QUANTIFICATION AND OPTIMIZATION OF ASPERGILLUS FUMIGATUS DNA DETECTION IN BLOOD SAMPLE USING REAL TIME PCR

Background and purpose: Invasive aspergillosis (IA) is a serious opportunistic infection caused by various species of Aspergillus in immnucompromissed individuals. Basically, rapid and early diagnosis prevents IA progression. The purpose of this study was to quantify and optimize detection of Aspergillus fumigatus DNA in blood samples using Real Time PCR.
Materials and methods: Five milliliter blood samples of healthy volunteers were spiked with various dilutions of conidial suspension of Aspergillus fumigatus (10⁶ to 10¹). DNA was extracted from blood using glass beads and QI Amp DNA Blood Mini Kit. The primers and hybridization probes were designed to amplify the 18S rRNA genes using the Light Cycler system and Fluorescence Resonance Energy Transfer.
Results: All PCR reactions were negative with other species of fungal DNA with 100% specificity. Melting curve analysis showed the species- specific melting temperature patterns on 69.14^oC which helps differentiating Aspergillus fumigatus. The lower limit was determined 10¹ CFU (100 fg) or 10 conidia or cells per PCR reaction which indicates a high sensitivity.
Conclusion: Real-time PCR method employing hybridization probes is a highly specific and sensitive approach to determine the fungal load for early diagnosis and monitoring treatment.