New England Biolabs, E3019S, Luna® SARS-CoV-2 RT-qPCR Multiplex Assay Kit

The Large size of this product (NEB #E3019L) was discontinued on December 15, 2024. The Small pack size will remain available. Related Categories Luna® qPCR & RT-qPCR,, PCR, qPCR & Amplification Technologies Applications Probe-based qPCR & RT-qPCR,, qPCR & RT-qPCR,, Two-Step RT-qPCR FAQ Q: Do different SARS-CoV-2 variants impact the effectiveness of the Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit? A: Omicron Update The Luna SARS-CoV-2 RT-qPCR assay (NEB #E3019) is an RUO multiplex assay based on the US CDC-designed SARS-CoV-2 N1 and N2 genes. The new COVID-19 variant, Omicron, contains one point mutation in a region of the N-gene that is targeted by the N1 probe (C28311U). Direct testing of purified RNA representing both the wild-type (Wuhan) and mutant SARS-CoV-2 (Omicron) N1 templates resulted in substantially equivalent results and 100% detection of both samples at 10 copies per reaction (27/27 replicates tested). Additionally, a subpopulation of the Omicron BA.5 variant contains an additional point mutation at the 3′ end of the N1 probe (A28330G). Testing of purified RNA representing both the wild-type (Wuhan) and mutant BA.5 template (C28311U, A28330G) also resulted in substantially equivalent results. Other Variants NEB has developed a web tool (Primer Monitor) to continually monitor registered primer sets for overlapping variants in sequences from GISAID. Using this tool, we identified a prominent variant from some countries with a SNP close to the 3’ end of the 2019-nCoV-2_N2 forward primer included in the Luna kit, which detects the CDC SARS-CoV-2 N1, N2 targets and human RNase P gene with modifications (click for details) (A). To evaluate the impact of this SNP on the N2 target detection, we prepared two N gene RNA fragments containing the wild type and mutant N2 targets, respectively, by in vitro transcription and quantitated them using the SARS-CoV-2 RNA Control 2 from Twist Bioscience. Using the primer/probe set included in the Luna kit, we observed an average 4.2 Cq delay for the mutant N2 target (B). The limit of detection (LOD) for the mutant N2 target was 25 copies/reaction. Though all the reactions containing 10 copies of the mutant RNA generated amplification signal, only 11 of 24 had a Cq ≤ 40, the cut-off for detection (C). Q: What components are included in the Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit? A: The Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit contains all the necessary components to conduct multiplex RT-qPCR assays as specified in the details below. The Luna Probe One-Step RT-qPCR 4X Master Mix with UDG (NEB #M3019) is supplied at 4X concentration and contains all the necessary components for One-Step RT-qPCR. It is also formulated with a unique passive reference dye that is compatible across a variety of instrument platforms, dUTP/UDG for carryover prevention and a nonfluorescent visible dye for monitoring reaction setup. The SARS-CoV-2 Primer/Probe mix provided in 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. Q: How many test samples can be run on a 96-well plate? A: The Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit allows for simultaneous detection of two regions of the SARS-CoV-2 virus N gene plus an internal control. We recommend running one positive control, and one negative control per plate, enabling accommodation of up to 94 test samples on a 96-well plate. Q: Does the Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit enable carryover prevention/contamination reduction? A: Yes, the kit features the Luna Probe One-Step RT-qPCR 4X Master Mix with UDG (NEB #M3019), which is formulated with a mixture of dTTP and dUTP. This enables the incorporation of dU into the reaction products without sacrificing amplification efficiency. Antarctic Thermolabile Uracil DNA glycosylase (UDG) in the mix digests RT-qPCR products containing dU, preventing contamination from carryover amplification products. Because RT-qPCR can generate significant quantities of DNA, best practices include the use of various strategies to reduce contamination, such as the use of environmental controls and not opening vessels post amplification. Q: Can I set up the SARS-CoV-2 assay at room temperature? A: The Luna Probe One-Step RT-qPCR 4X Mix with UDG (NEB #M3019) provided in the kit contains Luna WarmStart® RT paired with Hot Start Taq, enabling room temperature setup. For best results, the reagents from the kit should be kept on ice during the reaction setup and stored at -20°C. Q: How much sample can be added to the reaction as input for the SARS-CoV-2 assay? A: For 96-well assays, we typically recommend 2-10 μl of extracted nucleic acid in a 20 μl reaction for best results. For manual 384-well assays, we recommend up to 5 μl of extracted nucleic acid in a 10 μl reaction. In some 384-well workflows, 5 μl reactions (up to 2 μl sample input) can also be used. Q: What targets do the SARS-CoV-2 Primer/Probe mix detect? How were these primers chosen? A: 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 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 optimized internal control (IC) primer and probe set, designed to amplify the human RNaseP gene, is also provided in the primer mix. Amplification of the IC is observed in the Cy5 channel. Q: Is the sequence of the RNase P primer/probe set the same as the CDC? A: 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. Q: What is the SARS-CoV-2 Positive Control included in the kit? A: The SARS-CoV-2 Positive Control (N2117) is a plasmid that contains the SARS-CoV-2 N-gene (GenBank: MN908947.3). The concentration at 12.5X is approximately 1,000,000 copies/uL and each lot is assessed by qPCR. Please note that we do not recommend use of the Positive Control concentration as an absolute copy number for quantitative experiments. Q: How are results interpreted in the Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit? A: Sample 2019-nCoV N1 (HEX) 2019-nCoV N2 (FAM) Internal Control (Cy5) Interpretation Positive Control Cq<27 Cq<27 ND QC passed Negative Control ND ND ND QC passed Test Sample ND ND ND Invalid Test (Internal Control Failure) Cq<40 ND Cq<40 Inconclusive - Retest ND Cq<40 Cq<40 Inconclusive - Retest Cq<40 Cq<40 Cq<35 or ND* Positive ND (or Cq>40) ND (or Cq>40) Cq<35 Negative * It is important to note that when a test sample contains viral RNA yet only a few or no somatic cells, it is possible to observe positive N1 and N2 amplification signals (Cq < 40) and negative RNase P signal (Cq > 35) due to target competition. This sample should be considered as Positive. Q: How stable is the Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit primer mix? A: The SARS-CoV-2 Primer/Probe Mix (N1/N2/RP) is stable and robust under standard handling and usage conditions. For best results, materials should be stored at -20°C when not in use. Stability testing using up to 30 freeze/thaw cycles has shown no negative effect on the primer/probe mix performance. Q: What types of samples/materials are compatible with the Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit? A: This kit has been extensively evaluated using purified total RNA or total nucleic acid eluted in nuclease-free water or common elution buffers. Material eluted in AVE buffer should be kept to 13 μl (65% v/v) or less of the final reaction volume. Material eluted in TE or similar elution buffer should be kept to 5 μl (25% v/v) or less of the final reaction volume. Material in PBS buffer should be kept to 2 μl (10% v/v) or less of the final reaction buffer. Q: What instruments are compatible with the Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit? A: Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit Compatible Instruments Applied BioSystems® 7500 Fast Applied BioSystems® QuantStudio 6/7 Applied BioSystems® QuantStudio 12K Bio-Rad CFX384/CFX96 Bio-Rad IQ™5 Cepheid Smartcycler™/Smartcycler™ II Eppendorf Mastercycler® Qiagen Rotor-Gene® Q/ Rotor-Gene® 6000 Roche LightCycler® 480 Roche LightCycler® 1536 Roche LightCycler® Nano Q: What are the sequences for the primers and probes included in the kit? A: Primer Name Forward Primer Reverse Primer Probe 2019-nCoV_N1 5′ GAC CCC AAA ATC AGC GAA AT 3′ 5′ TCT GGT TAC TGC CAG TTG AAT CTG 3′ 5′ HEX-ACC CCG CAT TAC GTT TGG TGG ACC-Q 3′ 2019-nCoV_N2 5′ TTA CAA ACA TTG GCC GCA AA 3′ 5′ GCG CGA CAT TCC GAA GAA 3′ 5′ 6-FAM-ACA ATT TGC CCC CAG CGC TTC AG-Q 3′ RNase P 5′ AGA TTT GGA CCT GCG AGC G 3′ 5′ CAA CTG AAT AGC CAA GGT GAG C 3′ 5′ Cy5-TTC TGA CCT GAA GGC TCT GCG CG-Q 3′ Note: Q = quencher Q: Can I prepare a plate in advance? A: Typically, we recommend running RT-qPCR reactions as soon as possible after reactions are set up. However, based on testing using purified total RNA in 96-well assays (20 ul reaction volume), Luna RT-qPCR reactions can be set up and protected from light, for up to 8 hours at 25°C and still maintain comparable performance to control reactions. Similar results were observed when testing 384-well assays (10 ul reaction volume). In preparing 96-well assays (20 ul reaction volume) in advance with everything except sample input, we have observed similar performance when the reaction premix (step 4 in the protocol/manual) is stored for over 48 hours at 4°C (protected from light) compared to control reactions run immediately after reaction set up.