Division 3 Equipment and environmental conditions

The diagnostic test used for OsHV-1 μvar detection and identification by PCR requires the equipment and environmental conditions classically used for PCR assays as follows:

• A closed hood equipped with an UV producing system to eliminate potential contaminations when preparing PCR mix.

• Two complete sets of pipettes (2 μl; 20 μl; 200 μl and 1 000 μl), the first one for DNA extraction, and the second one for PCR mix preparation.

• Three different pipettes: one pipette (2 μl) to dispense samples in PCR mix, one pipette (20 μl) for EB sampling and another pipette (20 μl) to load PCR products in agarose gels.

• Filter pipette tips (2 μl; 20 μl; 200 μl and 1 000 μl) for DNA extraction, PCR mix preparation and sample dispensing.

• Pipette tips (20 μl) to collect EB and to load amplification products in agarose gel.

• A thermal cycler to perform amplifications.

• A horizontal electrophoresis system for PCR products electrophoresis.

• An UV table to observe PCR products after agarose gel electrophoresis.

• A system to acquire pictures of the gels.

The manipulator must wear a lab coat and some gloves during all the different steps described bellow. Lab coat and gloves must be changed preferably after each main step: DNA extraction, preparation of PCR mix, sample dispensing, amplification and gel loading.

It is recommended to perform these different steps in different rooms. More particularly, amplification and gel loading/electrophoresis should take place in a room separate from DNA extraction, PCR mix preparation and DNA dispensing.