PCR Amplification

The example of a Carlavirus-specific PCR assay (12) is set out below. However, there are numerous PCR assays available for potato viruses that may also be carried out from the cDNA produced using the method previously described.

The two primers used for the PCR were the NotI Poly dT primer (as used for production of cDNA) and a Carlavirus-specific primer (12), Carla-Uni, 5'-GGAGTAACYGAGGTGATACC-3' (where Y stands for a C or G nucleotide).

Make up the PCR master mix as follows:

Ingredient

Volume per PCR

H2O

14.8 |L

10X PCR buffer

2.5 |L

MgCl2 (25mM)

2.5 | L

dNTPs (10mM)

0.5 |L

NotI poly dT primer (5 pmol/pL)

1.0 |L

Carlavirus primer (5 pmol/pL)

1.0 |L

Taq DNA polymerase (5 units/pL)

0.2 | L

Transfer 22.5 ^L of this master mix to each of the PCR tubes, and add 2.5 ^L of cDNA to the reaction. Seal the tubes and perform amplification under the following conditions; 95°C for 5 min; 94°C for 30 s, 50°C for 1 min, 72°C for 1 min (35 cycles for each); and 72°C for 5 min.

After PCR amplification, perform electrophoresis of the products through a 1.5% (w/v) agarose gel in 1X TBE buffer and then stain with ethidium bromide at 0.5 ^g/mL (13).

Examine the gel for a band of approx 120 bp (dependent on the virus used), which indicates the presence of a Carlavirus. Include positive and negative controls to ensure that the assay is working and that contamination has not occurred (Fig. 1).

The range of potato viruses that can be detected include potato virus S, potato virus M (12), and potato latent virus (10). Further members of the Carlavirus genus can be detected in other plant species (12).

0 0

Post a comment