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Applied and Environmental Microbiology, June 2005, p. 3131-3136, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.3131-3136.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Quantitative Real-Time PCR Assays for Detection of Human Adenoviruses and Identification of Serotypes 40 and 41

Narayanan Jothikumar,1,2,3* Theresa L. Cromeans,1,2,3 Vincent R. Hill,1 Xiaoyan Lu,1 Mark D. Sobsey,3 and Dean D. Erdman1

Centers for Disease Control and Prevention, Atlanta,1 Atlanta Research and Education Foundation, Decatur Georgia,2 University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-74313

Received 23 June 2004/ Accepted 3 January 2005

A quantitative real-time TaqMan PCR assay for detection of human adenoviruses (HAdV) was developed using broadly reactive consensus primers and a TaqMan probe targeting a conserved region of the hexon gene. The TaqMan assay correctly identified 56 representative adenovirus prototype strains and field isolates from all six adenovirus species (A to F). Based on infectious units, the TaqMan assay was able to detect as few as 0.4 and 0.004 infectious units of adenovirus serotype 2 (AdV2) and AdV41, respectively, with results obtained in less than 90 min. Using genomic equivalents, the broadly reactive TaqMan assay was able to detect 5 copies of AdV40 (which had zero mismatches with the PCR primers and probe), 8 copies of AdV41, and 350 copies of AdV3 (which had the most mismatches [seven] of any adenovirus serotype tested). For specific detection and identification of F species serotypes AdV40 and AdV41, a second real-time PCR assay was developed using fluorescence resonance energy transfer (FRET) probes that target the adenovirus fiber gene. The FRET-based assay had a detection limit of 3 to 5 copies of AdV40 and AdV41 standard DNA and was able to distinguish between AdV40 and AdV41 based on melting curve analysis. Both the TaqMan and FRET PCR assays were quantitative over a wide range of virus titers. Application of these assays for detection of adenoviruses and type-specific identification of AdV40 and AdV41 will be useful for identifying these viruses in environmental and clinical samples.

* Corresponding author. Mailing address: Centers for Disease Control and Prevention, 4770 Buford Highway, Mailstop F-36, Atlanta, GA 30341. Phone: (770) 488-3776. Fax: (770) 488-4253. E-mail: JIN2{at}cdc.gov.

Applied and Environmental Microbiology, June 2005, p. 3131-3136, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.3131-3136.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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