Skip to main content
U.S. flag

An official website of the United States government

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you’ve safely connected to the .gov website. Share sensitive information only on official, secure websites.

MPXV (Monkeypox) Synthetic DNA PCR Standards

Summary

*** The next shipment of MPXV materials will be 10/3/2022 *** We will continue to accept requests during this time, but there will be a slight delay in processing due to end-of-fiscal-year inventory. 

If you are in need material for an emergency, contact MPXV-RGTM [at] nist.gov.

Description

MPXV RGTM wrapped linear insert

A new material from NIST can aid in the evaluation and development of selected qPCR assays for the monkeypox 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.

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 monkeypox virus (MPXV) in humans on several continents and states that the situation is a global emergencyAs MPXV 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 monkeypox diagnostics.

NIST rapidly developed a research grade test material consisting of linearized plasmid DNA containing 9 PCR target regions of the monkeypox virus genome. The nine target regions are listed in Table 1. Note that proprietary commercial assays that target other regions of the MPXV genome will not amplify. Please check your primers against the plasmid sequence.

This plasmid does not contain any complete coding regions (CDSs) from the monkeypox 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 monkeypox 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 monkeypox assays, and may calibrate other in-house or commercial monkeypox controls. We have characterized this material by size, UV/Vis absorbance, fluorescence, multiple droplet digital PCR (ddPCR) assays, and qPCR methods. 

The NIST monkeypox 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 monkeypox 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.

Table 1. Monkeypox 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 MPXV 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 MPXV​ 

US Army
https://doi.org/10.1038/labinvest.3700143 

N3R​ 

conserved for specifically detecting MPXV​ 

US Army
https://doi.org/10.1038/labinvest.3700143​ 

E9L-OPX​3 

CDC Assay, all MPXV​ 

CDC
​https://doi.org/10.4269/ajtmh.2010.09-0716 

B6R​ 

CDC Assay; selective for MPXV​ 

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 complete this form

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 Monkeypox Tests

Diagnostics World News: NIST Develops Monkeypox Reference Materials, Sees Growing Role in Outbreak Response

Created June 28, 2022, Updated September 16, 2022