Mar 31, 2026
Version 1
Multiplex PCR-based method for whole-genome sequencing of measles virus from wastewater V.1
This protocol is a draft, published without a DOI.
- Sipho Gwala1,
- Natasha Singh1,
- Victor Mabasa1,
- Joshua Levy2,
- Kerrigan McCarthy1,3,
- Mukhlid Yousif1,3
- 1National Institute for Communicable Diseases;
- 2Department of Translational Medicine, The Scripps Research Institute, La Jolla, CA, United States;
- 3School of Public Health, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
Protocol Citation: Sipho Gwala, Natasha Singh, Victor Mabasa, Joshua Levy, Kerrigan McCarthy, Mukhlid Yousif 2026. Multiplex PCR-based method for whole-genome sequencing of measles virus from wastewater. protocols.io https://dx.doi.org/
Manuscript citation:
https://doi.org/10.64898/2026.02.21.26346782
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
We use this protocol and it's working
Created: March 31, 2026
Last Modified: March 31, 2026
Protocol Integer ID: 314183
Keywords: genome sequencing of measles virus, full measles virus genome, measles virus, efficient enrichment of mev, multiplex pcr, genome sequencing, high genome coverage suitable for molecular epidemiology, using wastewater sample, wastewater the protocol, wastewater sample, high genome coverage, enabling efficient enrichment, mev, wastewater, degraded rna, sequencing workflow
Funders Acknowledgements:
Gates Foundation
Disclaimer
The cDNA synthesis and amplification are based on the SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific) and Q5 High-Fidelity 2X Master Mix (New England Biolabs) kits, respectively.
Abstract
The protocol relies on multiplex PCR to generate overlapping amplicons spanning the full measles virus genome, enabling efficient enrichment of MeV genetic material from low-input and degraded RNA. Amplified products are compatible with Illumina sequencing workflows and support high genome coverage suitable for molecular epidemiology and phylogenetic analysis. This protocol has been optimized for sensitivity and reproducibility using wastewater samples and can be completed within 1-2 days.
Materials
SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific)
Q5 High-Fidelity 2X Master Mix (New England Biolabs)
Nuclease-free water
Thermal cycler
Ice
200µL PCR plate or tubes
Virus enrichment and nucleic acids extraction
Wastewater samples can be processed for virus concentration and nucleic acid extraction using a previously published protocol https://www.protocols.io/print/enrichment-of-intact-virus-from-wastewater-using-d-hqy3b5xyp. Briefly, samples are subjected to a virus enrichment step to enrich viral particles from the wastewater matrix using magnetic beads, followed by extraction of total nucleic acids using a a magnetic beads-based method. The procedure is optimized to maximize recovery of viral RNA while minimizing the impact of environmental inhibitors commonly present in wastewater.
Anneal primer to template RNA
6m
Prepare the RNA-primer mix by adding the following components to a200 µL PCR reaction tube.
| A | B | |
| Reaction component/ reagent | Volume (µL) | |
| 50µM random Haxamers | 1 | |
| 10mM dNTPs mix (10mM each) | 1 | |
| Template RNA | 11 |
Briefly vortex and centrifuge the reaction components/ reagents.
Heat the RNA-primer mix at 65 °C for 00:05:00 in a thermocycler, and thereafter incubate on ice for at least 00:01:00
6m
Prepare the reverse transcription reaction mix
Add the following components to a 200µL PCR reaction tube.
| A | B | |
| Reaction component/ reagent | Volume (µL) | |
| 5× SSIV Buffer | 4 | |
| 100mM DTT | 1 | |
| Ribonuclease inhibitor | 1 | |
| SuperScript IV reverse Transcriptase | 1 |
The 5× SSIV buffer is essentially composed of salts. It must be pre-warmed and mixed thoroughly before use.
Cap the tube, mix, and then briefly centrifuge its components.
Incubate the reaction components using the following conditions on a thermal cycler.
| A | B | |
| Time | Temperature (°C) | |
| 10 | 23 | |
| 10 | 52.5 | |
| 10 | 80 | |
| ∞ | 4 |
Multiplex tilling PCR
The multiplex PCR panel for measles virus amplicon sequencing consist of two primer pools that amplify the virus genome and can be accessed at https://labs.primalscheme.com/detail/artic-measles/400/v1.0.0/.
Prepare two master mixes in separate PCR tubes for Pools 1 and 2 as follows:
| A | B | |
| Reaction component/ reagent | Volume (µL) | |
| Q5 High-Fidelity 2× Master Mix | 12.5 | |
| Pool 1 or 2 Primer Mix (10µM) | 7.3 | |
| cDNA | 3 | |
| Nuclease-free water | 2.2 |
Amplify the cDNA using the following thermal cycling profile:
| A | B | C | D | |
| Step | Time | Temperature (°C) | No. of cycles | |
| Polymerase activation | 30 sec | 98 | - | |
| Denaturation | 15 sec | 98 | 45 | |
| Annealing | 5 min | 65 | ||
| Extension | ∞ | 4 | - |
Combine amplicons from Pool 1 and Pool 2, then verify the presence of ~400 bp fragments using gel electrophoresis