Principles of the Assay:

The assay is designed to target the well conserved regions of N protein genes, the mutation sites of E484Q and P681R in the S gene.

The specific primers and probes set are designed, the different fluorophores labels are used to multiplex detection of N gene and the mutation sites of E484Q and P681R in the S gene. Nucleic acids are extracted from designated samples with adequate methods and materials. After reverse transcription, the presented target sequences will be amplified by respective specific primers and detected by TaqMan probes. During the extension phase of each PCR cycle, the Taq polymerase cleave reporter dye off the 5′ end of the probe, separate dye from the quencher, producing fluorescent signals. The increasing fluorescence signals are detected over the PCR course and data analyzed.

A specific primer and probe set is designed for a human housekeeping gene GAPD which serves as the internal control (IC) for this assay. GAPD is consistently expressed in human cells that are present in intended samples for the assay. The occurrence of false negatives due to issues in sample quality and/or extraction process can be monitored and controlled effectively.

Reagent Storage, Shipment, and Handling

All reagents should be stored at the temperature between -25°C to -15°C in a non-frost-free freezer for use before the expiration date. Freeze/Thaw more than three times should be avoided during the kit usage period. The reagents should be shipped at the temperature between -25°C to 8°C.