The Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit is optimized for real-time qualitative detection of SARS-CoV-2 nucleic acid using hydrolysis probes. In a single tube, RNA is first converted to cDNA by a reverse transcriptase, then a DNA-dependent DNA polymerase amplifies the cDNA, enabling quantitation via real-time or quantitative PCR (qPCR). 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 PCR cycle. 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.
Figure 1: Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit components
Figure 2: The Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit detects two regions of the N gene and the human RNase P gene in a single reaction
A. The two SARS-CoV-2 sequences are based on those provided by the CDC, but modified to contain different fluorophores (N1: HEX, N2: FAM).
B. The RNase P internal control includes a Cy5 labeled probe and a re-designed reverse primer. This primer spans an exon-exon junction to avoid amplification of human genomic DNA which contains a 2.4 kb intron.
The SARS-CoV-2 Primer/Probe Mix 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)]. The probes 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 RNase P gene, is also included 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.
The Luna Probe One-Step RT-qPCR 4X Mix with UDG (NEB #M3019) included in the kit enables higher amounts of input material and supports sample pooling strategies, with minimal loss of sensitivity or specificity. It contains all necessary components for one-step RT-qPCR and 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 thermolabile UDG and dUTP for carryover prevention and a non-fluorescent visible tracking 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.
Figure 3: The Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit demonstrates a lower limit of detection than
TaqPath™1-Step RT-qPCR Master Mix, CG
LOD comparison using: Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit for multiplex RT-qPCR targeting 2019-nCoV_N1 target (HEX) and 2019-nCoV_N2 target (FAM), according to reaction and cycling conditions provided in the E3019 product manual, and TaqPath 1-Step RT-qPCR Master Mix, CG for singleplex RT-qPCR targeting 2019-nCoV_ N1 (FAM) and 2019-nCoV_N2 (FAM), according to the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel guidelines. Performance was evaluated using Synthetic Twist SARS-CoV-2 RNA Control 2 diluted in 10 ng of Jurkat total RNA. Data was collected on an Applied Biosystems® 7500 Fast real-time instrument (96-well, 20 µl reactions). Under these conditions, the Luna Kit has an LOD of 5 copies/reaction for both targets while the LOD using TaqPath is 10 copies/reaction for these targets.
Figure 4: Luna SARS-CoV-2 RT-qPCR Multiplex Assay kit enables sample pooling of purified RNA
A pool containing five samples was prepared by combining 2 µl of one mock positive (10 copies of the Twist Synthetic SARS-CoV-2 RNA Control diluted in 10 ng of Jurkat total RNA) and 2 µl each of 4 mock negative samples (10 ng Jurkat total RNA only). Multiplex performance for the pooled sample (10 µl) was compared to an individual sample (2 µl, a total of 10 copies of N gene and 10 ng of Jurkat total RNA). Data was collected on an Applied Biosystems 7500 Fast Real-Time instrument (96-well, 20 µl reactions). The amplification curves for the N1 and N2 targets indicate similar Cq values from a positive sample whether assayed individually or as part of a pool. As expected, the RNase P signal from the pooled sample has an earlier Cq compared to the single sample since it contains 5 times the amount of human total RNA.
Figure 5: Limit of Detection (LOD) of the Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit
The Luna SARS-CoV-2 RT-qPCR LOD was determined by multiplex RT-qPCR targeting 2019-nCoV_N1 (HEX) and 2019-nCoV_N2 (FAM) from various control samples of SARS-CoV-2 RNA. Performance was evaluated using (A) Twist Synthetic SARS-CoV-2 RNA Control 2 diluted in 10 ng of Jurkat total RNA. Data was collected by two users on Applied Biosystems (ABI) 7500 Fast Real-Time instrument, ABI QuantStudio™ 6 Flex Real-Time PCR system, and Bio-Rad CFX instrument (96-well, 20 µl reactions). The LOD for the Twist RNA is 5 copies/reaction on all three instruments. Performance was also evaluated using (B) Genomic SARS-CoV-2 RNA from ATCC (ATCC® VR-1986D™), NIST (Fragment 1 – Includes SARS-CoV-2 sequence: 25949-29698 of isolate USA-WA1/2020), and SeraCare AccuPlex™ SARS-CoV-2 Verification Panel V2 (0505-0132) RNA extracted with Monarch Total RNA Miniprep Kit (NEB# T2010). All RNA samples in this panel were diluted in 10 ng of Jurkat total RNA and tested on an ABI 7500 Fast Real-Time instrument. The LOD is 2.5 GE (genomic copy equivalent) for the SARS-CoV-2 genomic RNA from ATCC, 1x107-fold dilution for the NIST SARS-CoV-2 Test Material and 15 copies for the SeraCare AccuPlex reference material (assuming 100% recovery of input material from the RNA extraction procedure).
Figure 6: Improved performance on the SARS-CoV-2 N2 target from the Luna Probe One-Step RT-qPCR 4X Mix with UDG compared to the TaqPath 1-Step RT-qPCR Master Mix, CG
A. Singleplex RT-qPCR targeting 2019-nCoV_1 (HEX) and 2019-nCoV_N2 (FAM) was performed using the Luna Probe One-Step RT-qPCR 4X Mix with UDG, according to reaction and cycling conditions provided in the E3019 product manual.B. Singleplex RT-qPCR targeting 2019-nCoV_N1 (FAM) and 2019-nCoV_N2 (FAM) was performed using TaqPath 1-Step RT-qPCR Master Mix, CG, as outlined in the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel guidelines. Performance was evaluated over a 5-log range of Twist Synthetic SARS-CoV-2 RNA Control 2 diluted in 10 ng of Jurkat total RNA. Data was collected on an Applied Biosystems 7500 Fast Real-Time instrument (96-well, 20 µl reactions). Under these conditions, both Luna and TaqPath mixes perform well with the N1 target. For the N2 target, even though 10 copies is in a detectable range for the TaqPath mix, substandard linearity is consistently observed, while the Luna mix exhibits strong linearity and faster Cq’s.
The Luna Probe One-Step RT-qPCR 4X Mix with UDG features Hot Start Taq DNA Polymerase 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.
Figure 7: Room temperature stability of Luna RT-qPCR Mix enables workflow flexibility
Singleplex (SP) and multiplex (MP) RT-qPCR targeting 2019-nCoV_N1 (HEX) and 2019-nCoV_N2 (FAM) was performed using the Luna Probe One-Step RT-qPCR 4X Mix with UDG, according to reaction and cycling conditions provided in the NEB #E3019 product manual. Singleplex RT-qPCR targeting 2019-nCoV_N1 (FAM) and 2019-nCoV_N2 (FAM) was performed using TaqPath 1-Step RT-qPCR Master Mix, CG, according to the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel guidelines. Performance was evaluated over a 5-log range (100,000-10 copies) of Synthetic SARS-CoV-2 RNA Control 2 diluted in 10 ng of Jurkat total RNA. RT-qPCR reactions were incubated at room temperature for 0, 2, 5 and 24 hours before running on an Applied Biosystems 7500 Fast real-time instrument (96-well, 20 μl reactions). Using synthetic Twist RNA, consistent performance is observed with up to 24 hours of room temperature incubation with the Luna probe One-Step RT-qPCR Mix, while TaqPath showed a Cq delay ≥1 at 2 hours with declining performance as incubation time increased.
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