Real-time LightCycler polymerase chain reaction and melting temperature analysis for identification of clinically important Candida spp.
Ziauddin Khan, Abu Salim Mustafa, Fasahat Fakhar Alam
J Microbiol Immunol Infect. 2009;42:290-295.
Received: September 30, 2008 Revised: October 8, 2008 Accepted: November 7, 2008
Corresponding author: Ziauddin Khan, Department of Microbiology, Faculty of Medicine, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait. E-mail: firstname.lastname@example.org
Background and purpose:
Invasive candidiasis is a major fungal infection occurring in patients who have prolonged hospital admissions. The rapid detection and confirmation of candida spp. in clinical specimens is essential for efficient management and improved prognosis of these patients. The purpose of this study was to develop a real-time LightCycler polymerase chain reaction (PCR) assay for the identification of Candida spp. commonly associated with invasive infections.
Using the LightCycler PCR system, the targets of genomic DNA isoalted from the reference strains of 6 Candida spp. were amplifiied using genus- and species-specific primers, and detected in real-time employing SYBR Green fluorescent dye. The identity of Candida spp. was established by melting curve analysis. A similar analysis was performed with clinical isolates (n=72) previously identified by conventional methods.
The melting curve analysis of amplified DNA from the reference strains could differentiate between Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, Candida krusei, and Candida dubliniensis. The specificity of the real-time PCR assay was validated by testing 72 clinical isolates of Candida spp. with 100% concordance, as compared with conventional identification methods. The notable findings of the study were differentiation of C. krusei from all other Candida spp. tested and of C. dubliniensis from C. albicans by melting temperature analysis; the latter 2 species share common phenotypic characteristics of germ-tube formation and chlamydospore production, so are often misidentified.
Real-time PCR using LightCycler and melting curve analysis are reliable methods for rapid identification of 6 Candida spp. frequently associated with candidemia and invasive candidiasis.
Candida; Laboratory techniques and procedures; Polymerase chain reaction; Transition temperature