Molecular identification of Candida lusitaniae using a PCR-REA method

A pair of primers selected from the costant region of the ERG11 gene of C. albicans, was used to amplified a 350 bp segment from the genomic DNA of C. lusitaniae strain. The PCR product was cloned in plasmid pCR-TOPO (Invitrogen) and sequenced (GenBank submitted). The sequence of the fragment was compared with the published gene sequences of C. albicans, C. tropicalis, C. krusei, C. glabrata, C. guillermondi, C. dubliniensis, C. parapsilosis and C. kefyr. The homology range observed was 62% to 97% among all species. The amplicon obtained from C. lusitaniae was therefore subjected to REA using Sau3A, HincII and Not I according to a precedent study in which restriction enzyme analysis (REA) of PCR product was used to identify Candida strains at the species level. The REA pattern obtained allowed to differenziate C. lusitaniae from the other species of Candida except from C. glabrata whose patterns were very similar. Thus we tested another restriction enzyme, RsaI, with all species of Candida previously described. The results showed that only C. lusitaniae and C. krusei had a recognition site for this enzyme but the patterns were different from each other . The use of Rsa I for PCR-REA, in addition to the other enzyme, makes the identification of C. lusitaniae possible. An early and rapid identification of this species in clinical specimens is of great importance because C. lusitaniae is an emerging human pathogen that can cause severe infections in immunocompromised hosts.