Version 1 2022-08-04

MPXV Synthetic DNA Plasmid Guidance 

NIST Research Grade Test Material 10223

Sample Description

Each unit of RGTM 10223 contains approximately 110,000 copy/μL linearized plasmid DNA suspended 

in 250 μL of 1× TE buffer + approximately 5 ng/μL yeast tRNA to act as a carrier. The plasmid contains 

PCR targets for MPXV assays. The concentration was estimated using digital PCR assays (dPCR). 

Figure 1. Vector (top) and insert (bottom) schematics. RGTM 10223 is provided as a linearized DNA that 

was cut at the ScaI site.
Version 1 2022-08-04

One unit of material contains two tubes – one with plasmid and one with dilution buffer. NIST is 

currently evaluating the stability of the diluted plasmid using this buffer, and will update with new data 

when available.

Purpose • Act as a positive control for MPXV PCR assays

• Serve to generate standard/calibration curve for MPXV PCR assays

Storage

Store material at 4 °C. DO NOT FREEZE.

Notices and Warnings

This plasmid does not contain any complete coding regions 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. 

Please review the Safety Data Sheet provided with the material. The Safety Data Sheet can also be 

downloaded from the website https://www.nist.gov/programs-projects/mpxv-monkeypox-synthetic-dnapcr-standards. 

Instructions for Use

Vortex briefly (approximately 5-10 s) and spin to recollect the mixed contents. Recap the vial 

immediately after use to avoid altering the concentration or introducing contaminants.

The included tRNA dilution buffer should be used for diluting this material (e.g. positive controls, 

dilution curves). This buffer will maintain the stability of the DNA in solution such that there is no 

observable change in the copy number for years. To date (7/22/22) a 1:100 dilution of this material has 

been stable. You may also use the dilution buffer as a no-template control (NTC).

Follow the procedure specific to your assay. 

Estimated Values

The copy number concentration was estimated using droplet digital PCR. “Assay” is a NIST-designation 

and may not match other naming conventions.

Target Assay

Mean concentration 

(copy number / μL) Coefficient of Variation

E9L E9L-NIST 1.10E+05 <10 %

B2R B2R-NIST 1.10E+05 <10 %

G2R_G G2R_G-NIST 1.11E+05 <10 %

For the 3 targets were tested, no significant difference in concentration was observed. This indicates that 

the linearized plasmid contains a single copy of each target. The full sequence of the insert can be found 

on our website in the “Access Data” section. 

Target Selection

We selected targets were based upon published assays of relevant targets for MPXV. The plasmid 

contains only the sequence fragment for the PCR assay, typically ~100-200 bp. Each fragment is 

separated by a short sequence to help identify the target as synthetic.
Version 1 2022-08-04

Target Assay Primer/Probe Sequences* Source

E9L E9L_NVAR

F- TCAACTGAAAAGGCCATCTATGA 

P- TET-CCATGCAATATACGTACAAGATAGTAGCCAAC

R- GAGTATAGAGCACTATTTCTAAATCCCA 

doi:10.1016/j.jcv.2006.03.012 

OPX E9L_OPX 

F- TCAAATATTGATCGTCCAACGA

P- FAM-TAACATCCGTCTGGAGATATCCCGTTAGA-BHQ1

R- TGGATGAATTTCTCAATATTAGTTGG

doi:10.4269/ajtmh.2010.09-0716 

G2R_G G2R_G 

F- GGAAAATGTAAAGACAACGAATACAG

P- FAM-AAGCCGTAATCTATGTTGTCTATCGTGTCC-BHQ1 

R- GCTATCACATAATCTGGAAGCGTA

doi:10.1016/j.jviromet.2010.07.012 

G2R_WA G2R_WA 

F- CACACCGTCTCTTCCACAGA

P- FAM-AACCCGTCGTAACCAGCAATACATTT-BHQ1

R- GATACAGGTTAATTTCCACATCG

doi:10.1016/j.jviromet.2010.07.012 

C3L C3L 

F- TGTCTACCTGGATACAGAAAGCAA

P- FAM-CCCATATATGCTAAATGTACCGGTACCGGA-BHQ1

R- GGCATCTCCGTTTAATACATTGAT

doi:10.1016/j.jviromet.2010.07.012 

F3L F3L

F- CTCATTGATTTTTCGCGGGATA

P- 6FAM-CATCAGAATCTGTAGGCCGT-MGBNFQ

R- GACGATACTCCTCCTCGTTGG

doi:10.1038/labinvest.3700143 

N3R N3R 

F- AACAACCGTCCTACAATTAAACAACA

P- 6FAM-TATAACGGCGAAGAATATACT-MGBNFQ

R- CGCTATCGAACCATTTTTGTAGTCT

doi:10.1038/labinvest.3700143 

B6R B6R 

F- ATTGGTCATTATTTTTGTCACAGGAACA 

P- MGB/DarkQuencher-AGAGATTAGAAATA-FAM

R- AATGGCGTTGACAATTATGGGTG

doi:10.1016/j.jcv.2006.03.012 

B2R B2R multiple doi:10.1038/s41586-019-0928-6 

* These are the original assays as published; NIST has not tested or verified their utility.

The plasmid length is 3376 nt and consists of a cloning vector with ampicillin resistance and the insert. 

The insert size is 1155 nt. The sequence of the insert was confirmed using Sanger sequencing. 

Feedback

Since NIST produced the material for no-charge, we ask you to provide feedback on the material. Your 

feedback regarding use of this material will help us further test, develop, and improve these materials for 

future development. Please provide your feedback by August 31, 2023. After that date, please contact us 

directly using MPXV-RGTM@nist.gov. 

Feedback form

NIST Additional Information

NIST generated assays for dPCR on each target to ensure they could all be measured using the same 

reaction conditions. These may not match the published assays. Those assays will be available at a later 

date.

NIST Contact Information

General Inquiries (MPXV-RGTM@nist.gov)