Mar 30, 2018

Public workspaceRespiratory picornavirus genotyping conventional nested RT-PCR ("Wisdom VP42 assay") V.1

DOI
dx.doi.org/10.17504/protocols.io.nz7df9n
  • 1The University of Queensland
Ian M Mackay
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DOI: dx.doi.org/10.17504/protocols.io.nz7df9n
Protocol Citation: Ian M Mackay, Claire Y. T. Wang, Katherine E Arden 2018. Respiratory picornavirus genotyping conventional nested RT-PCR ("Wisdom VP42 assay"). protocols.io https://dx.doi.org/10.17504/protocols.io.nz7df9n
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: March 25, 2018
Last Modified: March 30, 2018
Protocol Integer ID: 11039
Keywords: s
Abstract
This is my preferred, previously published [Ref 1], rhinovirus (RV) and enterovirus (EV) genotyping assay when seeking to characterize the genotypes of respiratory picornavirus identified after use of a screening real-time RT-PCR to test nucleic acid extracts from clinical samples.
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
STEP MATERIALS
ReagentSensiFAST Probe no ROX one-step kitBiolineCatalog #BIO-76005
ReagentMyTaq HS DNA PolymeraseBiolineCatalog #BIO-21113
ReagentSensiFAST Probe no ROX one-step kitBiolineCatalog #BIO-76005
ReagentMyTaq HS DNA PolymeraseBiolineCatalog #BIO-21113
Protocol materials
ReagentMyTaq HS DNA PolymeraseBiolineCatalog #BIO-21113
ReagentSensiFAST Probe no ROX one-step kitBiolineCatalog #BIO-76005
ReagentMyTaq HS DNA PolymeraseBiolineCatalog #BIO-21113
ReagentSensiFAST Probe no ROX one-step kitBiolineCatalog #BIO-76005
ReagentSensiFAST Probe no ROX one-step kitBiolineCatalog #BIO-76005
ReagentMyTaq HS DNA PolymeraseBiolineCatalog #BIO-21113
Oligonucleotide sequences...
Oligonucleotide sequences...
 NameSequence (5'-3')
RT-PCR
(Round 1)		HRV_HEV VP42 OSCCGGCCCCTGAATGYGGCTAA
 HRV_HEV VP42 OASACATRTTYTSNCCAAANAYDCCCAT
PCR
(Round 1)		HRV_HEV VP42 IS ACCRACTACTTTGGGTGTCCGTG
 HRV_HEV VP42 IAS TCWGGHARYTTCCAMCACCANCC
  1. Expected amplicon sizes: Round 1: ~380 base pairs; Round 2: 
  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
ReagentSensiFAST Probe no ROX one-step kitBiolineCatalog #BIO-76005
ReagentMyTaq HS DNA PolymeraseBiolineCatalog #BIO-21113
Reaction set-up
Reaction set-up
ReagentVol (μl) 1xFinal reaction concentration
Nuclease-free water1.4N/A
SensiFAST no ROX One-Step Mix (2X)101X
Primers (μM)16600nM
MgCl203mM
RNase inhibitor0.4Unknown
RT/Taq (?U/μl)0.2Unknown
Template2N/A
  1. Both mixed to this final concentration
  2. Dispense 18µL to each reaction tube.
  3. Add 2µL of template (extracted RNA, controls or NTC [nuclease-free water] )
  4. Total reaction volume is 20µL
ReagentVol (μl) 1xFinal reaction concentration
Nuclease-free water8.7N/A
MyTaq Reaction Buffer (5X)41X
Primers (μM)13.8380nM
MgCl21.44.75mM
MyTaq HS DNA Polymerase (5U/uL)0.2Unknown
1st round amplicon2N/A
  1. Both mixed to this final concentration
  2. Dispense 18µL into each reaction tube
  3. NB: a 1:100 pre-dilution can be made first
  4. Total reaction volume is 20µL
Amplification
Amplification
45°C20 min1X
94°C2 min1X
   
94°C18 sec|
50°C21 sec| 35X
72°C90 sec|
   
72°C7 min1X
4°C∞ 
95°C1 min1X
   
94°C18 sec|
50°C21 sec|  35X
72°C90 sec|
   
4°C∞