TITLE: Hydrogen peroxide decontamination of eDNA dedicated material
AUTHORS: Marine Vautier, Cyrielle Galiegue

[DESCRIPTION]
The purpose of this protocol is to detail the material decontamination steps to be performed to remove all traces of DNA from environmental DNA (eDNA) dedicated material.

The use of concentrated hydrogen peroxide (H2O2) solution requires careful handling under a ventilated hood with personal protective equipment (gloves and protective gown and goggles).

Note: The autoclave only sterilises equipment and does not eliminate possible traces of DNA. Therefore, materials intended for eDNA sampling must follow a decontamination protocol such as the one proposed here.

[BEFORE_START]
Preparation of the material:
- Concentrated hydrogen peroxide solution H2O2 - to be diluted to a final concentration of 10%.
- Prepare all material to be decontaminated (removal of DNA contamination).
- Prepare a waste container for the diluted hydrogen peroxide solution.
-Personal protective equipment (gloves and protective gown and goggles).

Pre and post equipment cleaning:
- Specific workstation with ventilated hood

The following precautions should be taken:
- Wear personal protective equipment (gloves and protective gown and goggles) throughout the handling.
- All operations must be carried out under a special work station with a ventilated hood.


[GUIDELINES]
The main steps of the protocol are:

Material preparation
Material decontamination
Waste management

[STEPS]
SECTION: Material preparation
1. Pre and post equipment cleaning.

Preparation of all material to be decontaminated.

Provide a waste container to dispose of the hydrogen peroxide solution.

Personal protective equipment (gloves and protective gown and goggles) and ventilated hood.

SECTION: Material decontamination
2. The aim of this decontamination protocol is to eliminate all traces of DNA present on the material to be used for eDNA analysis.

Note: Handle under ventilated hood and wear personal protective equipment (gloves and protective gown and goggles).

Switch on the hood ventilation.

Dilute the concentrated hydrogen peroxide solution H2O2 with distilled water to a final concentration of 10%.

In the case of bottles or containers:

- It is possible to pour the concentrated hydrogen peroxide solution directly into the bottle or container and top up with distilled water to dilute the solution. Make sure that the bottle is not completely full so that it can be shaken well.
- Close and shake the bottle or container vigorously.
- Empty the diluted hydrogen peroxide into another bottle to be decontaminated (if applicable) or dispose of in a suitable waste container. Up to ten bottles can be decontaminated with the same hydrogen peroxide solution.
- Top up with distilled water before shaking again to remove any traces of hydrogen peroxide.
- Repeat this rinsing process 3 times.
- Dry and store the material in a clean room.

Note: After decontamination, do not touch the neck of the bottle and the inside of the caps, and ideally, dry in an incubator at 60/80°C.

For small equipment (filtration equipment, tubes...):

- Take a retention tray and immerse the small equipement in the diluted hydrogen peroxide solution for at least one hour.
- Thoroughly rinse  with distilled water (3 times).
- Dry and store the material in a clean room.

Note : Ideally, dry in an incubator at 60/80°C.

At the end of handling, provide a suitable waste container to dispose of the diluted hydrogen peroxide solution 10% H2O2.

 


SECTION: Waste Management
3. The waste container containing the hydrogen peroxide solution must be labelled and disposed of as hazardous liquid waste. Under no circumstances should this waste be released to the environment.