Development of multiplex PCR diagnostic kits for detection of candida species in oral rinse solution

AIM: Various amount of microogranism live in healthy human flora containing yeast fungi. Disturbance in the balance of colonization can cause yeast colonisation or intraoral candidiasis. Candida species (Candida albicans, Candida tropicalis, Candida krusei, Candida parapsilosis and Candida dubliniensis) are the most frequent species known in oral microflora. Antifungal treatment and epidemiological aspects are very important in the correct assessment of Candida. In the presented research the design of a new Real-time PCR method is aimed in order to detect the medically important Candida species by polymerase reaction (PCR). MATERIAL METHOD: 200 students aged between 19 and 36 from Çukurova University Faculty of Medicine participated in this study. Each student rinsed their mouth with 15 ml of distilled water, samples were centrifuged 10 times at 3000 rpm. The pellets were suspensed in 200 µL sterile distilled water and inoculated into CHROMAgar Candida medium. Genomic DNA isolation was done with the Fungal Mini preparation kit. CHROMAgar Candida medium, MALDI TOF MS and RT-PCR was used for the identification of isolations. Two pairs of specific primers were used for gene amplification. Analysis was performed targeting ITS-2 sequences of 7 different Candida reference strains. In the presented study, a new method of RT PCR-MCA method that works with real-time polymerase chain reaction (RT-PCR) and melting curve analysis (MCA) was developed for the detection of Candida species. Results: A total of 71 yeasts were isolated from 52 (26%) clinical specimens. Of these 71 yeasts, 64 (90.1%) were Candida spp. and the isolated species, by frequency, were 22 C. albicans (34.4%), 16 C. parapsilosis (25%), 9 C. dubliniensis (14.1%), 6 C. lusitaniae (9.4%), 5 C. kefyr (%) 7.8), 2 C. lambica and 4 (6.9%) other Candida species [1 C. krusei, 1 C. tropicalis, 1 C. inconspicua and 1 C. pararugosa]. MALDI-TOF MS data for a total of 64 Candida isolates were compared with CAC and two separate RT-PCR results. On CAC medium, 1 strain of C. tropicalis formed a green colony and was incorrectly identified as C. albicans. Six Candida species isolated in the study were identified by MCA method by RT-PCR method, but two species, C. albicans and C. kefyr, could not be distinguished from each other according to Tm temperature. In addition, two isolates identified as C. lambica by MALDI-TOF MS could not be amplified by RT-PCR. Conclusion : It may take 2-3 days for Candida species to be identified and differentiated from each other by traditional methods. Melting temperature (MCA) analysis of Candida species after real-time PCR within one day is a simple, inexpensive and fast method. However, in this study, it was understood that it was not sufficient for the definitive diagnosis of Candida species. Key words: Candida, chromogenic agar, identification, MALDI-TOF MS, PCR, Real time PCR