Clc bio main workbench 7

Nucleotide sequences were aligned and primers were designed with the help of: CLC bio Main Workbench 7.7 (Qiagen Bioinformatics, Aarhus, Denmark), Blastn and Primer-Blast (NCBI), Clustal Omega (EMBL), PrimerQuest (Integrated DNA Technologies, Leuven, Belgium), Oligoevaluator (Sigma-Aldrich), FigTree v1.4.3 (Andrew Rambaut, University of Edinburgh). Statistical analysis was performed using Prism (GraphPad Software, La Jolla, CA). Qualitative variables definite as percentage were compared using Fischer’s exact test or Pearson’s Chi-square test. Quantitative variables were compared using Mann-Whitney U test. A two-tailed P value less than 0.05 was considered significant. Adobe Photoshop CS6 and Illustrator CS6 were used for image processing and alignment.

Oligonucleotide primers (Table S3) were designed using CLCbio Main Workbench 7.7 (Qiagen Bioinformatics, Aarhus, Denmark). The specificity of each primer was assessed using Primer-BLAST software (http://blast.ncbi.nlm.nih.gov/Blast.cgi). As reported^{37 (link)}, partially conserved regions of the EV genome were chosen: 5′UTR (untranslated region), 2 C (membrane- and RNA-binding, helicase), 3Dpol (RNA-dependent RNA polymerase). For poliovirus type-1, -2, -3, primers to the VP1-coding region were devised. Published primer pairs to the 5′UTR region were also used^{12 (link), 39 (link), 64 (link)}. pUC57 plasmids containing the whole 5′UTR sequence of CV-A6 (Hyogo9426 strain; A species), CV-B3 (Nancy strain; B species), PV-1 (Mahoney strain; C species), EV-D68 (SZ04/CHN/2015 strain; D species) where obtained from GenScript (Piscataway, NJ) and used to evaluate the sensitivity of PCR assays in terms of copy number/reaction. Twenty-six primer pairs (Table S3) were tested against select EV reference strains and clinical/environmental isolates belonging to the A, B, C, and D species in parallel with appropriate negative controls (Table S2). Positive results were confirmed by Sanger sequencing. Amplicons of viral isolates were identified based on best matches in public databases.