Department of Hematology and BMT center, Chinese PLA General Hospital, 28 Fuxing Road, Beijing 100853, China.
BMC Infect Dis. 2013 Jun 1;13:255. doi: 10.1186/1471-2334-13-255.
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).
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.
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.
The use of qPCR with whole blood to detect and verify the 28S-ITS2 sequence is a specific and useful way to diagnose IA.
侵袭性曲霉病（IA）是免疫功能低下个体发病和死亡的重要原因。本研究旨在通过实时定量聚合酶链反应（qPCR），寻找诊断曲霉病的理想靶 DNA 序列。
从曲霉属、假丝酵母属和细菌种属中提取基因组 DNA，并用 qPCR 验证部分核糖体 DNA 28S-ITS2 序列。从 72 例发热血液病患者中采集乙二胺四乙酸抗凝血液样本，从血浆和全血中提取总 DNA 进行曲霉 qPCR。采用受试者工作特征曲线进行分析。所有病例均采用修订后的欧洲癌症研究与治疗组织/侵袭性真菌感染合作组和美国国立过敏与传染病研究所真菌病研究组（EORTC/MSG）诊断标准进行评估。
qPCR 对 15 种曲霉属种产生阳性结果，而对假丝酵母属种、细菌株和人 DNA 产生阴性结果。检测限为每微升 DNA 一个拷贝。分析灵敏度和特异性分别为 6 个拷贝的 DNA 和 100%。标准曲线表明 qPCR 可可靠地用于曲霉属检测，且全血中获得的 DNA 拷贝数明显高于血浆（P<0.001）。在截值≥25 拷贝/μL 时，28S-ITS2 qPCR 诊断 IA 的灵敏度和特异性分别为 90.9%和 73.4%。
使用全血进行 qPCR 检测和验证 28S-ITS2 序列是诊断 IA 的一种特异性和有用方法。
