2.4 Viral DNA extraction, Polymerase Chain Reaction and
nucleotide sequencing
Viral DNA was extracted from a 30% stool suspension by using the TRIzol
method (Invitrogen, Waltham, MA) according to the manufacturer’s
instructions. PCR assay with Platinum® Taq DNA Polymerase kit
(Invitrogen, USA) was used for amplification, using hexon gene specific
primers for AdV detection (Allard et al., 1990) and penton gene specific
primers for genotyping (Fujimoto et al., 2012). The nucleotide sequences
were determined by using the Big Dye Terminator cycle sequencing kit
v3.1 (Applied Biosytems, Foster City, CA, USA) and an ABI 3130XL genetic
analyzer. The specificity of the nucleotide sequences obtained was
confirmed using BLAST
(www.ncbi.nlm.nih.gov/blast)
and phylogenetic analysis was carried out using the neighbor joining
method with 1000 bootstrap replicates (Tamura et al., 2013). The
nucleotide sequences of the strains examined in the study have been
deposited in the Gen Bank (Accession numbers OP699436- OP699483;
OP939983- OP939984; OP939989- OP939995) for partial penton and hexon
gene (Accession numbers OP939985-OP939988).