Apr 03, 2018

Public workspaceHuman rhinovirus screening conventional RT-PCR ("Gama assay") V.4

DOI
dx.doi.org/10.17504/protocols.io.n7gdhjw
  • 1The University of Queensland
Ian M Mackay
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DOI: dx.doi.org/10.17504/protocols.io.n7gdhjw
Protocol Citation: Ian M Mackay, Katherine E. Arden 2018. Human rhinovirus screening conventional RT-PCR ("Gama assay"). protocols.io https://dx.doi.org/10.17504/protocols.io.n7gdhjw
Manuscript citation:
Reference 1.
Polymerase chain reaction amplification of rhinovirus nucleic acids from clinical material
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC338719/
 
Reference 2.
Amplification of rhinovirus specific nucleic acids from clinical samples using the polymerase chain reaction. https://www.ncbi.nlm.nih.gov/pubmed/2544679 
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 used this protocol in our group and it worked
Created: April 03, 2018
Last Modified: April 03, 2018
Protocol Integer ID: 11208
Abstract
This is a very handy, previously published [Ref 1 and 2], rhinovirus (RV) screening and genotyping assay which I and many others have used on many sample extracts, mostly originating from acutely ill paediatric patients, spanning well over a decade's worth of collection dates.
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.
This assay produces a useful backup subgenomic sequencing target for use primarily should the 'Wisdom VP42' protocol fail to amplify a product for sequencing. It is worth noting that these primers span a target region that can produce confusing results. Recombination within the 5' end of the genome of some members of the genus Enterovirus can lead to 5'UTR sequences generated by RV-C genotypes that appear to be RV-As (see the figure below in which green RV-Cs appear in what is expected to be the red RV-A branch of the phylogenetic tree). 

Materials
MATERIALS
ReagentSensiFAST Probe no ROX one-step kitBiolineCatalog #BIO-76005
STEP MATERIALS
ReagentSensiFAST Probe no ROX one-step kitBiolineCatalog #BIO-76005
ReagentSensiFAST Probe no ROX one-step kitBiolineCatalog #BIO-76005
Protocol materials
ReagentSensiFAST Probe no ROX one-step kitBiolineCatalog #BIO-76005
ReagentSensiFAST Probe no ROX one-step kitBiolineCatalog #BIO-76005
ReagentSensiFAST Probe no ROX one-step kitBiolineCatalog #BIO-76005
ReagentSensiFAST Probe no ROX one-step kitBiolineCatalog #BIO-76005
Oligonucleotide sequences
Oligonucleotide sequences
NameSequence (5'-3')
GAMA_OL26_01.1GCACTTCTGTTTCCCCC
GAMA_OL26_02.1CGGACACCCAAAGTAG
  1. Expected amplicon size ~380 base pairs
  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 and Ref 2
Reagents
Reagents
ReagentSensiFAST Probe no ROX one-step kitBiolineCatalog #BIO-76005
Reaction set-up
Reaction set-up
Reagent Vol (μl) x1Final reaction concentration
Nuclease-free water1.8N/A
SensiFAST no ROX One-Step Mix(2X)101X
 Primers (2μM)14400nM
MgCl2 (25mM)1.65mM
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
Amplification
Amplification
  • This protocol has been used successfully on Applied Biosystems PCR System 2400 and GeneAmp® PCR System 2700 thermal cyclers
45°C20min1X
94°C2min1X
   
94°C20sec| 40X
55°C20sec|
72°C50sec|
   
72°C10min1X
15°C ∞
Amplicon detection
Amplicon detection
  • 5μl of amplicon is loaded into the well of a 1.5% agarose gel (TAE or TBE buffer according to your own protocols) and electrophoresed for at least 20min (depending on gel size) at 100-120V.
  • Be sure to include at least one known positive sample and a no-template control and load at least one well per gel row with a 100 base-pair ladder (or whatever may be in use at your lab for use with this sized amplicon).


Figure 1. Two examples experiments showing positive results using
the Gama assay (1 band per experiment) along with 100-bp ladder).