Multiplex RT-PCR for Rotavirus Genotyping

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)].

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Gutierrez M.B., Fialho A.M., Maranhão A.G., Malta F.C., de Andrade J.D., de Assis R.M., Mouta S.D., Miagostovich M.P., Leite J.P, & Machado Fumian T. (2020). Rotavirus A in Brazil: Molecular Epidemiology and Surveillance during 2018–2019. Pathogens, 9(7), 515.

Publication 2020

One Step RT-PCR kit ExoSAP clean-up kit

Agarose Equine Genes Genotypes Multiplex pcr Nucleotide sequence Primers Rt pcr Strains

Corresponding Organization : Fundação Oswaldo Cruz

Top 5 similar protocols

Variable analysis

independent variables

RT-qPCR assay for RVA-positive samples
One-step multiplex RT-PCR for G- and P-genotyping
Use of forward conserved primers VP7uF or VP4uF and specific reverse primers for G and P types
Sanger sequencing for characterizing nucleotide sequences of non-typeable samples or equine-like G3 strains
Use of consensus primers directed to conserved regions within the VP4 and VP7 genes

dependent variables

G-genotypes (G1, G2, G3, G4, G9, and G12)
P-genotypes (P[4], P[6], P[8], P[9], and P[10])
Nucleotide sequences of specific RVA strains

control variables

Use of the Qiagen One Step RT-PCR kit
Adherence to the recommendations by the Centers for Disease Control and Prevention, USA, for primer selection
Use of agarose gel analysis for assigning G- and P-genotypes based on amplicon sizes
Use of the ExoSAP clean-up kit for purifying amplicon fragments prior to Sanger sequencing
Utilization of the FIOCRUZ Institutional Platform for DNA sequencing (PDTIS)

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