人A组G2P[4]型轮状病毒的分离培养和鉴定

Isolation, culture and identification of a group A human rotavirus G2P[4] strain

目的对我国轮状病毒A组G2P[4]流行株进行分离培养和鉴定。方法采用轮状病毒抗原胶体金法筛查56份病毒性腹泻患儿粪便标本,对阳性样本VP7和VP4基因测序确定其G和P基因型别,并接种至MA-104细胞,持续传代。运用SDS-PAGE电泳、实时荧光定量PCR、间接免疫荧光、嗜斑实验和电镜等技术进行鉴定。结果检出轮状病毒阳性样本46份,测序确定G2P[4]型2株,细胞培养盲传5代,嗜斑纯化后G2P[4]型毒株RNA PAGE电泳图呈DS-1株轮状病毒的(4:2:3:2)形态,电镜下呈车轮状形态,轮廓清晰,病毒滴度随时间变化逐步上升。结论成功从粪便样本中分离和培养出G2P[4]型A组轮状病毒。

Objective Isolation,culture and identification of the prevalent strain of rotavirus G2P[4]in China.Methods The stool samples of 56 children with viral diarrhea were tested for rotavirus antigen by colloidal gold method.The positive samples were inoculated into MA-104 cells and maintained continuous propagation.The strains were identified by SDS-PAGE,real-time PCR,indirect immunofluorescence,plaque test and electron microscopy.Results A total of 46 rotavirus positive samples were detected.Sequencing identified 2 strains of G2P[4].After blind passage of 5 generations and plaque purification,the PAGE electrophoregram of the RNA of G2P[4]strain showed patterns similar to DS-1 strain of rotavirus(4:2:3:2).The virus showed a clear morphology of wheel under electron microscope.The growth kinetic curve of the virus increased gradually with time.Conclusions Successful isolation and culture the strain of group A rotavirus G2P[4]from fecal samples.