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Summary
Update: CDC NWSS has recently shared two published CDC diagnostic assays, non-variola orthopoxvirus (NVO) and MPXV Clade II virus. Targets for these 2 assays are included on the NIST RGTM 10223 plasmid.
Description
A new material from NIST can aid in the evaluation and development of selected qPCR assays for the mpox virus. We are offering a unit free of charge in exchange for your feedback, which will help us improve and further develop the material.
Questions? Contact MPXV-RGTM [at] nist.gov (MPXV-RGTM[at]nist[dot]gov).
NIST supports the nation's surveillance of emerging diseases with tools that assure users and manufacturers of diagnostic tests that their methods and assays are working properly.
The World Health Organization is monitoring cases of mpox virus in humans on several continents and states that the situation is a global emergency. As mpox cases rose, we immediately pivoted the NIST measurement infrastructure that has long supported biotechnology and biopharmaceutical manufacturing to address the critical need for control materials for molecular mpox diagnostics.
NIST rapidly developed a research grade test material consisting of linearized plasmid DNA containing 9 PCR target regions of the mpox virus genome. The nine target regions are listed in Table 1. Note that proprietary commercial assays that target other regions of the mpox genome will not amplify. Please check your primers against the plasmid sequence.
This plasmid does not contain any complete coding regions (CDSs) from the mpox genome. It should be handled according to your institution’s practices regarding synthetic nucleic acid or it should be handled using BSL-1 practices. View the safety data sheet (PDF).
This linearized plasmid DNA can assist in the validation of qPCR assays for the detection of mpox virus (target regions included are listed in Table 1). The DNA, characterized for concentration using digital PCR methods, may be used as a positive control, to assess limits of detection for mpox assays, and may calibrate other in-house or commercial mpox controls. We have characterized this material by size, UV/Vis absorbance, fluorescence, multiple droplet digital PCR (ddPCR) assays, and qPCR methods.
The NIST Mpox Research Grade Test Material (RGTM 10223) differs from a NIST SRM in that it is not as highly characterized or traceable to the SI, but is homogeneous and undergoing continual stability testing.
The materials are not intended to be subjected to extraction processes.
The material consists of linear plasmid DNA (approx. 3.4 kb) containing assay targets from the mpox genome in a background of 5 ng/μL yeast tRNA (stored at 4 °C, BSL-1; DO NOT FREEZE). Vials contain approximately 250 μL at 110,000 plasmid copy/μL. Each unit of RGTM will be shipped along with the tRNA buffer for in-lab dilutions. Users are strongly encouraged to dilute the sample (purple cap) using the supplied buffer (green cap) by 100-fold (to approximately 1000 copy/μL), as this will keep the material stable and extend the supply.
- Linear plasmid – Total length: 3376 nt; Includes 9 PCR assay target regions separated by spacer. Target sequences were taken from the references listed in Table 1.
- For the dilution buffer recipe, see p6 of https://nvlpubs.nist.gov/nistpubs/SpecialPublications/NIST.SP.260-221.pdf. The buffer is approximately 10 mg of yeast tRNA (ThermoFisher catalog #AM7119*) per 1 L of 1x TE pH 8 in a clean container (or the TE bottle itself). This has worked well to stabilize DNA materials down to 5 copy/μL.
* Certain commercial products or company names are identified here to describe our study adequately. Such identification is not intended to imply recommendation or endorsement by the National Institute of Standards and Technology, nor is it intended to imply that the products or names identified are necessarily the best available for the purpose.
Table 1. Mpox target sequences
| Target | Purpose | Source |
| E9L-NVAR | CDC Assay, all MPXV | CDC https://doi.org/10.1016/j.jcv.2006.03.012 |
| G2R_G | Assay with G2R_G primers and probe: detects all Mpox strains | CDC https://doi.org/10.1016/j.jviromet.2010.07.012 |
| G2R_WA | Assay with G2R_WA primers and probe: detects Western African clade viruses | CDC https://doi:10.1016/j.jviromet.2010.07.012 |
| C3L | Assay with C3L primers and probe: detects Congo Basin clade viruses | CDC https://doi:10.1016/j.jviromet.2010.07.012 |
| F3L | conserved for specifically detecting Mpox | US Army https://doi.org/10.1038/labinvest.3700143 |
| N3R | conserved for specifically detecting Mpox | US Army https://doi.org/10.1038/labinvest.3700143 |
| E9L-OPX3 | CDC Assay, all Mpox | CDC https://doi.org/10.4269/ajtmh.2010.09-0716 |
| B6R | CDC Assay; selective for Mpox | CDC https://doi.org/10.1016/j.jcv.2006.03.012 |
| B2R | Poxin gene associated with toxin | https://doi.org/10.1038/s41586-019-0928-6 |
TO RECEIVE RGTM 10223
NIST is making this material available free of charge.
This is not a diagnostic test for personal use. It is for the evaluation of clinical assays by researchers and laboratory professionals.
If you would like a free unit of RGTM 10223, please go here.
We will ship a unit to you. Please download the guidance sheet (PDF) with details about the material including storage and instructions for use.
TO PROVIDE FEEDBACK
Please complete this form to provide feedback regarding use of this material. Sharing your experience will help us further test, develop, and improve the materials for future development.
Example of feedback that we request:
- Affiliation
- Feedback on the material volume and concentration
- Description of how the material was used
- Concentration value estimates from quantitative and/or digital PCR methods
- Feedback on the material's impact on your work
Thank you in advance for your participation in the development of this new material from NIST.
TO ACCESS DATA FOR RGTM 10223
The fasta file for the insert can be accessed here (.zip file).
IN THE NEWS
NIST highlight: NIST Develops Genetic Material for Validating Mpox Tests
Diagnostics World News: NIST Develops Mpox Reference Materials, Sees Growing Role in Outbreak Response
This page was updated on Dec. 8, 2022, to reflect the World Health Organization’s decision to change the name of the virus from “monkeypox” to “mpox.”