Description
The oligonucleotide primers and probes for specific detection of SARS-CoV-2 are selected from regions of Open Reading Frame 1ab (ORF1ab) and the nucleocapsid gene (N) of the SARS-CoV-2 genome. The kit includes primers/probes that are specific for the ORF1ab gene (probe labeled with FAM) and N gene (probe labeled with VIC) of SARS-CoV-2. In addition, the kit also contains primers and a probe (labeled with CY5) for the human RNase P gene as an endogenous internal control for specimen integrity, nucleic acid isolation, amplification and detection. RNA isolated and purified from upper and lower respiratory tract specimens is reverse transcribed to cDNA and amplified in a Real-time PCR instrument using one-step Master Mix. Probes consist of a reporter dye at the 5’ end and quenching dye at the 3’ end. The fluorescent signals emitted from the reporter dye are absorbed by the quencher. During PCR amplification, probes hybridized to amplified templates are degraded by the Taq DNA polymerase with 5’-3’ exonuclease activity, thereby separating the reporter dye and quencher and generating fluorescent signals that increase with each cycle. The PCR instrument automatically draws a real-time amplification curve for each optical channel based on the signal change, and calculates cycle threshold (Ct) values (the point at which fluorescence is detectable above background) that are interpreted by the operator to determine the presence/absence of SARS-CoV-2 RNA