Division 5 WSSV Polymerase Chain Reaction (PCR)

The method to be used for the detection of WSSV shall be the protocol for the detection of WSSV by nested PCR set out in the following paragraphs which amplifies a 1 447 bp and an 848 bp amplicon of the 18s rRNA gene in the first and second round PCRs respectively.

The 1st round PCR reaction shall be set up in a 50 μl volume which contains final concentrations of 1 × GoTaq Buffer (Promega), 5 mM MgCl₂, 1 pmol/μl WSSV 146 F1 primer, 1 pmol/μl WSSV 146 R1 primer (Table 1), 0,25 mM dNTPs, 1,25U Taq polymerase and 2,5 μl of DNA. Each sample shall be run in duplicate alongside a negative extraction control, a negative PCR control, (2,5 μl H₂O added instead of DNA) and a positive control. The positive control shall be diluted WSSV plasmid made and validated for use in-house (available from the EURL).

The 2nd round WSSV PCR reaction shall be set up in the same way as the first round but using the WSSV 146 F2/R2 primer set and using a second positive control to check that this stage of the PCR has worked.

Both 1st and 2nd round PCRs shall be run using the following cycling conditions on a DNA Engine Tetrad 2 Peltier Thermal Cycler (or equivalent). An initial denaturation step of 94 °C for two minutes followed by 94 °C for 30 seconds, 62 °C for 30 seconds, 72 °C for 30 seconds repeated for 30 cycles, a 72 °C extension step for two minutes and held at 4 °C.