Method for rapid typification of microorganisms and set of reagents used

A process for rapid bacterial typing by Pulsed Field Gel Electrophoresis (PFGE) performed in 7 to 13 hours and including the separation of bacterial DNA restriction fragments by electrophoresis in minichambers of CHEF (Contour Clamped Homogeneous Electric Field) system, electrophoresis performed in 1.5% agarose gels and 0.5×TBE buffer (44.5 mM Tris, 44.5 mM Boric Acid, 1 mM EDTA, pH 8.3);

• process wherein the improvement comprises a step for preparing, in agarose miniplugs, intact and immobilized DNA molecules from bacterial cells of the species Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus, wherein said step of preparation further comprises steps of:
• i) washing, embedding and incubating the bacterial cells and the DNA molecules in a plurality of lytic and protease enzymes-free solutions, composed by chemical reagents;
• wherein said solutions are:
• (1) NaCl salt, at a concentration of 0.15M, and the metal chelating agent EDTA, at a concentration of 0.01M and pH 8.0;
• (2) NaCl salt at a concentration of 0.15M;
• (3) low melting agarose at a concentration of 1.5% suspended in NaCl solution at a concentration of 0.15M;
• (4) low melting agarose at a concentration of 1.5% suspended in the solution that contains NaCl salt at a concentration of 0.15M and the metal chelating agent EDTA at a concentration of 0.01M and pH 8.0;
• (5) the metal chelating agent EDTA at a concentration of 0.1M, two anionic detergents Sarkosyl and Nonidet P-40 both at a concentration of 1%, and 0.01M Tris base, pH 8.0;
• (6) the metal chelating agent EDTA at a concentration of 0.1M, the two anionic detergents Sarkosyl and Nonidet P-40 both at a concentration of 1%, 0.01M Tris base, and 4M Urea, pH 9.5;
• (7) the metal chelating agent EDTA at a concentration of 0.1M and 0.01M Tris base, pH 8.0;
• ii) embedding said bacterial cells in agarose miniplugs by means of a flexible and sterilizable mold which has a lid and various square depressions stamped in one of its surfaces;
• mold that is flexible enough to be bent for detaching said miniplugs from it and is reusable after its sterilizing;
• and
• iii) casting in the mold said agarose miniplugs containing cells of one of said bacterial species, detaching said miniplugs from the mold, and incubating and washing the miniplugs with the solutions composed by chemical reagents.