2.3 G and P Genotyping
Each RVA-positive stool specimen confirmed by sequencing was performed with the semi-nested multiplex RT-PCR to detect six G genotypes and six P genotypes with primers shown in table 1. A detailed explanation is as follows: first round PCR reaction for G genotypes was amplified with primers of VP7-forward (F) and VP7-reversed (R) 24; and then G genotypes was performed with 2 μl first round PCR product and a mixture of 20 mmol of each specific primer for G1-G4, G8, G9 and VP7-R primer with the length of amplified products being 618, 521, 682, 452, 754 and 179 bp, respectively 25. Similarly, the first-round PCR reaction for P genotypes was conducted with primers of VP4-forward and VP4-reversed. P genotyping was performed with 2 μl of first-round PCR product and a mixture of 20 mmol each of internal primers for P[4], P[6], P[8], P[9], P[10], P[11], and VP4-F primer, with the length of the amplified products being 362, 146, 224, 270, 462, and 191 bp, respectively26. Thermal cycling of G or P genotypes was performed as follows: 94°C for 2 min, followed by 40 cycles at 94°C for 30 sec, 44°C for 30 sec, 72°C for 30 sec, and with a final extension at 72°C for 5 min. All amplified PCR products were electrophoresed on 2.0% agarose gels containing Super GelBlue (Shanghai BioScience) and visualized using an automated gel image analysis system (Shanghai Tanon Life Science).
All RVA G8 genotype amplicons were purified and sequenced for phylogenetic analysis using first-generation sequencing technologies (Shanghai Sangon Biotech). Phylogenetic trees were constructed using the maximum-likelihood method (Kimura two-parameter substitution model with 1000 bootstrap replicates for branch support) in MEGA (v6.0) software. The nucleotide sequences of the G8 genotype detected in this study were compared with the sequences of corresponding reference strains available in the GenBank database. The nucleotide sequences of the RVA strains and the accession numbers used were as follows: G1: HM560972; FJ348346; GU183204; EY033973. G2: U73941; KC152925; FJ460820. G3: FJ460831. G4: FJ386453; EF011974; GU183208. G4: FJ386453; EF011974; GU183208. G8: LC133530; LC169879; LC169956; LC169967; LC386070; MG996097; MN058740; MT410500; GU984761; D01054; AB749175; DQ005109; DQ005120; KJ748481; KP883176; KX632259; KX655456; KX655467; LC406824; MG181903; MK434791; MN921185; DQ995179; EF218677; JQ988904; MT232573; FJ611782; JN831225; KJ411440; MF940612; MG214342. G9: MW254156; MW384390; LC477377.