【摘要】 目的从妇幼保健院腹泻患儿临床样本中分离鉴定一野生型人A组轮状病毒株,并对其毒力进行测试。方法采集水样粪便样品,应用A组轮状病毒特异性抗体采用点杂交(Dot blot)方法检测,感染vero细胞并用TRIzol法提取病毒RNA,SDS-PAGE电泳分析病毒RNA迁移型式,以轮状病毒VP7通用引物扩增特异性鉴定,体内毒力分析。结果Dot blot显示粪便中轮状病毒阳性,用通用引物PCR能扩增出轮状病毒特异条带,RNA电泳迁移型式为4:2:3:2。感染病毒株的vero细胞病变显著,实验鼠水样性腹泻严重,生长缓慢。结论分离获得的病毒株属野生型人A组轮状病毒,具有较强的感染性和致病性。更多还原

【Abstract】 Objective To isolate and characterize a native human Group A rotavirus(h RA) from stool samples in Women’s and Children’s Hospital and test its virulence.Methods The watery stool sample was collected from a suspected h RA infected infant with severe diarrhea and dehydration,and the stool sample was subjected to detection by dot blot assay.Next,the fecal suspension was employed to infect VERO cells,and cell supernatants were collected by centrifugation and virus stocks were prepared by ultrafiltration.The ds RNA of virus was isolated from virus stocks using TRIzol reagent.The total ds RNA was subjected to migration pattern assay and reversely transcribed to c DNA.A h RA-specific consensus primer for VP7 was used to amplify VP7 DNA fragment.Finally,7-day newborn C57 mice were challenged with virus stocks by gavages.Results The stool sample from a suspected h RA infected infant was rotavirus positive,and a specific VP7 DNA fragment could be amplified by PCR.RNA denaturing urea PAGE assay showed a migration pattern of 4:2:3:2.In vivo,the virus could infect and lead to severe diarrhea and growth blockade in the newborn C57 mice.Conclusion Taken together,we successfully isolated a wide-type human Group A rotavirus,and this isolated h RV showed a high efficacy of infection in newborn C57 mice.更多还原