Sep 19, 2024
Hepatitis E and Enterovirus Multiplex Nested RT-PCR
- Shannon Fitz1,
- Alex Shaw1,
- Dilip Abraham2,
- michael Owusu3
- 1Imperial College London;
- 2CMC Vellore;
- 3Kwame Nkrumah University of Science and Technology
- GEMS - Genomic Environmental Microbial Surveillance
Protocol Citation: Shannon Fitz, Alex Shaw, Dilip Abraham, michael Owusu 2024. Hepatitis E and Enterovirus Multiplex Nested RT-PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzjd74lx1/v1
Manuscript citation:
Hepatitis E primers extracted from: Wang B, Harms D, Papp CP, Niendorf S, Jacobsen S, Lütgehetmann M, Pischke S, Wedermeyer H, Hofmann J, Bock CT. Comprehensive Molecular Approach for Characterization of Hepatitis E Virus Genotype 3 Variants. J Clin Microbiol. 2018 Apr 25;56(5):e01686-17. doi: 10.1128/JCM.01686-17. PMID: 29514938; PMCID: PMC5925713.
Enterovirus primers: designed by Dr Alex Shaw, unpublished.
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: September 19, 2024
Last Modified: September 19, 2024
Protocol Integer ID: 107998
Keywords: PCR, Hepatitis E, Enterovirus, Multiplex PCR, Nested PCR, enterovirus multiplex nested rt, enterovirus, hev-37-f, hev-38-f, hev-39-r, hev-27-r, hev, outer hev, pcr for multiplex, inner hev, gargcyatggtbgagaarg hev-39-r, pcr, cggtaccyttgtrcgcctg rev7-5ncr, caagcacttctgttwccc rev6-5ncr, rev7-5ncr, for2-5ncr, rev6-5ncr, for1-5ncr, ggttycgygcyattgaraarg hev-27-r
Funders Acknowledgements:
The Gates Foundation
Grant ID: INV-049092 PA4273
Abstract
For multiplex, nested amplification of Hepatitis E (HEV) and Enterovirus (EV) samples.
Primer sets and target amplicon size
Outer HEV: HEV_38_f and HEV_39_r (538 bps)
HEV_38_f: GARGCYATGGTBGAGAARG
HEV_39_r: GCCATRTTCCARACRGTRTTCC
Inner HEV: HEV_37_f and HEV_27_r (306 bps)
HEV_37_f: GGTTYCGYGCYATTGARAARG
HEV_27_r: TCRCCRGARTGYTTCTTCC
Outer EV: For1_5NCR and Rev7_5NCR (535 bps)
For1_5NCR: CGGTACCYTTGTRCGCCTG
Rev7_5NCR: ATTGTCACCATAAGCAGCC
Inner EV: For2_5NCR and Rev6_5NCR (388 bps)
For2_5NCR: CAAGCACTTCTGTTWCCC
Rev6_5NCR: CCAAAGTAGTCGGTTCCGC
Materials
DreamTaq PCR Master Mix (2X)Thermo FisherCatalog #K1072 SuperScript III One-Step RT-PCR System with Platinum TaqInvitrogen - Thermo FisherCatalog #12457-026
Approximate total cost per sample: £8.01
8 primers required (cost excluded from estimate as primers do not need to be ordered each time)
Superscript cost per sample: ~£7.56
Dreamtaq cost per sample: ~£0.45 per sample
Extra equipment required:
Vortex, mini centrifuge, thermocycler
Protocol materials
DreamTaq PCR Master Mix (2X)Thermo FisherCatalog #K1072
SuperScript III One-Step RT-PCR System with Platinum TaqInvitrogen - Thermo FisherCatalog #12457-026
Troubleshooting
Hepatitis E and Enterovirus Multiplex Nested PCR
First round PCR for combined enterovirus and hepatitis E samples
SuperScript III One-Step RT-PCR System with Platinum TaqInvitrogen - Thermo FisherCatalog #12457-026
Prepare master mix on ice per number of samples/controls + 10%. Flick master mix gently to mix, and spin down. Aliquot 20 ul into each well, then add 5 ul of template.
| A | B | |
| Reagent | Volume for 1 rxn (ul) | |
| 2x reaction mix | 12.5 | |
| HEV_38_f primer | 0.5 | |
| HEV_39_r primer | 0.5 | |
| For1_5NCR primer | 0.5 | |
| Rev7_5NCR primer | 0.5 | |
| Nuclease free water | 4.5 | |
| SSIII taq | 1 | |
| Template | 5 ul | |
| Total volume | 25 ul |
Cycling conditions for first round PCR
Flick samples gently to mix, spin down tubes. Amplify samples using the following conditions:
| A | B | C | |
| Number of cycles | Temperature (°C) | Time | |
| 1 | 50 | 30 minutes | |
| 1 | 94 | 5 minutes | |
| 40 | 94 | 30 seconds | |
| 52 | 30 seconds | ||
| 68 | 45 seconds | ||
| 1 | 68 | 5 minutes | |
| 10 | Hold |
Second round PCR
DreamTaq PCR Master Mix (2X)Thermo FisherCatalog #K1072
Prepare on ice per number of samples/controls + 10%. Vortex briefly, and spin down. Aliquot 24 ul of master mix into each well, then add 1 ul of template.
| A | B | |
| Reagent | Volume per 1 reaction (ul) | |
| Dreamtaq | 12.5 | |
| HEV_37_f | 0.5 | |
| HEV_27_r | 0.5 | |
| For2_5NCR | 0.5 | |
| Rev6_5NCR | 0.5 | |
| Nuclease free water | 9.5 | |
| Template (amplicon from first round PCR) | 1 | |
| Total | 25 ul |
Cycling conditions for second round PCR
Vortex samples briefly, spin down, and amplify using the following conditions:
| A | B | C | |
| Number of cycles | Temperature (oC) | Time | |
| 1 | 95 | 10 minutes | |
| 40 | 95 | 30 seconds | |
| 52 | 30 seconds | ||
| 72 | 45 seconds | ||
| 1 | 72 | 5 minutes | |
| 10 | Hold |
Check all samples and controls on an agarose gel or a tapestation.
Protocol references
Wang B, Harms D, Papp CP, Niendorf S, Jacobsen S, Lütgehetmann M, Pischke S, Wedermeyer H, Hofmann J, Bock CT. Comprehensive Molecular Approach for Characterization of Hepatitis E Virus Genotype 3 Variants. J Clin Microbiol. 2018 Apr 25;56(5):e01686-17. doi: 10.1128/JCM.01686-17. PMID: 29514938; PMCID: PMC5925713.