The Ribosomal DNA Restriction Fragment Length Polymorphism (RFLP) patterns of isolated Candida Spp. from King Chulalongkorn Memorial Hospital

Abstract

This study is to differentiate Candida spp. by using PCR-RFLP profiles of ITS-rDNA region. The ITS1 and ITS2 primers were used to amplify the region between ITS1 and 2 and then the obtained PCR product was digested by restriction enzymes. One hundred twenty clinical isolates of Candida were recruited. The result showed that the size of PCR product from C. glabrata (875 bp) and C. guilliermondii (608 bp) was distinct. In contrast, the PCR yield from other species revealed almost the same size of 500 bp approximately (C. albicans 536 bp, C. tropicalis 523 bp, C. parapsilosis 521 bp, C. dubliniensis 541 bp, C. krusei 510 bp). Hence, further experiment of PCR-RFLP using single restriction enzyme Hae III, Dde I and Tru9 I was performed. Based on the reference strains, it was possible to differentiate 120 isolates by PCR-RFLP as follows: 55C. albicans (45.8%), 26 C. tropicalis (21.7%), 19 C. parapsilosis (15.8%), 9 C. glabrata (7.5%), 6 C. dubliniensis (5.0%), 3 C. guilliermondii (2.5%) and 2 C. krusei (1.6%). The conventional and PCR-RFLP identification was concordant for 114 of 120 Candida isolates with 95% identity. The left six isolates were identified as C. albicans by phenotypic method and did not agree with their phenotypic identifications and C. dubliniensis by PCR-RFLP profiles. The PCR-RFLP profiles using single digestion by Hae III, Dde I and Tru9 I revealed the similar patterns to those from reference strains except the profiles from 40 samples of the enzyme Tru9 I digestion. These forty isolates were identified as C. albicans by other method and enzymes digestion. Then, the RFLP of the PCR product with Mbo I was performed. The results showed that all the RFLP profiles were identical to C. albicans reference strain profile. This polymorphism might due to the mutation of the base position at Tru9 I recognition site. In conclusion, the method of PCR-RFLP in the region between ITS1 and 2 is the rapid method with high accuracy and useful in differentiate Candida important yeasts.