Introduction: Adenoviruses (AdV) are DNA viruses that belong to the Adenoviridae family. At least 52 serotypes have been described with several genotypes within the same serotype. The high diversity of these viruses remains a big challenge for the development of a generic, sensitive and specific real time PCR assay.

In this sense, real time assays are more challenging because at least three areas of the target gene should match the sequences (primers and probe) to obtain an accurate detection signal.

Few mismatches in the studied area may give false negative results.

Objective: To optimize an already well studied assay and gene for AdV detection by Real Time PCR

Materials and Methods: A well conserved region of the hexon gene has been selected and studied for PCR methods since 1997. Modification of the original primers and designment of different probes was achieved. Initial probes for species A/C, B, D, E and F were optimized for this purpose.

The optimization included: a) redesignment of probes, b) annealing temperatures difference of at least 2ºC, c) distance between primers and probes d) avoid a guanine (G) adjacent to the reporter dye. The commercial kit (Adenovirus r-gene, Argene Inc) included the master mix with the optimized primers and probes.

Clinical validation included respiratory and ocular samples positive for AdV. Representative serotypes from all species were evaluated. Extraction was performed using QIAamp (Qiagen) extraction columns. A SmartCycler II (Cepheid) was used for the assay.

Results: All AdV strains tested with the optimized assay were detected with the commercial Real Time PCR. These included ATCC and clinical isolates strains from different countries.

All respiratory and ocular samples evaluated were correctly detected by the commercial Real Time PCR.

Results with the new probes showed higher fluorescence level and better crossing threshold allowing a better sensitivity compared to the original ones.

Conclusion: The commercial Real Time PCR assay (Adenovirus r-gene, Argene Inc) was able to detect in a sensitive and reliable way all AdV strains from clinical samples with optimal performance characteristics.