Nov 27, 2019
Version 2
Respiratory picornavirus genotyping conventional nested RT-PCR ("Wisdom VP42 assay") V.2
Version 1 is forked from Respiratory picornavirus genotyping conventional nested RT-PCR ("Wisdom VP42 assay")
- 1Public Health Virology, Forensic and Scientific Services
External link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2786677/
Protocol Citation: Ian M Mackay, Judy A Northill 2019. Respiratory picornavirus genotyping conventional nested RT-PCR ("Wisdom VP42 assay"). protocols.io https://dx.doi.org/10.17504/protocols.io.9tyh6pw
Manuscript citation:
Reference 1. Screening Respiratory Samples for Detection of Human Rhinoviruses (HRVs) and Enteroviruses: Comprehensive VP4-VP2 Typing Reveals High Incidence and Genetic Diversity of HRV Species C. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2786677/
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: November 27, 2019
Last Modified: November 27, 2019
Protocol Integer ID: 30296
Keywords: enterovirus, rhinovirus, EV-D68, EV-A71, RV-C, RV-A, RV-B, picornavirus, respiratory, subgenomic, conventional RT-PCR, RT-PCR, nested PCR
Abstract
This is my preferred, previously published [Ref 1], rhinovirus (RV) and enterovirus (EV) genotyping assay when seeking to identify the genotype of a respiratory picornavirus detected in a clinical sample extract. It is employed after use of a screening real-time RT-PCR has identified a respiratory picornavirus.
I have not confirmed that it can detect every single RV genotype but I do know that it detects many from each of the three RV species (Human rhinovirus A, Human rhinovirus B and Human rhinovirus C) as well as at least some Human enterovirus (EV) genotypes.
The assay picks up EVs due to the shared genetic similarities in the 5'UTR target region. EVs can be discriminated using subgenomic sequencing (see VP42 typing assay protocol), or simply described as 'respiratory EVs' since there is no specific-specific vaccine or treatment available anyway.
This is a robust primary subgenomic sequencing assay. It is more sensitive than any VP1 protocols because it targets more conserved primer target sites. It produces a more reliable typing result than does the 5'UTR region alone.
Materials
MATERIALS
MyFi MixBiolineCatalog #BIO-25049
SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA PolymeraseThermo FisherCatalog #12574026
STEP MATERIALS
SuperScript III One-Step RT-PCR System with Platinum TaqInvitrogen - Thermo FisherCatalog #12457-026
MyFi MixBiolineCatalog #BIO-25049
Protocol materials
SuperScript III One-Step RT-PCR System with Platinum TaqInvitrogen - Thermo FisherCatalog #12457-026
MyFi MixBiolineCatalog #BIO-25049
MyFi MixBiolineCatalog #BIO-25049
SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA PolymeraseThermo FisherCatalog #12574026
MyFi MixBiolineCatalog #BIO-25049
SuperScript III One-Step RT-PCR System with Platinum TaqInvitrogen - Thermo FisherCatalog #12457-026
Oligonucleotide sequences...
Oligonucleotide sequences...
| Name | Sequence (5'-3') | ||
| RT-PCR (Round 1) | HRV_HEV VP42 OS | CCGGCCCCTGAATGYGGCTAA | |
| HRV_HEV VP42 OAS | ACATRTTYTSNCCAAANAYDCCCAT | ||
| PCR (Round 1) | HRV_HEV VP42 IS | ACCRACTACTTTGGGTGTCCGTG | |
| HRV_HEV VP42 IAS | TCWGGHARYTTCCAMCACCANCC |
- Expected amplicon sizes: Round 1: ~380 base pairs; Round 2:
- The naming used here is my in-house adaptation (FYI: 01 - forward / sense; 02 - reverse / antisense; .x - version of the design of this particular named oligonucleotide). If you prefer to be true to the original publication, please see Ref 1
Reagents
Reagents
SuperScript III One-Step RT-PCR System with Platinum TaqInvitrogen - Thermo FisherCatalog #12457-026
MyFi MixBiolineCatalog #BIO-25049
Reaction set-up
Reaction set-up
ROUND 1 REACTION MIX
- Ideally, set up more reaction mixes than you'll need for both rounds at the same time
- Strips of 8x 0.2ml tubes are good for this use
- Freeze in a frost-free freezer at -20°C until needed
| Reagent | Vol (μl) 1x | Final reaction concentration | |
| Nuclease-free water | 4.08 | N/A | |
| Reaction Mix (2X) | 10.00 | 1X | |
| HRV_HEV VP42 OS 200pmol/µl | 0.06 | 600nM | |
| HRV_HEV VP42 OAS 200pmol/µl | 0.06 | 600nM | |
| SuperScript® III RT/ Platinum® Taq Enzyme Mix | 0.20 | Unknown | |
| Template | 5.00 | N/A |
- Dispense 15µL to each reaction tube
- Total reaction volume will be 20µl
| Reagent | Vol (μl) 1x | Final reaction concentration | |
| Nuclease-free water | 7.92 | N/A | |
| MyTaq Reaction Buffer (2X) | 10.00 | 1X | |
| HRV_HEV VP42 IS 200pmol/µl | 0.038 | 380nM | |
| HRV_HEV VP42 IAS 200pmol/µI | 0.038 | 4.75mM | |
| 1st round amplicon | 2.00 | N/A |
- Dispense 18µL into each reaction tube
- Total reaction volume will be 20µl
Amplification: ROUND 1, RT-PCR
Amplification: ROUND 1, RT-PCR
- This reaction has been run using a SimpliAmp (Applied Biosystems) thermal cycler
- Transfer 5µl of nucleic acid extract (extracted RNA, controls or NTC [nuclease-free water]) to defrosted reaction tubes and cycle
| 55°C | 20 min | 1X | |
| 94°C | 2 min | 1X | |
| 94°C | 30 sec | | | |
| 50°C | 45 sec | | 4X | |
| 68°C | 50 sec | | | |
| 94°C | 30 sec | | | |
| 40°C | 45 sec | | 25X | |
| 68°C | 50 sec | | | |
| 68°C | 5 min | 1X | |
| 15°C | ∞ |
Amplification: ROUND 2, PCR
Amplification: ROUND 2, PCR
- This reaction has been run using a SimpliAmp (Applied Biosystems) thermal cycler
- Transfer 2µl of 1:100 pre-diluted Round 1 amplicon into defrosted 0.2ml reaction tubes and cycle
| 95°C | 1 min | 1X | |
| 95°C | 30 sec | | | |
| 50°C | 45 sec | | 4X | |
| 72°C | 50 sec | | | |
| 95°C | 30 sec | | | |
| 40°C | 45 sec | | 25X | |
| 72°C | 50 sec | | | |
| 72°C | 5 min | 1X | |
| 15°C | ∞ |
Amplicon visualisation
Amplicon visualisation
- electrophorese 2-5µl amplicon on a 1.5% agarose gel in 0.5X TBE buffer