2.8. Southern or dot blot hybridization of kDNA PCR products of biopsies, isolates and Leishmania reference strains

The kDNA PCR products of clinical samples, Mexican isolates and reference strains, were Southern or dot blotted onto nylon membranes and were hybridized with the cloned fragments of kDNA used as probes: B4Rsa, which hybridizes specifically to members of the L. donovani complex; 9.2 and 9.3, specific for the L. mexicana complex; and B18, specific for members of the L. braziliensis complex. The probes were labeled with DIG Random Primer DNA labeling kit (Boehringer Mannheim) and either visualized colorimetrically with NBT and BCIP (Boehringer Mannheim) or labeled with [32P]d ATP, using the Prime-it™ Random Primer DNA labeling kit (Stratagene). The hybridization conditions were described elsewhere [14, 17].
