Terminal restriction fragments length polymorphism method (t-RFLP) is a modified classical RFLP method. The main difference is based on carrying out PCR using a fluorescently labeled primer or primers. The result of such modification is that after digestion of amplicons only terminal fragments of PCR product (containing fluorescent dye) are visualized. T-RFLP method has found application in environmental microbiology and epidemiology. It is mostly used for genotyping and study of microbial diversity and bacterial community structure in different environmental samples. In this study a method to differentiate six Candida species using PCR t-RFLP technique was elaborated. This work shows the new application of t-RFLP in molecular diagnostic of fungi. Homocitrate synthase gene was chosen as a molecular target. This gene encodes enzyme involved in the first reaction in lysine biosynthesis pathway. The protein is characteristic for fungi and several Archaea. Specific forward primer was labeled with fluorescein. During this study new sequence for homocitrate synthase gene fragment was obtained for C. krusei and submitted to NCBI GenBank. Three endonucleases were chosen for digestion of amplicons: DraI, RsaI and BglII. Electrophoretic pattern of bands obtained for three enzymes gives unequivocal result for determinig the Candida species. Designed method is flexible for differentiation of each combination of investigated Candida species and only one or two enzymes may be used for diagnostic purpose.
Authors
Additional information
- Category
- Inne
- Type
- supllement, wydanie specjalne, dodatek
- Language
- angielski
- Publication year
- 2015
