Species-specific identification of human adenoviruses by a multiplex PCR assay

Abstract

A multiplex PCR assay was developed by using primers to the fiber gene that could differentiate human adenovirus (Ad) species A through F in a single amplification reaction. The assay correctly identified the species of all 49 recognized Ad prototype strains as well as 180 geographically and temporally diverse Ad field isolates. Ad serotype 6 (Ad6) (species C), Ad16 (species B), Ad31 (species A), and Ad40 and Ad41 (species F) could also be distinguished by amplicon size within each respective species. In comparison, a previously described Ad species-specific multiplex PCR assay that used primers to the Ad hexon gene gave equivocal results with several serotypes of species B, whereas our multiplex assay amplified all species B serotypes equally well. Our multiplex PCR assay will permit rapid, accurate, and cost-effective classification of Ad isolates.

FIG. 1

Ethidium bromide-stained agarose gel showing PCR products from five different combinations of Ad species-specific primers. Lanes, from left to right:, respectively: M, molecular weight marker III (Boehringer Mannheim); species A-F, species A to F; A-D, F, species A, B, C, D, and F; B, D, E, species B, D, and E; B, C, E, species B, C, and E; B, C, D, species B, C, and D; Neg, template-free negative control. Numbers on the left are in base pairs.

FIG. 2

Ethidium bromide-stained agarose gel showing PCR products of 49 Ad prototype strains. Lanes, from left to right, respectively: M, molecular weight marker III (Boehringer Mannheim); P, pooled control DNAs of representative Ad serotypes of species A to F; 1 to 49, individual Ad serotypes; N, pooled control DNA without indicated Ad species. Numbers on the left are in base pairs.

FIG. 3

Ethidium bromide-stained agarose gel showing PCR products of the hexon (A) and fiber (B) multiplex assays. Lanes: M, molecular weight marker VI (Boehringer Mannheim); 1, isolate 98034069; 2, V-2064A; 3, 99018072; 4, V-2181; 5, V-2079A; 6, V-2167A; 7, RU-8176; 8, prototype Ad3; 9, Ad7; 10, Ad16; 11, Ad21; 12, Ad11; 13 Ad14; 14, Ad34; 15, Ad35; N, negative control. Both assays were performed with the same Ad DNA extracts, and identical results were obtained in two separate amplification reactions. Numbers on the left are in base pairs.