Probe-based qPCR/RT-qPCR monitors an increase in fluorescence upon 5→ 3 exonuclease cleavage of a quenched, target-specific probe to measure DNA amplification at each cycle of a PCR. At a point where the fluorescence signal is confidently detected over the background fluorescence, a quantification cycle or Cq value can be determined. Cq values can be used to evaluate relative target abundance between two or more samples.
In the Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit, Hot Start Taq DNA Polymerase is combined with a novel WarmStart-activated
reverse transcriptase, allowing dual control of enzyme activity via reversible, aptamer-based inhibition. This temperature-dependent activation helps to prevent undesirable non-specific priming and extension prior to thermocycling, providing added security for setting up reactions at room temperature. The engineered WarmStart Luna Reverse Transcriptase also possesses higher thermostability than many other RTs, allowing an optimal reaction temperature of 55°C.
The Luna Probe One-Step RT-qPCR Master Mix is supplied at 4X concentration and contains all the necessary components for One-Step
RT-qPCR. It is formulated with a unique passive reference dye that is compatible across a variety of instrument platforms, including those that require a high or low ROX reference signal. The Reaction Mix also features dUTP/UDG for carryover prevention and a nonfluorescent visible dye for monitoring reaction setup. This visible dye does not overlap spectrally with fluorophores commonly used in qPCR and does not interfere with real-time detection.
The SARS-CoV-2 Primer/Probe mix provided with this kit contains primers and probes specific to two regions of the SARS-CoV-2 virus
N-gene [based on sequences provided by the Centers for Disease Control and Prevention (CDC)]. However, they have been modified to
contain different fluorophores (N1, HEX; N2, FAM) to enable simultaneous observation on two different channels of a real-time
instrument. To ensure the integrity of the input material and absence of inhibition, an internal control (IC) primer and probe set, designed to amplify the human RNaseP gene, is also provided in the primer mix. The reverse primer of this target has been modified from the CDC design to target an exon/exon boundary to reduce background amplification from possible contaminating genomic DNA. Amplification of the IC is observed in the Cy5 channel. A positive control (PC) template (SARS-CoV-2 N-gene cloned into a plasmid) is also provided.