3.3.1. PCR amplification of repetitive elements

Polymerase chain reaction (according to Genersch and Otten, 2003):

  1. Carry out PCR reactions in a final volume of 25 μl consisting of 1 × reaction buffer and a final concentration of 2.5 mM MgCl2, 250 μM of each dNTP, 10 μM of primer, and 0.3 µg of Hot start Taq polymerase. Five to ten µl of template DNA is added to the reaction.
  2. The cycling conditions are: an initial activation step at 95°C for 15 min, 35 cycles at 94°C for 1 min, 53°C for 1 min, and at 72°C for 2.5 min, and a final elongation step at 72°C for 10 min.
  3. Analyse five µl of the PCR reaction by electrophoresis on 0.8 % agarose gel in TAE or TBE buffer.
  4. Stain the amplified bands by incubation of the agarose gel in ethidium bromide (0.5 μg/ml in water) for 30 min.
  5. Visualize under UV light and photograph using a digital camera.
  6. Compare obtained fingerprints visually or using specific analysis programs.

Independent PCR reactions should be performed using primers ERIC1R/ERIC2, BOXA1R and MBO-REP1 (Table 3).

Modifications of the present protocol, such as those reported by Alippi et al. (2004) or Antúnez et al. (2007) also resulted useful, allowing the differentiation of P. larvae genotypes.

Interpretation of the results:

  • ERIC1R-ERIC2 primers: four different genotypes (ERIC I, II, III and IV) can be distinguished (Genersch et al., 2006).
  • BOXA1R primers: three different patterns can be found in Europe (A, a and ) (Genersch and Otten, 2003) while four patterns can be found in America (A, B, C and D) (Alippi et al., 2004).
  • MBO REP1 primers: four band patterns can be found in Europe (B, b, β, Б) (Peters et al., 2006).