Coronavirus qPCR Design Case Study & Related Products to Support SARS-CoV-2 Research

Coronavirus qPCR Primer and Probe Design For SARS-CoV-2

We are working hand in hand with public health authorities and customers to support the fight against novel coronavirus (SARS-CoV-2). In response to the community’s need for SARS-CoV-2-specific reagents and protocols, our scientists have designed qPCR assays for SARS-CoV-2 target detection (see Table 1, below). Research Use Only. Not for Use in Diagnostic Procedures.

Special Notes on Assay Design & Reactivity

2019-nCoV shares 95% homology with SARS viruses, creating a high risk of cross-reactivity. Please keep the following notes in mind while designing your experiments using Table 1:

  1. For all assays, no cross reactivities with human or bacterial genome/mRNA sequences were identified in GenBank.
  2. The two SYBR Green assays, assays 1 and 2, do not identically match any other virus; however, they may recognize SARS viruses with 2–3 mismatched bases.
  3. The Dual-Labeled Probe assays, assays 3–6, can be used in parallel to mitigate issues of cross-reactivity.
    1. Assays 3 and 4 do not identically match any other virus but may recognize other viruses (mostly SARS) with several mismatched bases. These assays can reliably detect your sequence of interest, as each falls in a highly-conserved region, but they may also capture false positives.
    2. Assays 5 and 6 have no reactivity with any other virus, including SARS, so they should specifically detect only your sequence of interest; however, these assays fall in a highly variable region, so they may not capture all variants.
    3. Therefore, assays 3 and 4 can be used to detect positives, while assays 5 and 6 can be used to eliminate false positives.
       

Table 1. SARS-CoV-2 qPCR Detection Assays
 

Assay Detection Method Forward Primer Reverse Primer Probe Probe Sequence
1 SYBR® Green GCCTCTTCTCGT
TCCTCATCAC
AGCAGCATCACCG
CCATTG
   
2 SYBR® Green AGCCTCTTCTCG
TTCCTCATCAC
CCGCCATTGCCAG
CCATTC
   
3 Dual-Labeled Probe CCGCAAGGTTCT
TCTTCGTAAG
TGCTATGTTTAGT
GTTCCAGTTTTC
A AAGGATCAGTGCC
AAGCTCGTCGCC
4 Dual-Labeled Probe GGCTTACCGCAA
GGTTCTTC
TGCTATGTTTAGT
GTTCCAGTTTTC
A AAGGATCAGTGCC
AAGCTCGTCGCC
5 Dual-Labeled Probe CAGGTATATGCG
CTAGTTATCAGAC
CCAAGTGACATAG
TGTAGGCAATG
B AGACTAATTCTCC
TCGGCGGGCACG
6 Dual-Labeled Probe GCAGGTATATGC
GCTAGTTATCAG
ACACTGGTAGAAT
TTCTGTGGTAAC
B AGACTAATTCTCC
TCGGCGGGCACG

SARS vs. SARS-CoV-2 Positive Control Assay

To better differentiate between SARS viruses and SARS-CoV-2, we designed the following SARS positive control assay (see Table 2, below). Please keep the following notes in mind while designing your experiments using Table 2:

  1. This assay falls in a highly-conserved region of the SARS genome, enabling detection of multiple SARS variants.
  2. This assay does not recognize any human, bacterial, or other viral sequences, including SARI.

Table 2. SARS Positive Control qPCR Assay
 

Assay Detection Method Forward Primer Reverse Primer Probe Probe Sequence
SARS Positive Control Dual-Labeled Probe GCTGTGTGTTTGC
CTATGTTGG
ACGTTCACGACTC
AGTATCTCAAG
SARS Control AGCGTGCCTACTG
GGTTCCTCGTGC

Order SARS-CoV-2 Primers and Probes

Predesigned Primers & Probes

Our qPCR assay designs above are available for immediate ordering from our KiCqStart® product lines.
 

Our KiCqStart Dual-Labeled Probes are labeled with our own license and royalty-free dark quencher, Onyx Quencher (OQ). OQ is also available with custom probe orders. To learn more about OQ beyond what is on the landing page, please contact oligotechserv@merckgroup.com.

Custom Primers & Probes

We have a commercial license to supply probes labeled with Black Hole Quencher® (BHQ®) & BlackBerry® Quencher (BBQ®) to detect SARS-CoV-2.

To request a quote for custom primers and probes, please email sirnarequest@milliporesigma.com.

Positive Controls

Custom

The SARS-CoV-2 virus has a positive-sense ssRNA genome. We offer in vitro transcribed ssRNA against regions of this genome, which can serve as positive controls. We can synthesize custom sequences from 150 to 420 nucleotides (to ensure successful manufacturing, please avoid sequences with inverted repeats and repetitions). For Research Use Only. Not for Use In Diagnostic Procedures.

To request a quote or additional help, please email sirnarequest@milliporesigma.com

Predesigned

Single stranded RNA (ssRNA) Reference Materials intended for the use as a positive quality control sample for coronavirus SARS-CoV-2 in real-time RT-PCR assays.
 

Description Product No.
Single stranded RNA (ssRNA) fragments of SARS-CoV-2 EURM019

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

RT-PCR and RT-qPCR Genetic Analysis and Supporting Reagents

We are committed to providing researchers with the reagents and technical expertise for timely support of the SARS-CoV-2 outbreak. Explore our offering of world-class RT-PCR, RT-qPCR and supporting reagents that are suitable for SARS-CoV-2 vaccine research.

Transcriptor One-Step RT-PCR Kit

Utilizing hot start one-step RT-PCR technology, Transcriptor One-Step RT-PCR Kit includes a mixture of enzymes, including Taq DNA polymerase and a proofreading polymerase, to ensure sensitive and robust reverse transcription. Except for template and primers, Transcriptor One-Step RT-PCR Kit provides all the components required for one-step RT-PCR.

KAPA PROBE FAST One-Step  

KAPA PROBE FAST One-Step is designed for high-throughput, fast-cycling, one-step RNA quantification. KAPA PROBE FAST One-Step is suitable for use with all fluorogenic probe-based technologies, including hybridization probes (e.g., FRET), hydrolysis probes (e.g., TaqMan®) and displacement probes (e.g., molecular beacons).

Titan™ One Tube RT-PCR System

Incorporating a mixture of three enzymes, Titan™ One Tube RT-PCR System utilizes reverse transcriptase AMV for first-strand cDNA synthesis and the Expand™ High Fidelity enzyme blend, consisting of Taq DNA polymerase and a polymerase with a proofreading activity, for amplification of cDNA by PCR.

Quantitative RT-PCR ReadyMix™

In addition to containing Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) for first-strand cDNA synthesis, the Quantitative RT-PCR ReadyMix™ is conveniently blended with JumpStart Taq DNA polymerase, 99% pure deoxynucleotides, buffer, stabilizers, and ships as a 2x concentrate.

KiCqStart® One-Step Probe RT-qPCR ReadyMix™

Suitable for all dual-labeled probe chemistries, KiCqStart® One-Step Probe RT-qPCR ReadyMix™ comes with all required components for RT-qPCR, except for RNA template and probe. KiCqStart® One-Step Probe RT-qPCR ReadyMix™ is available with or without ROX™ reference dye.

SYBR® Green Quantitative RT-qPCR

Providing a highly sensitive method for the quantitative analysis of gene expression, this kit combines the advantages of Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) and JumpStart Taq in a convenient ReadyMix.

Table 3. RT-PCR, RT-qPCR and Supporting Reagents
 

Description Product No.
Transcriptor One-Step RT-PCR Kit TOSRTRO
KAPA PROBE FAST One-Step KK4752
Titan™ One Tube RT-PCR System 11855476001
Quantitative RT-PCR ReadyMix™ QR0200
KiCqStart® One-Step Probe RT-qPCR ReadyMix™ KCQS07
KiCqStart® One-Step Probe RT-qPCR ReadyMix™, Low ROX™ KCQS08
KiCqStart® One-Step Probe RT-qPCR ReadyMix™, ROX™ KCQS09
Nuclease-Free Water, Molecular Grade W4502
SYBR® Green Quantitative RT-qPCR QR0100

Proficiency Testing

In support of quality assurance efforts, the SARS-CoV-2 proficiency testing kit is designed for use with assays like real-time RT-PCR which detect the viral RNA by nucleic acid amplification. Reports provide evaluation of qualitative results as well as quantitative properties such as Cq(Ct) values and efficiencies.
 

Description Product No.
SARS-CoV-2 Proficiency Testing Kit
(containing 5 Samples - SARS-CoV-2 Sample A through SARS-CoV-2 Sample E)
PE5006-1KT

RNA Interference

Our MISSION® esiRNA COVID-19 panel (51 genes with 2 controls) provides RNAi researchers with a proven, cost-effective, and simple way to perform post-transcriptional silencing of putative genes involved in the intracellular life cycle of SARS-CoV-2. Included in the panel are genes that were found to be involved with the infection caused by other coronaviruses, such as SARS-CoV and MERS. This panel is available as a customizable esiFLEX 96-well array at quantities up to 5.0 μg, which is sufficient for screening common human cell lines. For Research Use Only. Not for Use In Diagnostic Procedures.
 

Human Genes in COVID-19 esiRNA Panel  
ACE2 TMPRSS2 ITGA3 ITGAV ITGB1 ITGB3 ITGB5 ITGB6
ITGB8 RPS3 DPP4 ADAM17 CD209L VEGFR1 VEGFR2 VEGFR3
HIF1A PACS1 GBP5 COPB2 COPB1 GBF1 PKR CDK6
MTHFD1 ATP6V1B2 ATP6V1F ATP6V0D1 CLTA CLTC COPZ1 COPG1
SEC23B SEC61B ARF1 ARF4 AGT AGTR1 MME MEP1A
MEP1B PRCP XPNPEP2 FAM3B TMPRSS4 ETV1 PTEN FKBP5
ETV4 SLC45A3 NKX3-1          

Included with the panels is the following:

  • Positive control: Human Eg5 (KIF11) to ensure transfection & experimental set up optimization
  • Negative control: RLUC to distinguish sequence-specific silencing from non–specific effects

To request a quote or additional help, please email sirnarequest@milliporesigma.com

We will continue to monitor the situation and work closely with customers to support response efforts to this WHO global emergency. Please pass along our primer and probe sets for qPCR detection of SARS-CoV-2 and biochemicals for Vaccine and Diagnostics research (Download PDF).

Contact us for expedited support in SARS-CoV-2 vaccine development.