McRAPD

The McRAPD approach has been developed and first published by our research group, for details see Plachy et al., 2005 (PubMed, reprint request).

Briefly, it relies on the possibility to analyze melting (denaturation) of a mixture of species-specific RAPD products. RAPD (Random Amplified Polymorhic DNA, also termed AP-PCR, i.e. Arbitrarily primed-PCR) is widely used for strain typing of many types of organisms. However, it has been scarcely used for species identification, although it has been demonstrated to show very good performance in this area (see e.g. Liu et al., 1996; Steffan et al., 1997). Most probably there are two reasons for this fact. Because of its extreme sensitivity to variation in reaction conditions, RAPD is known to show low inter-laboratory reproducibility. In addition, it requires laborious analysis and comparison of resulting fingerprints.

In our project we have overcome these drawbacks. Firstly, we have optimised the reaction conditions resulting in very low intra-species variability, albeit still satisfactorily for inter-species discrimination. This allows us to significantly reduce the problems with inter-laboratory reproducibility. We have also shown, that in vast majority of isolates it is possible to omit gel electrophoresis and laborious analysis of fingerprints by analyzing the melting of RAPD products and automatically comparing the resulting melting curves (McRAPD). We have also upgraded the technique with the availability of the High resolution melter HR-1 (Idaho Technology) and are continuing to expand its availability with the Rotor Gene 6000 (Corbett Life Science), which also offers the High-Resolution Melting Analysis (HRMA) option.

Therefore, we would like to invite everybody to cooperate in developing a shared database of McRAPD results. To facilitate such cooperation, we are providing the community with the following resources:

• how does it work altogether?
• optimised McRAPD protocol
• representative (average) melting curves for pathogenic yeast species
• software tool for automatic analysis and comparison of results

In case of interest, you are welcome to contact the principal investigator Dr. Vladislav Raclavsky, phone +420 58 563 9515, fax +420 58 563 2417.