Detection of Candida species in environmental sources

The identification and classification of yeasts has been based on morphological, physiological and biochemical properties. In recent years, different molecular biological techniques have been developed for yeast identification. A simple �·widely used but sUfficiently sensitive method is restriction analysis of amplified fragments (PCR-RFLP). Previous studies have produced reference records for Candida species and other yeast. However this reference library is not complete. In this study, PCR using ITS1 and ITS4 primers to amplify the ITS1, ITS2 and the 5.88 rRNA gene, and RFLP using C'ol, Haelll and Hinfl were used with 19 yeasts isolated from 21 soil and grass samples. Among these yeasts, PCR products varied between 400 bp to 650 bp. The restriction patterns could not all be identified due to limitations in the siz~ of the reference . database. Real-time PCRbased methodology has proved to be a sensitive detection system to both identify and quantify the level of microorganisms. In this study, real-time PCR using the F152 and R152 primer pair, targeting amplification of the 188 rONA, allowed for the differentiation of Candida and Saccharomyces, based on the Tm peak of the resultant products whilst the sensitivity of real-time PCR was as low as 500 femtogram of DNA whereas the sensitivity of conventional PCR was 500 picogram of DNA. Real-time PCR could be used to rapidly detect the presence of Candida species in environmental samples to a concentration of approximately 5 cellslcm3 �� The real-time PCR assay described detects Candida species in about 3 hours, which is faster than conventional culture methods. DNA isolation kits proved to be able to get rid of inhibitors in dirty samples but also inhibit the probe function in this study. The kits were only applicable for use with SYBR Green assay. By using DNA isolation kits, there were less positive real-time PCR results for Candida than cultivation followed by manual DNA extraction methodology. This investigation illustrates that the Tm values from SYBR Green assay may act as useful screening technique for Candida identification. The Cp value from probe assay is a confirmation technique as Cp values above 24 can be eliminated. The samples with Cp values below 24 were iden.tified as Candida species whilst those with Cp values above 24 were identified as other yeasts. PCR-RFLP can be used as an identification method for a number of Candida species but for Candida albicans and Candida tropicalis the results are inconclusive. Candida albicans and Candida tropicalis cannot be distinguished by RFLP or real-time PCR whilst morphological test such as germ tube production were also inclusive. However, sequence results clearly distinguished between these two species.EThOS - Electronic Theses Online ServiceGBUnited Kingdo