TITLE: Preparation, storage and use of the positive and negative controls for DDNS
AUTHORS: Joyce Akello, Manasi Majumdar, Alex Shaw, Catherine Troman, Erika Bujaki, Javier Martin, Nick Grassly

[DESCRIPTION]
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).


[STEPS]
SECTION: CVA20 positive control reconstitution
1. Reconstitution of the positive control

 

 

SECTION: CVA20 positive control reconstitution
1.1. Working in the MSCII, reconstitute the vial containing the lyophilised CVA20 (freeze dried material) by adding 1mL of nuclease free water (NFW)

 

SECTION: CVA20 positive control reconstitution
1.2. Vortex briefly to ensure that the material completely dissolves in water giving a colourless liquid

 

SECTION: CVA20 positive control reconstitution
1.3.  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.

 


SECTION: Use of the positive extraction control (CVA20) and negative extraction control during RNA extraction
2.     Prepare the CVA20 positive extraction control for RNA extraction as follows:
 

SECTION: Use of the positive extraction control (CVA20) and negative extraction control during RNA extraction
2.1.  Retrieve a 30 µl aliquot of the CVA20 from the -20°C freezer.
SECTION: Use of the positive extraction control (CVA20) and negative extraction control during RNA extraction
2.2. Allow to thaw at room temperature whilst in the MSCII cabinet
SECTION: Use of the positive extraction control (CVA20) and negative extraction control during RNA extraction
2.3. Briefly centrifuge for 5 secs. 
SECTION: Use of the positive extraction control (CVA20) and negative extraction control during RNA extraction
2.4. Add 270 µl of NFW and pipette up and down to mix the solution. The solution is now ready for immediate RNA extraction .

 

 

SECTION: Use of the positive extraction control (CVA20) and negative extraction control during RNA extraction
3. Prepare the negative extraction control as follows:
SECTION: Use of the positive extraction control (CVA20) and negative extraction control during RNA extraction
4.  Perform RNA extraction of the samples in parallel with the positive and negative controls. 
SECTION: Use of the positive extraction control (CVA20) and negative extraction control during RNA extraction
3.1. Aliquot 300 µl of nuclease free water (NFW) into a sterile 1.5ml Eppendorf tube (DNase/RNase free) labelled ExNTC (extraction negative control)

 

SECTION: Use of the positive extraction control (CVA20) and negative extraction control during RNA extraction
5. The elutes / purified RNA from the controls and samples following RNA extraction can now be processed according to the DDNS protocol.