The One qPCR Enzyme Mix includes Reverse Transcriptase, Recombinant Ribonuclease Inhibitor in an optimized formulation, also include Bioingentech® Taq DNA polymerase and all reagents for an optimized RT-qPCR. The SARS CoV-2 (N-ORF1ab-RdRp gene) primer and probe mix provided in the kit hybridize in specific and conserved zones of two genes. The first one is N gene which facilitate its replication and process the virus particle assembly and release, ORF1ab gene which generates a replicase polyprotein involved in the virus transcription and replication, on the other hand, and RdRp gene which generate a vital enzyme for the replication and transcription cycle of the virus: RNA dependent RNA polymerase.
These genes can be detected through your real time platform by the 5’ nuclease PCR detection method. During PCR amplification, forward and reverse primers hybridize to the SARS CoV-2 target cDNA generated. Fluorogenic probes are included in the same reaction mixture. N gene is amplified and detected in CY5 channel; ORF1ab gene is amplified and detected in FAM channel and RdRp gene is amplified and detected in CY3.
An additional primer/probe set to detect the human RNase P gene (RP) as internal control to monitor PCR inhibition is also included in the panel labeled with a 5-reporter and a 3-quencher. During PCR amplification, the probe is cleaved and the reporter dye and quencher are separated. As a result, a fluorescence increase can be detected on a range of real time PCR platforms through HEX/VIC channel.
Figure 1: Clinical tests of the One Step qPCR-realtime™ SARS CoV-2 N-CY5, ORF1ab-FAM and RdRp-CY3 Genes detection Kit were performed with more than 5000 samples from asymptomatic and symptomatic patients.
Total RNA was isolated followed by a One Step RT-PCR for the detection of the SARS CoV-2 N gene, SARS CoV-2 ORF1ab, SARS CoV-2 RdRp gene and the human RNase P gene.