Evaluation of a Manual DNA Extraction Method Combined with In-House Aspergillus Real-Time PCR.

Abstract

Background: To improve the molecular diagnostic yield for Aspergillus spp. from respiratory samples, we developed and evaluated a new DNA extraction method directly from respiratory samples combined with in-house Aspergillus real-time PCR.

Methods: We developed a method using beads and resin, where a sample is centrifuged to separate the supernatant and pellet. The pellet undergoes bead beating, is mixed with the supernatant, washed with AL buffer and distilled water, followed by the addition of Chelex-100 resin and boiling to extract the DNA template from the supernatant. As a comparator method, the Qiagen kit was used. To evaluate the efficiency of DNA extraction, nucleic acids extracted by the two methods were tested using in-house Aspergillus real-time PCR, and the cycle threshold (Ct) values were compared. The evaluation was conducted using contrived sputum samples with reference strains and 100 clinical respiratory specimens.

Results: Using contrived sputum samples at various concentrations, for A. niger and A. flavus, the Ct values were significantly lower when nucleic acids were extracted using the beads plus resin method compared to the Qiagen kit. For clinical samples, the beads plus resin method demonstrated a higher sensitivity of 88% (44/50) for the pan-Aspergillus primer/probe set compared to 76% (38/50) with the Qiagen kit, and significantly lower Ct values were obtained with our method (p < 0.001).

Conclusions: We developed a DNA extraction method that outperforms a commercial kit, enabling effective molecular detection of Aspergillus spp. directly from respiratory specimens to aid in diagnosing invasive pulmonary aspergillosis.