SYBR Green RT-PCR assay

The ΔΔCt method of relative quantification using RT-PCR with SYBR Green detection was used for this study. To detect SARS-CoV-2 target genes, comparative quantitative RT-PCR was performed using SYBR Green fluorescent dye, which binds double-stranded DNA by intercalating between the DNA bases. The 7500 Fast Real-Time PCR system and software (Applied Biosystems, California, USA) were used.

The target SARS-CoV-2 genes included envelope (E), open reading frame (ORF1a), nucleocapsid (N), and RNA-dependent RNA polymerase RdRp. In addition, RNase P, Ribonuclease P, which is a type of ribonuclease that cleaves RNA that is commonly used as an internal control with human clinical specimens to confirm the presence of nucleic acid. Primers targeting the N, E, RdRp and RNase P genes were designed and synthesized in-house by the Oligonucleotide Unit of KFSHRC (Table 1 ). SYBR Green RT-PCR assays using these primers generated in-house were conducted for all patient samples that were included in this study. To amplify cDNA in the patient samples, a master mix was prepared with 12.5 μL of SYBR Green master mix, 0.5 μL of forward primer (20 μM), 0.5 μL of reverse primers (20 μM), 2 μL of cDNA, and 4.5 μL of water (for a total of 20 μL). The amplification conditions were 50 °C for 20 s and 95 °C for 10 min. followed by 45 cycles of 95 °C for 3 s with 55 °C for 30 s. At the end of each reaction, the cycle threshold (Ct) was acquired at the level that reflected the best kinetic PCR parameters.

Real-time-PCR primer sets for SARS-CoV-2 detection using the SYBR Green-based assay.

IPC: internal positive control.

SYBR Green is released when a PCR product is denatured, which results in a rapid increase in absorbance intensity (fluorescence signal) followed by a decrease in the signal. When an amplified PCR product is specific, a melting curve generated using that product should provide a single peak that corresponds to that PCR product. Thus, to check the specificity of each PCR product, we conducted a melting curve analysis at the end of each PCR assay. Each product sample was analyzed once. For the melting curve, the fluorescence signal of each PCR product was monitored continuously as the temperature was increased to 95 °C for 15 s, decreased to 60 °C for 1 min and then increased again to 95 °C for 30 s and 60 °C for 15 s.