Quantification and Optimization of Candida albicans DNA in Blood Samples Using Real- Time PCR

Background: Candida albicans (C. albicans) is a major cause of candidaemia in people with impaired immunity. Blood culture is a “gold standard” for candidaemia detection but is time-consuming and relatively insensitive. We established a real-time PCR assay for C. albicans detection in blood by LightCycler PCR and melting curve analysis. Methods: Five milliliter blood samples from healthy volunteers were spiked with ۱۰۰-۱۰۶ C. albicans cells to determine the detection limit of our method. DNA was extracted from whole blood using glass beads and the QIAamp DNA Blood Mini Kit (Qiagen, Hilden Germany). DNA from C. albicans isolates were amplified with primers and inserted into Escherichia coli (E. coli) DH۵α.۱ cells with the TA cloning vector (Invitrogen). The plasmid was used for standardization and optimization. A quantitative PCR assay with the LightCycler amplification and detection system based on fluorescence resonance energy transfer (FRET) with two different specific probes was established. To assess the precision and reproducibility of real-time PCR the intra-assay precision was determined in six consecutive assays. Results: No cross-reactivity of the hybridization probes with the DNA of non-C. albicans species or human genomic DNA was observed, which confirmed its ۱۰۰% specificity. The minimum limit detected was one C. albicans cell or ۱۰۰ CFU/ml (۱۰ fg) per PCR reaction. The real-time PCR efficiency rate for Candida was high (E = ۱.۹۵). Melting curve analysis of C. albicans showed a specific melting peak temperature of ۶۵.۷۶ °C. Conclusion: The real-time PCR assay we developed is highly specific and sufficiently sensitive to detect the fungal load for early diagnosis of invasive candidiasis.

Keywords:

Candida albicans , Invasive candidiasis , Real-time PCR