Background
Invasive aspergillosis (IA) is an important cause of morbidity and mortality in immunocompromised individuals. This study was conducted to identify a desirable target DNA sequence for the diagnosis of aspergillosis using real-time quantitative polymerase chain reaction (qPCR).
Conclusions
The use of qPCR with whole blood to detect and verify the 28S-ITS2 sequence is a specific and useful way to diagnose IA.
Methods
Genomic DNA was extracted from Aspergillus, Candida, and bacteria species, and qPCR was applied to validate a partial ribosomal DNA 28S-ITS2 sequence. Ethylenediaminetetraacetic acid-anticoagulated blood samples were collected from 72 febrile hematological patients, while total DNA was isolated from plasma and whole blood for the Aspergillus qPCR. The
Results
Use of qPCR yielded positive results for 15 Aspergillus species but negative results for Candida species, bacterial strains, and human DNA. The limit of detection was one copy per microliter of DNA. Analytical sensitivity and specificity were six copies of DNA and 100%, respectively. The standard curve showed that qPCR was reliable for Aspergillus detection and that significantly more DNA copies were obtained from whole blood than from plasma (P < 0.001). At a cut-off value ≥ 25 copies/μL, the diagnostic sensitivity and specificity for IA using 28S-ITS2 qPCR were 90.9% and 73.4%, respectively. Conclusions: The use of qPCR with whole blood to detect and verify the 28S-ITS2 sequence is a specific and useful way to diagnose IA.