title: Comparison of different methods of isolation of DNA of commonly encountered Candida species and its quantitation by using a real-time PCR-based assay.
abstract: Molecular diagnosis based on genomic amplification methods such as real-time PCR assay has been reported as an alternative to conventional culture for early detection of invasive candidiasis. However, a major limitation of the molecular method is the difficulty associated with breaking fungal cell walls since the DNA extraction step still requires more than half of a working day. It has been suggested that PCR detection of free template DNA in serum is preferred over the use of whole blood for the diagnosis of systemic candidiasis. In this study, two conventional procedures (the first [the HLGT method] consists of boiling sera in an alkaline guanidine-phenol-Tris reagent, and the second [the PKPC method] uses proteinase K digestion, followed by organic extraction) and three commercially available kits for DNA isolation were evaluated for sensitivity, purity, cost, and use of template for most clinically important Candida species in a TaqMan-based PCR assay. To optimize these procedures, we evaluated the effect of adding 0.5% bovine serum albumin to DNA extracts and found that it decreased the effects of inhibitors. The QIAamp DNA blood kit did significantly shorten the duration of the DNA isolation but was among the most expensive procedures. Furthermore, the QIAamp DNA blood kit proved to be as sensitive as the HLGT DNA isolation method for PCR amplification from 52 serum samples from hematology or oncology patients with clinically proven or suspected systemic Candida infections. All PCR-positive samples showed approximately the same Candida species load by both procedures (100% correspondence), whereas one discordant result was obtained between PCR and blood culture.
authors.affiliation: Department of Microbiology and Infectious Diseases, Institut Jules Bordet, Rue Héger-Bordet 1, 1000 Brussels, Belgium.
authors.forename: Younes
authors.initials: Y
authors.lastname: Maaroufi
authors.forename: Naïma
authors.initials: N
authors.lastname: Ahariz
authors.forename: Mireille
authors.initials: M
authors.lastname: Husson
authors.forename: Françoise
authors.initials: F
authors.lastname: Crokaert
mesh_terms: D002175:Candida / Q000235:genetics*; D004271:DNA, Fungal / Q000097:blood / Q000302:isolation & purification*; D016469:Fungemia / Q000175:diagnosis*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic
chemical_list: D004271:DNA, Fungal; D011933:Reagent Kits, Diagnostic