Throat swabs or nasopharyngeal aspirations were placed in virus transport medium and submitted to the virus diagnostic laboratory as soon as they were obtained, year-round. Viral cultures of laboratory-confirmed adenoviruses were processed as described previously.8 (link) The adenoviruses were subcultured in A549 cells when 85% cytopathic effect was observed. The cells were then harvested for DNA extraction.
Viral DNA was extracted according to a modified procedure as previously described.9 (link) DNA sequencing of hexon and fiber genes of respiratory adenovirus was carried out as previously described.7 (link) Briefly, the Loop1 region of the hexon gene was amplified with primer pair HXL1F (5′-CGTGTGCAGTTYGCCCG) and HXL1R (5′-ACAGCCTGATTCCACAT). The Loop2 region of the hexon gene was amplified with primer BL (5′-TTGACTTGCAGGACAGAAA) and BR (5′-CTTGTATGTGGAAAGGCAC). PCR mixtures consisted of 1U of DNA polymerase (KOD Plus Polymerase, Toyobo), 1 mM MgSO4, 0.2 mM dNTP, 300 pm of each primer, and 1 to 2 μL of template from the original purified DNA solution in a 50-μL reaction volume. DNA sequencing analysis of PCR products was performed using Sanger method.
Viral DNA was extracted according to a modified procedure as previously described.9 (link) DNA sequencing of hexon and fiber genes of respiratory adenovirus was carried out as previously described.7 (link) Briefly, the Loop1 region of the hexon gene was amplified with primer pair HXL1F (5′-CGTGTGCAGTTYGCCCG) and HXL1R (5′-ACAGCCTGATTCCACAT). The Loop2 region of the hexon gene was amplified with primer BL (5′-TTGACTTGCAGGACAGAAA) and BR (5′-CTTGTATGTGGAAAGGCAC). PCR mixtures consisted of 1U of DNA polymerase (KOD Plus Polymerase, Toyobo), 1 mM MgSO4, 0.2 mM dNTP, 300 pm of each primer, and 1 to 2 μL of template from the original purified DNA solution in a 50-μL reaction volume. DNA sequencing analysis of PCR products was performed using Sanger method.
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