title: Quantification and optimization of Candida albicans DNA in blood samples using Real- Time PCR.
abstract: 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 10(0)-10(6) 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 10(0) 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.
  
  
  CONCLUSIONS
  The real-time PCR assay we developed is highly specific and sufficiently sensitive to detect the fungal load for early diagnosis of invasive candidiasis.
authors.affiliation: Department of Medical Parasitology and Mycology, Invasive Fungi Research Center (IFRC), Mazandaran University of Medical Sciences, Sari, Iran ; Social Security Organization, Golestan, Iran.
authors.forename: Mojtaba
authors.initials: M
authors.lastname: Nabili
authors.affiliation: Department of Medical Parasitology and Mycology, Invasive Fungi Research Center (IFRC), Mazandaran University of Medical Sciences, Sari, Iran.
authors.forename: Mohsen
authors.initials: M
authors.lastname: Ashrafi
authors.affiliation: Department of Internal Medicine, Cell and Molecular Biology Research Center, Mazandaran University of Medical Sciences, Sari, Iran.
authors.forename: Ghasem
authors.initials: G
authors.lastname: Janbabaie
authors.affiliation: Department of Medical Parasitology and Mycology, Invasive Fungi Research Center (IFRC), Mazandaran University of Medical Sciences, Sari, Iran.
authors.forename: Mohamad Taghi
authors.initials: MT
authors.lastname: Hedayati
authors.affiliation: Hematology-Oncology Research Center and Stem Cell Transplantation Research Center (HORCSCT), Tehran University of Medical Sciences, Iran.
authors.forename: Kamran
authors.initials: K
authors.lastname: Ali-Moghaddam
authors.affiliation: Department of Medical Parasitology and Mycology, Invasive Fungi Research Center (IFRC), Mazandaran University of Medical Sciences, Sari, Iran.
authors.forename: Tahereh
authors.initials: T
authors.lastname: Shokohi
keywords: Candida albicans; Invasive candidiasis; Real-time PCR