Rapid and quantitative detection of human adenovirus DNA by real‐time PCR

塔克曼 实时聚合酶链反应 病毒学 病毒血症 生物 聚合酶 底漆(化妆品) 聚合酶链反应 分子生物学 病毒 基因 化学 遗传学 有机化学 水热
作者
Albert Heim,Carmen Ebnet,Gabi Harste,Patricia Pring‐Åkerblom
出处
期刊:Journal of Medical Virology [Wiley]
卷期号:70 (2): 228-239 被引量:789
标识
DOI:10.1002/jmv.10382
摘要

Abstract Rapid diagnosis of human adenovirus (HAdV) infections was achieved by PCR in the recent years. However, conventional PCR has the risk of carry‐over contamination due to open handling with its products, and results are only qualitative. Therefore, a quantitative “real‐time” PCR with consensus primer and probe (dual fluorescence labelled, “TaqMan”) sequences for a conserved region of the hexon gene was designed and evaluated. Real‐time PCR detected all 51 HAdV prototypes. Sensitivity of the assay was ≤15 copies/run and the linear range of quantitation 1.5 × 10 1 to 1.5 × 10 8 copies/run. TaqMan PCR gave identical results compared to an established conventional one‐step PCR protocol in 218 (38 positive and 180 negative) of 234 clinical samples including blood, serum, eye swabs, and feces, and had divergent results in 16 samples (15 positive only in TaqMan PCR, all with low copy numbers, and one positive only in conventional PCR), indicating a higher sensitivity of TaqMan PCR. Adenovirus viremia was detected by TaqMan PCR in 4 of 27 (14.8%) paediatric and 8 of 93 (8.6%) adult stem cell transplant recipients but only in 5 of 306 healthy controls (blood donors, 1.6%). Virus loads of pediatric patients (median 1.7 × 10 5 ) were significantly higher than in adult patients (median 2.3 × 10 3 ) and than in controls (all samples ≤1.7 × 10 3 copies/ml). A few immunosuppressed children had very high virus loads (up to 1.1 × 10 10 copies/ml), which were associated with symptoms of disseminated disease. In conclusion, real‐time PCR is a sensitive and quantitative procedure for the detection of adenovirus infections. J. Med. Virol. 70: 228–239, 2003. © 2003 Wiley‐Liss, Inc.
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