Detection for kobuvirus and other emerging diarrhea viruses
The presence of MuKV was detected by PCR using universal primers according to the previous description, UNIV-kobu-F/UNIV-kobu-R, targeting a 217-bp fragment of partial 3D region for all kobuvirus strains (Reuter et al., 2009). Two sets of primers for a longer 3D gene (461bp) and complete VP1 gene (831bp) of MuKV were also used in order to examine the nucleotide sequences among the 217-bp 3D gene-positive samples, in line with the previous research (You et al., 2020). Each PCR mixture was conducted in a total volume of 25μl, including 12.5μl Green Master Mix (Promega, USA), 8.5μl sterilized H2O, 2μl cDNA template and 1.0μM each of the primers. The mixtures were amplified by 95℃ for 2min, 40 cycles of 95℃ for 30s, 56℃ for 1min, 72℃ for 1min, with a final elongation step at 72℃ for 5min. The amplified products were analyzed by 1.0% agarose gels electrophoresis and further sequenced using ABI Prism 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Moreover, kobuviruses have found to be related to gastroenteritis and considered as one of the emerging diarrhea viruses in recent years, containing Bocavirus, Salivirus, Cosavirus, Bufavirus, Parechovirus and Tusavirus (Finkbeiner et al., 2008); we thereby screened for other emerging diarrhea viruses among these samples according to PCR assays previously reported (Kapoor et al., 2010; Kapoor et al., 2008; Phan et al., 2014; Shan et al., 2010; Yang et al., 2016).