Oct 21, 2025

Public workspaceDual-target detection of Mpox virus

DOI
https://dx.doi.org/10.17504/protocols.io.n2bvj3b8plk5/v1
  • Alan O'Dwyer1,
  • Tracy D Lee1,
  • Michael Chan1,
  • Branco Cheung1,
  • Frankie Tsang1,
  • John R Tyson1,2,
  • Kathleen L Kolehmainen1,
  • Natalie A Prystajecky1,2,
  • Agatha N Jassem1,2
  • 1British Columbia Centre for Disease Control Public Health Laboratory, Vancouver, British Columbia, Canada;
  • 2Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada
BCCDC Public Health Laboratory
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DOI: https://dx.doi.org/10.17504/protocols.io.n2bvj3b8plk5/v1
Protocol Citation: Alan O'Dwyer, Tracy D Lee, Michael Chan, Branco Cheung, Frankie Tsang, John R Tyson, Kathleen L Kolehmainen, Natalie A Prystajecky, Agatha N Jassem 2025. Dual-target detection of Mpox virus. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj3b8plk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
This protocol is currently in use at British Columbia Centre for Disease Control Public Health Laboratory.
Created: December 01, 2023
Last Modified: October 21, 2025
Protocol Integer ID: 91715
Keywords: target detection of mpox virus, detecting mpox virus, mpox virus, mpxv gp113 gene, tnf receptor deletion, targeting tnf receptor gene, tnf receptor gene, enabled pcr assay, undetectable by the g2rg tnf, hbg multiplex qpcr for the detection, human betaglobin, g2rg tnf, human betaglobin as endogenous control, targeted qpcr, secondary mpxv target, existing g2rg target, mpxv, cases of mpox, hbg multiplex qpcr, mpox, g2rg target in order
Abstract
This procedure provides instructions for detecting Mpox virus and human beta-globin using a laboratory developed fast-enabled PCR assay on a Quantstudio 7 real-time PCR instrument.

To address the 2022 Mpox virus (MPXV) outbreak a real-time PCR targeting TNF receptor gene (G2RG) as described in the Journal of Virological Methods 2010 publication by Li et al. (1) was assessed and multiplexed with human betaglobin as endogenous control.

In September 2022, the CDC released an alert on its Laboratory Outreach Communication System stating that three cases of Mpox were detected in California with a significant deletion in the TNF receptor gene. These cases were undetectable by the G2RG TNF-targeted qPCR (2). In response, a secondary MPXV target was required to multiplex with the existing G2RG target in order to detect any future cases with a TNF receptor deletion. Several targets were trialed and the MPXV GP113 gene was selected.

This validation report presents the data supporting the use of a G2RG/GP113/HBG multiplex qPCR for the detection of Mpox virus.
Guidelines
This procedure describes testing of extracted DNA. Sample extraction is not included.
Prepare PCR master mixes in a Clean Room.
Perform all manipulations of samples and DNA in a genomic level PCR laboratory
Materials
Samples: DNA extracts from suspected Monkeypox cases.


Materials
ReagentsEquipmentSupplies
TaqMan Fast Advanced Master Mix 2x (Cat No. 4444557)ABI QuantStudio 7 Pro real time PCR instrumentApplied Biosystems Fast Optical 96-well plates
Primers and probes (see below)Appropriate pipettesPipette tips
IDTE 1x TE Buffer pH 8.0MicroAmp Optical Adhesive Film and Applicator
UltraPURE DNAse/RNAse Free waterGloves & gowns
Materials




PRIMERS
NameSequence (5' -> 3')TargetAmplicon lengthAuthor
G2RG-FmodATGGAAARTGTAAAGACAACGAATACTNF receptor gene92 bpLi et al. 2010
G2RG-Rmod2GCTATCACATAATCTGRAAGCGTA
GP113-FTATTGGTACCGTCTTCCGACAACDS of S-S bond formation pathway protein83 bpTracy Lee 2022
GP113-RCATTGTACTGAATCCGCCTAAGC
HBG-FACCCAGAGGTTCTTTGAGTCCTTTHuman beta-globin gene82 bpHSV/VZV assay
HBG-RTGCCATGAGCCTTCACCTTAG
Primers - Supplied by Life Technologies and/or IDT




PROBES
NameSequence (5' -> 3')Dye/QuencherTargetAuthor
G2RG-PAAGCCGTAATCTATGTTGTCTATCGTGTCCFAM/ZENG2RGLi et al. 2010
GP113-PATCCGAGCATGTAGAAGAVIC-NFQ-MGBGP113Tracy Lee 2022
HBG-PCACTCCTGATGCTGTTATGNED-MGBHBGHSV/VZV assay
Probes - Supplied by Life Technologies and/or IDT





20X Mix oligo recipe
ReagentsStock Conc (µM)Final Conc (nM)Volumes for 100µL 20X mix
G2RG-Fmod100 µM90018
G2RG-Rmod218
G2RG-P2505
GP113-F4509
GP113-R9
GP113-P1503
HBG-F1002
HBG-R2
HBG-P1002
IDTE Buffer--32
TOTAL--100
20x mix - Oligo recipe for 100µL of 20x mix




Quality Control
  1. Positive Extraction Control
  2. Negative Extraction Control
  3. PCR Control (AmpliRun Monkeypox Virus DNA Control Cat# MBC146-R)
  4. No Template Control



Troubleshooting
Before start
Prepare a 20x oligo mix per the recipe in the materials section
Prepare master mix
Prepare the master mix cocktail according to the following table. Invert mixture gently several times to mix and quickly spin down.


Reagents1 x Rxn (µL)100x Rxn (µL)
TaqMan Fast Advanced MasterMix 2x101000
PCR grade water4400
20x Oligo Mix1100
Table 1: Mastermix recipe


Aliquot 15µL of master mix to each well of a MicroAmp Fast Optical 96 well reaction plate (0.1mL). Transfer plate to the PCR Genomic Room
Mpox PCR
In a Biological Safety Cabinet, transfer 5µL of sample extract and controls to the optical plate in order:
  1. Patient Sample Extracts
  2. Positive and Negative Extraction Controls
  3. MPXV Positive PCR control (Amplirun Monkeypox Virus DNA Control)
  4. Matrix (BG) control
  5. No Template Control (NTC)

Seal the plate with an optical plate film

Load the plate onto an ABI QuantStudio 7 Real-Time PCR instrument. Refer to Table 2 for fluorophore information. Refer to Table 3 for thermocycling conditions. Refer to Table 4 for recommended manual target threshold settings.


TargetReporterQuencher
G2RGFAMZEN
GP113VICNFQ
HBGNEDNone
Table 2 Probe dyes



StageTemp (°C)Time (Min)
40 cyclesInitial Denaturation950:20
Denaturation950:03
Annealing/Extension600:30
Table 3 Thermocycling conditions



TargetThreshold
G2RG0.2
GP1130.1
HBG0.03
Table 4 Manual target thresholds


Run the PCR and analyze results.
Protocol references
1. Li Y, Zhao H, Wilkins K, Hughes C, Damon IK. Real-time PCR assays for the specific detection of monkeypox virus West African and Congo Basin strain DNA. J Virol Methods. 2010;169(1):223-227. doi:10.1016/j.jviromet.2010.07.012

2. Lab Alert: MPXV TNF Receptor Gene Deletion May Lead to False Negative Results with Some MPXV Specific LDTs. CDC’s Laboratory Outreach Communication Systems (LOCS) – 09/02/2022