Protocol

DNA extraction

Prior to McRAPD, any extraction procedure yielding DNA suitable for PCR amplification can be used. Of course, low amount or insufficient purity of template DNA can interfere with the technique. In-house procedures should therefore be tested to provide reproducioble results before McRAPD is used for routine identification.

To accelerate the identification, we use a simple toothpick technique described by Steffan et al. (1997). This technique performs well when done by experienced staff. Commercial kits can be recommended when this technique fails to provide reproducible results.

Briefly:

• pick a part of a 24-hrs old colony on SGA using a sterile micropipettet barrier itp and transfer it into 5 ul (microliters) of freshly prepared lysing solution (1 M sorbitol, 5 mM MgCl2, 2 mM dithiothreitol, 12 U of Lyticase [Sigma-Aldrich])
• incubate the mixture at 37 °C for 30 min
• spin at 10,000 g for 5 min
• transfer the supernatant into a new tube and dilute it with TE buffer to 300 ul
• samples can be stored at -20 °C until further used, however, because of the relatively low DNA concentration and other contaminatintg compounds, performance can deteriorate with time.

Useful hints:

Test each batch of Lyticase for satisfactory performance. Be careful not to pick up traces agar when picking the colony - this will interfere with PCR amplification. When DNA yield is not satisfactory for amplification, dilution volume can be decreased to increase the DNA concentration (e.g. 50 ul instead of 300 ul).

PCR amplification

We use a Rapid Cycler 2 machine (Idaho Technology) to perform McRAPD. Results of amplification can differ on other platforms and the protocol may need to be optimised in order to use our database for identification.

Briefly:

• set up a 10 ul reaction mixture in LightCycler glass capillaries, which includes 1 ul of DNA extracted as described above, 0.4 uM primer 5'-ACG GGC CAG T-3' (Liu et al., 1996), 200 uM dNTPs (each), 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 0.1% Triton X-100, 2.0 mM MgCl2, 250 ng/ul BSA, 1x LCGreen I or LCGreen Plus (Idaho Technology), 2.5 U Taq polymerase
• perform amplification at the following conditions:
  • initial denaturation at 95 °C, 5 min
  • 45 cycles of denaturation at 95 °C, 5 s;
  • annealing at 48 °C, 10 s
  • slope 1 °C/s
  • extension at 72 °C, 40 s
  • followed by final extension at 72 °C, 5 min,

High-resolution melting analysis (HRMA)

We use a High Resolution Melter HR-1 (Idaho Technology). Results of HRMA can differ on other platforms and the protocol may need to be optimised in order to use our database for identification.

Briefly:

• heat the sample at ramping rate of 0.3 °C/s with acquisition of fluorescence data ranging from 75 to 95 °C,
• normalise the acquired data using the HR-1 analysis software with cursor No. 1 at 75.5 °C, No. 2 at 77.5 °C, No. 3 at 91.5 °C and cursor 4 at 93.5 °C
• export the graph data for further processing using our software tool

Contact us in case you need any detailed comments.