Exosap clean up kit

Norovirus-positive samples obtained by RT-qPCR were subjected to conventional RT-PCR for dual genotyping of polymerase and capsid regions. The reactions were performed using the Qiagen One-Step RT-PCR kit (Qiagen, Foster City, CA, USA) with primers Mon 432/G1SKR for GI and Mon 431/G2SKR for GII that target the ORF1/2 junction region [25 (link),26 (link)]. The generated amplicons fragments of GI (543 nucleotides (nt)) and GII (557 nt) were purified using the ExoSAP clean-up kit (ThermoFisher Scientific, Waltham, MA, USA) or the QIAquick Gel Extraction Kit (Qiagen, Foster City, CA, USA) and sent to the FIOCRUZ Institutional Platform for Sanger sequencing (PDTIS). Chromatogram analysis and consensus sequences were obtained using Geneious Prime 2020.1.2 (Biomatters Ltd., Auckland, New Zealand). Norovirus genotypes were firstly assigned based on the new nomenclature system using the two norovirus typing tools (https://www.rivm.nl/mpf/typingtool/norovirus and https://norovirus.ng.philab.cdc.gov).

RVA-positive samples obtained by RT-qPCR were G- and P-genotyped using a one-step multiplex RT-PCR. The reactions were performed using the Qiagen One Step RT-PCR kit (Qiagen), using forward conserved primers VP7uF or VP4uF and specific reverse primers for G types G1, G2, G3, G4, G9, and G12, or P types P[4], P[6], P[8], P[9], and P[10] as recommended by the Centers for Disease Control and Prevention, USA. The G- and P-genotypes were assigned based on different amplicon sizes [base pairs (bp)] using agarose gel analysis. Sanger sequencing was also used to characterize the nucleotide (nt) sequence of specific strains, such as non-typeable samples or the equine-like G3, using consensus primers directed to the conserved regions within the VP4 and VP7 genes. The amplicons fragments of 876 bp and 881 bp for VP4 and VP7, respectively, were purified using the ExoSAP clean-up kit (ThermoFisher Scientific) and sent to the FIOCRUZ Institutional Platform for DNA sequencing (PDTIS). All primers used for RVA genotyping were based on previously studies [32 (link),33 (link),34 (link)].

Norovirus-positive samples obtained by RT-qPCR were subjected to conventional one-step RT-PCR for dual genotyping of polymerase and capsid regions. The reactions were performed using the Qiagen One Step RT-PCR kit (QIAGEN, Hilden, Germany) with primers Mon 432/G1SKR for GI and Mon 431/G2SKR for GII, that amplifies the ORF1/2 junction region [33 (link),34 (link)]. The generated amplicons fragments [543 and 557 base pairs (bp) for GI and GII, respectively were purified using the ExoSAP clean-up kit (ThermoFisher Scientific, Waltham, MA, USA) or the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany). Purified DNA were Sanger sequenced in both directions at the FIOCRUZ Institutional Platform for DNA sequencing (PDTIS) on an ABI Prism 3730xl genetic analyzer (Applied Biosystems, Waltham, MA, USA).
Norovirus consensus sequences were obtained after nucleotide (nt) alignment and editing using Geneious prime 2021.2.2 (Biomatters Ltd., Auckland, New Zealand), and genotypes were confirmed in terms of closest homology sequence, using Basic Local Alignment Search Tool (BLAST v. 2.15.0). Norovirus genotypes were firstly assigned based on the new nomenclature system using the two norovirus typing tools https://www.rivm.nl/mpf/typingtool/norovirus and https://nrovirus.ng.philab.cdc.gov (accessed on 10 October 2023).