Reports of Biochemistry and Molecular Biology

en Quantification and Optimization of Candida albicans DNA in Blood Samples Using Real- Time PCR میکروب شناسی Microbiology 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 100-106 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) DH5α.1 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 100% specificity. The minimum limit detected was one C. albicans cell or 100 CFU/ml (10 fg) per PCR reaction. The real-time PCR efficiency rate for Candida was high (E = 1.95). Melting curve analysis of C. albicans showed a specific melting peak temperature of 65.76 °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. Candida albicans, Invasive candidiasis, Real-time PCR 42 47 http://rbmb.net/browse.php?a_code=A-10-1-19&slc_lang=en&sid=1 Mojtaba Nabili 10031947532846005965 10031947532846005965 No Department of Medical Parasitology and Mycology, Invasive Fungi Research Center (IFRC), Mazandaran University of Medical Sciences, Sari, Iran - Social Security Organization, Golestan, Iran Mohsen Ashrafi 10031947532846005966 10031947532846005966 No Department of Medical Parasitology and Mycology, Invasive Fungi Research Center (IFRC), Mazandaran University of Medical Sciences, Sari, Iran Ghasem Janbabaie 10031947532846005967 10031947532846005967 No Department of Internal Medicine, Cell and Molecular Biology Research Center, Mazandaran University of Medical Sciences, Sari, Iran Mohamad Taghi Hedayati 10031947532846005968 10031947532846005968 No Department of Medical Parasitology and Mycology, Invasive Fungi Research Center (IFRC), Mazandaran University of Medical Sciences, Sari, Iran Kamran Ali-Moghaddam 10031947532846005969 10031947532846005969 No 4Hematology-Oncology Research Center and Stem Cell Transplantation Research Center (HORCSCT), Tehran University of Medical Sciences, Iran Tahereh Shokohi shokohi.tahereh@gmail.com 10031947532846005970 10031947532846005970 Yes Department of Medical Parasitology and Mycology, Invasive Fungi Research Center (IFRC), Mazandaran University of Medical Sciences, Sari, Iran