Aug 30, 2024

Public workspacePreparation, storage and use of the positive and negative controls for DDNS V.2

DOI
dx.doi.org/10.17504/protocols.io.8epv5r594g1b/v2
  • 1Imperial College London;
  • 2Medicines and Healthcare products Regulatory Agency
  • Poliovirus Sequencing Consortium
Joyce Akello
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DOI: dx.doi.org/10.17504/protocols.io.8epv5r594g1b/v2
Protocol Citation: Joyce Akello, Manasi Majumdar, Alex Shaw, Catherine Troman, Erika Bujaki, Javier Martin, Nick Grassly 2024. Preparation, storage and use of the positive and negative controls for DDNS. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5r594g1b/v2Version created by Joyce Akello
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: August 29, 2024
Last Modified: August 30, 2024
Protocol Integer ID: 106726
Funders Acknowledgement:
Bill and Melinda Gates Foundation
Abstract
This protocol describes the laboratory process for the use of run controls (positive and negative controls) for performing direct detection of poliovirus by nanopore sequencing (DDNS assay).

The protocol outlines the key steps of how and when to use the run controls for DDNS assay including preparation, and storage. The run controls are used to demonstrate that the entire DDNS workflow starting from RNA extraction to obtaining a sequence is successfully performed.

Positive control (Coxsackievirus A20)  – Supplied by MHRA, contact Dr. Erika Bujaki at erika.bujaki@mhra.gov.uk.  and Dr. Thomas Wilton at thomas.wilton@mhra.gov.uk.  and copy Dr. Javier Martin at javier.martin@mhra.gov.uk).

Before start



CVA20 positive control reconstitution
CVA20 positive control reconstitution
Reconstitution of the positive control

Note
The CVA20 positive control must be reconstituted before use. No attempt should be made to weigh out the freeze dried material

Safety information
Infectious material –  Handle virus in a Class II Microbiological Safety Cabinet (MSCII)

Critical
Working in the MSCII, reconstitute the vial containing the lyophilised CVA20 (freeze dried material) by adding 1mL of nuclease free water (NFW)



Vortex briefly to ensure that the material completely dissolves in water giving a colourless liquid



Aliquot the solution into single-use volumes of 30 µl in  1.5mL sterile eppendorf tubes (DNase/RNase free) and store all aliquots at -200C for future use.

Note
The operator must label the tubes with the date the positive control was reconstituted.
Aliquots of the resuspended CVA20 can be store at -20°C for up to 5 weeks. Aliquoting will reduce the chance of source contamination as well as help to preserve the stability of the CVA20 solution by reducing the number of freeze-thaw cycles of the entire solution.    


Use of the positive extraction control (CVA20) and negative extraction control during RNA extraction
Use of the positive extraction control (CVA20) and negative extraction control during RNA extraction
Prepare the CVA20 positive extraction control for RNA extraction as follows:
Note
Include a positive extraction control and a negative extraction control for every RNA extraction batch with new preparations of solutions.

Retrieve a 30 µl aliquot of the CVA20 from the -20°C freezer.
Allow to thaw at room temperature whilst in the MSCII cabinet
Briefly centrifuge for 5 secs.
Add 270 µl of NFW and pipette up and down to mix the solution. The solution is now ready for immediate RNA extraction .



Note
It is the responsibility of the operator to check that the batch positive control is added to the process.

Prepare the negative extraction control as follows:
Aliquot 300 µl of nuclease free water (NFW) into a sterile 1.5ml Eppendorf tube (DNase/RNase free) labelled ExNTC (extraction negative control)



Perform RNA extraction of the samples in parallel with the positive and negative controls.
The elutes / purified RNA from the controls and samples following RNA extraction can now be processed according to the DDNS protocol.
Note
The elutes /purified RNA should be kept on ice after extraction and while working with it. If the eluted RNA is not for immediate use, store at - 80°C