VP7 and VP4 genotyping approach
Nucleic acid was extracted from stool or rectal swab specimens (19).
Specimens with the lowest RT-qPCR Ct value (corresponding to higher
viral load) were subsequently subjected to VP7/VP4 genotyping using
conventional RT-PCR and electrophoretyping with genotypes being
determined by banding pattern (29) with updated G12 primers (30)
(Supplemental Methods). A subset of specimens that could not be
genotyped due to polymorphisms at the primer binding sites or low viral
load, were subjected to an alternative sequence-based method of G/P
genotyping specifically designed for low viral load samples (15)
(Supplemental Methods). The sequences obtained were analyzed using
MEGAv7 software and genotypes were aligned to reference sequences
available in GenBank (31).