Genotype-based differentiation of the Cryptococcus neoformans serotypes by combined PCR-RFLP analysis of the capsule-associated genes CAP10 and CAP59

SUMMARY This report describes a method for indirect identification of the Cryptococcus neoformans serotype developed using combined restriction enzyme pattern analysis of two PCR-amplified portions of the capsule-associated genes CAP10 and CAP59. The method relies on the possibility to recognize the sequence conformation of nine serotype-related polymorphic sites by the analysis of four restriction profiles. A 610 nucleotides long trait of the CAP10 gene was digested with the enzymes Sty I or Sal I and a 597 nucleotides long trait of the CAP59 gene was digested with the enzymes Sal I or EcoRV+PstI. The resulting profiles, reported as a string of four numbers, defined for each strain an intrinsically coherent allelic profile closely correlated to the serotype. We analyzed by this method 172 C. neoformans strains obtained from different source. All the serotype A strains examined and all the strains of the B-C serotypes group were recognized by specific allelic patterns. The serotypes B and C could not be distinguished from each other. Of the serotype D strains, 84% were characterized by a unique allelic pattern, while the remainders 16% were genotipically indistinguishable from the AD serotype organisms among which differences in the ploidy number and evidence of recombination could be recognized