It is based on the fluorogenic 5’nuclease assay. During the elongation phase the DNA polymerase cleaves the probe at the 5’ end and separates the reporter dye from the quencher dye only when the probe hybridizes to the target DNA. This cleavage results in the fluorescent signal generated by the cleaved reporter dye, which is monitored real-time by the PCR detection system. The PCR cycle at which an increase in the fluorescence signal is detected initially (Ct) is proportional to the amount of the specific PCR product. Monitoring the fluorescence intensities in real time allows the detection of the accumulating product without having to re-open the reaction tube after the amplification.
The kit contains a specific ready-to-use system for the detection of Novel Coronavirus (2019-nCoV) by Reverse transcription Polymerase Chain Reaction (RT-PCR) in the real-time PCR system. The mastermix contains a Super Mix for the specific amplification of viral RNA. The reaction is done in one step real time RT-PCR, hence the kit is Multiplex. The first step is a reverse transcription (RT), during which the virus RNA is transcribed into cDNA. Afterwards, a thermostable DNA polymerase is used to amplify the specific gene fragments by means of polymerase chain reaction (PCR). Fluorescence is emitted and measured by the real time systems’ optical unit during PCR. The detection of amplified virus DNA fragment is performed in fluorimeter channel FAM, HEX/VIC/JOE and Cal Red 610/ ROX/TEXAS RED with the fluorescent quencher BHQ1.