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Amplification was performed using a thermal cycler (iQ5™ Real-Time PCR Detection System, Bio-Rad Laboratories). The PCR conditions were the same for HAdV and EV, and performed as follows: a denaturation step of 95 °C for 5 min followed by a two-step cycling protocol including a denaturation step at 95 °C for 15 s and an annealing/extension 60 s step at 55 °C for HAdV and 56 °C for EV. This step was repeated for 40 cycles in both cases, a melting-curve analyses were performed to verify product specificity (from 55 to 95 °C, 15 s each step). The sequences of the primers, their location in the viruses’ genomes and the conditions used for amplification are given in Table 1.

Table 1

Oligonucleotides and conditions used for amplification of AdV and EV in qPCR

VirusTarget geneOligonucleotide
PositionAnnealing Temp. (°C)Amplicon (bp)
NameSequencePolarity
AdVa Hexon VTB2-HadVCr 5'-GAGACGTACTTCAGCCTGAAT-3' Forward 106–126 55°C 102 
VTB2-HAdVCr 5'-GATGAACCGCAGCGTCAA-3' Reverse 190–207 
EVb 5'UTR ENT-F1 5'-CCTCCGGCCCCTGAATG-3' Forward 443–459 56°C 117 
ENT-R2 5'-ACACGGACACCCAAAGTAG-3' Reverse 541–559 
VirusTarget geneOligonucleotide
PositionAnnealing Temp. (°C)Amplicon (bp)
NameSequencePolarity
AdVa Hexon VTB2-HadVCr 5'-GAGACGTACTTCAGCCTGAAT-3' Forward 106–126 55°C 102 
VTB2-HAdVCr 5'-GATGAACCGCAGCGTCAA-3' Reverse 190–207 
EVb 5'UTR ENT-F1 5'-CCTCCGGCCCCTGAATG-3' Forward 443–459 56°C 117 
ENT-R2 5'-ACACGGACACCCAAAGTAG-3' Reverse 541–559 

aPrimer sequences reported by Wolf et al. (2010).

bPrimer sequences modified from those described by Tsai et al. (1993).

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