Detection of respiratory viruses in shelter dogs maintained under varying environmental conditions

Monteiro, Francielle Liz; Cargnelutti, Juliana Felipetto; Martins, Mathias; Anziliero, Deniz; Erhardt, Magnólia Martins; Weiblen, Rudi; Flores, Eduardo Furtado

RNA and DNA extraction from nasal swabs were performed using an RTP DNA/RNA virus extraction kit (Invitek, Hayward, CA, USA) according to the manufacturer's instructions. After RNA extraction, complementary DNA (cDNA) was synthesized using an enzyme Super Script III Reverse Transcriptase Kit (Life Technologies, Carlsbad, CA, USA). The PCR reactions were initially standardized to optimize the concentration of each reagent. Viruses obtained from two commercial vaccines were used as controls for CDV, CPIV and CAdV-2. For CaHV-1, nucleic acid extracted from the liver of a puppy naturally infected with CaHV-1 was used as a control. 17 Ultrapure water was used as a negative control in all reactions. The primers used in all reactions are described in Table 1 . All reactions were performed using a total volume of 25 L with 2 L of total DNA (100-200 ng) according to the PCR conditions described for each virus. Primers to CPIV were obtained using the Clone Manager 7 program (http://www.scied.com), and the sequences are shown in Table 1 For nucleotide sequencing, 90 L of each PCR product was purified using a PureLink ® Quick Gel Extraction and PCR purification Combo Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. Positive samples were randomly selected and sequenced in quadruplicates in an automatic sequencer ABI-PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The obtained sequences were analyzed using the Staden Package for consensus sequences achievement. 22 The matrix of sample identity with sequences deposited in GenBank was performed using the BioEdit