2.3 G- and P-genotyping of RVA by multiplex RT-PCR and DNA sequencing
The cDNAs obtained from the RVA-positive samples were used as templates for multiplex RT- PCR to identify G- and P-genotypes. VP7 and VP4 genes were first amplified using the primer pairs Beg9/End9 or VP7F/VP7R and Con3/Con2, respectively. G- and P-genotypes were then identified using the specific primers for different G genotypes (G1, G2, G3, G4, G9, G12) and P genotypes (P[4], P[6], P[8], P[9], P[10]), respectively.29-31 In addition, a one-step multiplex genotyping strategy was perform to identify RVA strains that were incompletely genotyped.32 Finally, DNA sequencing was performed to identify the G- and P-genotypes of the remaining RVA strains that had not been successfully typed by PCR-based methods.