Molecular diagnosis of Candida albicans using real-time polymerase chain reaction of a CaYST1 gene

AbstractCandida albicans is an opportunistic pathogen that causes infections in immuno-compromised individuals. Virulence of this organism is a function of a multiplicity of factors working jointly to overwhelm the host defenses. From a previous study, the in vitro adherence capabilities of seven isolates (collected from patients suffering vaginitis) to vaginal epithelial cells were tested. Isolate No. 2 recorded the highest adherence ability. In this part of work, Real-time PCR assay was applied for the identification of isolate No. 2. This isolate could be discriminated by species-specific primers, deduced from the intron nucleotide sequence of the C. albicans CaYST1 gene. To confirm that the isolated strain is a fungal strain, the universal fungal primers (ITS5 and ITS4) were used to amplify the complete internal transcribed spacer (ITS) region including the 5.8S ribosomal gene that is present in all fungi. The ITS region was successfully amplified from the tested yeast, provided a single PCR product of approximately 520 bp. In another run, a single pair of specific primers [Intron Nucleotide Sequence (INT1 and INT2)] was then used to amplify the intron nucleotide sequence of the CaYST1 gene, which present only in C. albicans. The generated amplified product gave the expected single band of 310 bp. The melting points of all PCR products were routinely determined. The results showed that the melting temperatures for the tested isolate and the reference strain of C. albicans (ATCC 10231) using ITS5 and ITS4 primers were 80.09°C and 83.99°C, respectively, while they were 80.78°C and 80.10°C using INT1 and INT2 when assayed individually. Based on their very close melting profiles, the tested isolate proved to be identical to C. albicans reference strain. These results supported the idea of using genes containing intron sequences to design species-specific primers for the identification of fungal strains by real time PCR