Quantification and optimization of detection Aspergillus fumigatus DNA in blood sample using Real Time PCR

Background and Ïnvasive aspergillosis (ÏÂ) is a serious opportunistic infection caused by various species of Âspergillus in immnucompromissed individuals. Basically، rapid and early diagnosis prevents ÏÂ progression. The purpose of this study was to quantify and optimize detection of Âspergillus fumigatus DNÂ in blood samples using Real Time PÇR. Five milliliter blood samples of healthy volunteers were spiked with various dilutions of conidial suspension of Âspergillus fumigatus (106 to 101). DNÂ was extracted from blood using glass beads and QÏÂmp DNÂ Blood Mini Kit. The primers and hybridization probes were designed to amplify the 18S rRNÂ genes using the LightÇycler system and Fluorescence Resonance Ënergy Transfer. Âll PÇR reactions were negative with other species of fungal DNÂ with 100% specificity. Melting curve analysis showed the species- specific melting temperature patterns on 69. 14oÇ which help to differentiate Âspergillus fumigatus. The lower limit was determined 101 ÇFÜ (100 fg) or 10 conidia or cells per PÇR reaction which indicates a high sensitivity. Çonclusion: Real-time PÇR method employing hybridization probes is a highly specific and sensitive approach to determine the fungal load for early diagnosis and monitoring treatment.