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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).    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 were analyzed using a receiver operating characteristic curve. All cases were evaluated using the revised European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) diagnostic criteria.    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.

bmc infectious diseases

Establishment and application of real-time quantitative PCR for diagnosing invasive Aspergillosis via the blood in hematological patients: targeting a specific sequence of Aspergillus 28S-ITS2