs was replaced in “X”.