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

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 <or=15 copies/run and the linear range of quantitation 1.5 x 10(1) to 1.5 x 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 x 10(5)) were significantly higher than in adult patients (median 2.3 x 10(3)) and than in controls (all samples <or=1.7 x 10(3) copies/ml). A few immunosuppressed children had very high virus loads (up to 1.1 x 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.