Product Class: Kit

Luna® SARS-CoV-2 RT-qPCR Multiplex Assay Kit 

For Research Use Only. Not for use in diagnostic procedures.


Catalog #E3019

Product Introduction

The Luna® SARS-CoV-2 RT-qPCR Multiplex Assay Kit is a real-time RT-PCR assay for the qualitative detection of SARS-CoV-2 nucleic acid. 

  • What is the impact of different SARS-CoV-2 variants on this kit, including Omicron? See the updated FAQ and view the Primer Monitor (more info here) for up-to-date information on variants mapped against common primer sets.
  • Multiplex detection of 2019-nCoV_N1 and 2019-nCoV_N2 targets and human RNase P gene enables high throughput workflows
  • Reduce background amplification from genomic DNA by use of a modified RNase P Internal Control reverse primer to target an exon-exon boundary
  • Increase sensitivity with 4X RT-qPCR mix allowing for more sample input
  • Luna WarmStart® RT paired with Hot Start Taq increases reaction specificity and robustness, enabling room temperature setup
  • Reduce risk of carryover contamination with thermolabile UDG and dUTP included in the master mix
  • Supports sample pooling with minimal loss in sensitivity
  • Learn how NEB is supporting COVID-19 research with a variety of RT-qPCR virus detection options

 

Using the Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit, up to 94 different samples can be assessed in a single 96-well plate. Anticipated results for each sample type are shown (in each fluorophore channel).

 

Product Information

Description

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.




 
This product is related to the following categories:
Luna® qPCR & RT-qPCR Products,
PCR, qPCR & Amplification Technologies Products,
This product can be used in the following applications:
qPCR & RT-qPCR

Properties & Usage

Features

  • Support for high throughput workflows, including 384-well plate test formats (compatible with low reaction volumes)
  • UDG and dUTP included in master mix for carryover prevention, reduced risk of contamination
  • Optimized primer/probe mix offers enhanced detection and sensitivity
  • Dual WarmStart/Hot Start enzyme formulation enables room temperature setup and stability up to 24 hours
 

Protocols, Manuals & Usage

Protocols

  1. Luna SARS-CoV-2 RT-qPCR Multiplex Assay Protocol (NEB #E3019)

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Application Notes

Tools & Resources

Web Tools

FAQs & Troubleshooting

FAQs

  1. Do different SARS-CoV-2 variants impact the effectiveness of the Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit?
  2. What components are included in the Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit?
  3. How many test samples can be run on a 96-well plate?
  4. Does the Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit enable carryover prevention/contamination reduction?
  5. Can I set up the SARS-CoV-2 assay at room temperature?
  6. How much sample can be added to the reaction as input for the SARS-CoV-2 assay?
  7. What targets do the SARS-CoV-2 Primer/Probe mix detect? How were these primers chosen?
  8. Is the sequence of the RNase P primer/probe set the same as the CDC?
  9. What is the SARS-CoV-2 Positive Control included in the kit?
  10. How are results interpreted in the Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit?
  11. How stable is the Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit primer mix?
  12. What types of samples/materials are compatible with the Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit?
  13. What instruments are compatible with the Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit? 
  14. What are the sequences for the primers and probes included in the kit?
  15. Can I prepare a plate in advance?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

Licenses

Notice to Purchaser: Nucleic acid-based aptamers for use with thermophilic DNA polymerases are licensed exclusively by New England Biolabs, Inc. from SomaLogic, Inc. (See Patent Nos. 7,947,447; 8,404,830; 8,975,388 and their foreign equivalents). New England Biolabs, Inc. gives the Buyer/User a non-exclusive license to use this product for Research Purposes Only. Commercial use of this product requires a license from New England Biolabs, Inc. Please contact busdev@neb.com for more information.

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