8.3.1. Qualitative PCR

Procedure (after McKee et al., 2003):

  1. Genomic DNA (5-30 ng) is amplified using a thermal cycler in a 50 µl reaction comprising:
    • 4 mM MgCl2 ,
    • 200 µM of each deoxyribonucleotide triphosphate,
    • 100 ng of primers MP1 and MP2 (Table 2),
    • 5 µl of 10 x PCR buffer (100 mM tris-HCl, pH 8.3; 500 mM KCL),
    • 2-5 µg of Taq DNA polymerase.
  2. Conditions for amplification consist of:
    • initial denaturation at 95o C for 2 min,
    • 40 cycles of denaturation (95o C, 30 s),
    • primer annealing (61o C, 15 s),
    • primer extension (72o C, 60 s),
    • final extension cycle (72o C, 5 min).
  3. Amplification products are analysed by electrophoresis (55 V, 1.5 h) through 1.0-1.5 % (wt / vol) agarose containing ethidium bromide. A 486 bp PCR product is produced from primers MP1 and MP2. To ensure test specificity; a second PCR following the same protocol (using primers MP1 and MP3) is conducted, and a specific 276 bp hemi-nested product is amplified from the 486 bp template.

 

Table 2. PCR-based methods for the detection of M. plutonius.

Table 2