M gene analysis of canine coronavirus strains detected in Korea

Jeoung, Seok-Young; Ann, So-Yun; Kim, Hyun-Tae; Kim, Doo

Viral RNA was extracted from fecal samples using an RNeasy mini kit (Qiagen, Germany) according to the manufacturer's protocol for animal cells. The target sequence for amplification was located in a segment of the gene encoding the transmembrane protein M of CCV. The following primers were prepared [22, 28] : The amplicons were separated on a 2% agarose gel and visualized by staining with ethidium bromide (10 μL/mL; Sigma, USA). The PCR amplicons were purified with a commercial kit (QIAquick Gel Extraction Kit; Qiagen), and sequenced using the dideoxynucleotide chain termination method with the CCV1/CCV2 primer pair and a commercial kit (Big Dye terminator cycle sequencing kit; Applied Biosystems, USA). The nucleotide and deduced amino acid sequences of the M gene from 22 Korean CCV strains were determined and compared to those for 10 reference coronaviruses obtained from GenBank (National Center for Biotechnology Information, USA), and aligned by the Clusteral W multiple sequence alignment algorithm using commercial software (DNASTAR 5.0, MegAlign; DNASTAR, USA) for phylogenetic analysis (Tables 1 and 2) . Table 4 ).