Quantitative detection and rapid identification of human adenoviruses

Abstract

We have established a method of quantitative detection and rapid identification of human adenoviruses (hAdVs). Using LightCycler PCR with a primer set, we were able to amplify 554 bp of the hexon gene from each of 51 prototype strains of hAdVs. The sensitivity of LightCycler PCR was 10 copies of hAdV DNA/reaction. When LightCycler PCR was performed using a set of primers, hAdV was positive for 74.4% (99 of 133) of conjunctivitis patients and for 27.3% (81 of 297) of respiratory infection patients. We also attempted to measure hAdV in the potentially contaminated eye drops used by patients, detecting 5.4 x 10(2) to 1.6 x 10(6) copies/ml of hAdV. We determined the 350-bp nucleotide sequence of the amplified hexon gene and compared it with the sequences of the 51 prototype strains. Phylogenetic analysis based on 350 bp of the hexon gene identified 99 positive conjunctival swabs as 24 cases of AdV type 3 (AdV-3), 14 cases of AdV-4, 1 case of AdV-8, 19 cases of AdV-19a, and 41 cases of AdV-37. The 81 sequences from pharyngeal or nasal mucus swabs were identified as 29 cases of AdV-2, 18 cases of AdV-1, 18 cases of AdV-5, 12 cases of AdV-4, 2 cases of AdV-37, 1 case of AdV-3, and 1 case of AdV-6. LightCycler PCR followed by phylogenetic analysis provides an effective tool for the rapid identification of hAdVs and for studying molecular epidemiology.

FIG. 1.

A standard curve plot of the 10-fold serial dilution of pAd8hxn. Linearity is observed throughout the range from 10¹ to 10⁸ copies/reaction.

FIG. 2.

Comparison of the nucleotide sequences of primers and those of 51 prototype strains. The nucleotide sequences of the pair of primers were well conserved in 40 serotypes. Only 11 serotypes showed one or two nucleotide substitutions in the primer region.

FIG. 3.

Evaluation of phylogeny-based identification. The 350-bp sequence of a partial hexon gene of the isolates was analyzed along with the sequences of prototype strains and the AdV-19a strain by the neighbor-joining method. The numbers at the nodes are percentages of 1,000 bootstrap pseudoreplicates containing the cluster distal to the node.

FIG. 4.

Phylogenetic analyses of AdVs from patients with conjunctivitis (A) and acute upper respiratory tract infection (B). The 350-bp sequence of a partial hexon gene of representative samples was analyzed by the neighbor-joining method together with the 51 prototype strains of hAdV and the AdV-19a strain. The numbers at the nodes are percentages of 1,000 bootstrap pseudoreplicates containing the cluster distal to the node.

FIG. 4.

Phylogenetic analyses of AdVs from patients with conjunctivitis (A) and acute upper respiratory tract infection (B). The 350-bp sequence of a partial hexon gene of representative samples was analyzed by the neighbor-joining method together with the 51 prototype strains of hAdV and the AdV-19a strain. The numbers at the nodes are percentages of 1,000 bootstrap pseudoreplicates containing the cluster distal to the node.