Detection of Aspergillus With Real-Time Fluorescent Quantitative PCR

Abstract

Objective: To establish a real-time fluorescent quantitative PCR (FQ-PCR) detection system for aspergillus in order to quickly, accurately detect common aspergillus pathogens in clinically invasive fungal infections.Methods: Standard strains of cultured fungi were used to prepare spore suspension samples with a concentration of 1,000,000 spores/mL. DNA samples were extracted with a one-step lysing heating method (LHM). Primers and probes for aspergillus fumigatus (A. fumigatus), aspergillus flavus (A. flavus), aspergillus niger (A. niger), and aspergillus terreus (A. terreus) were designed with the Primer Premier 5.0 and Beacon Designer 7.7. The most effective primer probe was obtained after an FQ-PCR detection system screening which tested the efficiency and amplification effect for extracting aspergillus DNA via a one-step LHM, and verified specificity, repeatability, and sensitivity of the reaction system.Results: No non-specific amplification was found. Coefficient of variation (CV) of Ct values in repetitive intra- and inter-assay experiments were less than 5 % (0.37 % - 3.53 %). All four aspergillum sensitivities reached at least 35 spores/mL after the established reaction system was detected.Conclusion: 1. Samples were prepared with spores cultured from aspergillus standard strains. Aspergillus DNA was extracted with an LHM method and was applied to the real-time fluorescent quantitative PCR detection. 2. Primers and probes for aspergillus fumigatus, aspergillus flavus, aspergillus niger, and aspergillus terreus were successfully designed and screened. Real-time FQ-PCR detection system for aspergillus was established. It was highly specific, sensitive and repeatable.