The GG COVID-19 Triplex Real-Time PCR test is a real-time reverse transcription polymerase chain reaction (rRT -PCR) test. The kit uses TaqMan based real-time PCR technique to conduct in vitro reverse transcription of SARS-CoV-2 RNA, DNA amplification and fluorescence detection.
RNA isolated and purified from upper and lower respiratory specimens is reverse transcribed to cDNA and subsequently amplified in one of the 2 commonly used real time RT-PCR machines and associated software. In the process, the probe anneals to a specific target sequence located between the forward and reverse primers. During the extension phase of the PCR cycle, the 5’ nuclease activity of Taq polymerase degrades the probe, causing the reporter dye to separate from the quencher dye, generating a fluorescent signal. With each cycle, additional reporter dye molecules are cleaved from their respective probes, increasing the fluorescence intensity.
There are 2 forms of Triplex assays:
1) RdRP and nucleocapsid (N) gene
2) RdRP and Open reading frame (Orf1ab) gene
The TaqMan probes for the two amplicons are labeled with FAM and Texas red fluorescent dyes respectively to generate target-specific signal. The assay includes an RNA internal control (human housekeeping gene) to monitor the processes from nucleic acid extraction to fluorescence detection. The IC probe is labeled with Hex (VIC) fluorescent dye to differentiate its fluorescent signal from SARS-CoV-2 targets. Fluorescence intensity is monitored at each PCR cycle in corresponding channels at a threshold of 0.05.