19份手足口病临床标本的病原学分离与鉴定

目的：为探索手足口病的病原并为该病的防治提供科学依据,对本省徐州地区采集的19份手足口病临床标本进行病原学分离与鉴定。方法：采集的患者咽拭子（6份）和肛拭子（13份）标本,接种Hep-2细胞进行病毒分离,经培养3代,对有明显致细胞病变效应并能连续传代的毒株,用肠道病毒通用引物real-time-RT-PCR方法鉴定确认为肠道病毒后,用肠道病毒分子分型引物（E187/E222）对肠道病毒VP1区进行扩增,将扩增产物测序获得的序列与GeneBank中肠道病毒核苷酸序列进行比对,确定毒株血清型别。结果：19份标本中分离到9株病毒,均为肠道病毒。对其VP1区部分核苷酸序列测定和比较分析后,鉴定出其中有6株为CoxB5,1株为CoxB3,2株为Echo6。结论：虽然肠道病毒71型（EV71）和柯萨奇病毒A16（CoxA16）是引起手足口病暴发流行的主要病原,但引起手足口病的肠道病毒存在多种血清型,CoxB5、CoxB3和Echo6等均能引起手足口病。因此,对手足口病的病原学检测,除EV71和CoxA16外,不能忽视对其他肠道病毒的病原学检测和鉴定。

Isolation and identification of pathogens from 19 hand-foot-mouth disease clinical specimens

Objective：In order to explore the cause of hand-foot-mouth disease（HFMD）and provide the scientific basis for the prevention and control of this disease,we isolated and identified pathogens from 19 HFMD clinical specimens collected from Xuzhou.Methods：Viruses were isolated from 6 throat swabs and 13 rectal swabs using Hep-2 cell line,and were cultivated at least three passages until cytopathic effects（CPE）were observed.The viral RNAs were extracted from cultured supernatant.RT-PCR was performed with two primer sets（universal enterovirus primers and E187/E222 for enterovirus VP1 sequence）and then the VP1 PCR products were sequenced and aligned to Genebank to determine the serotypes of the viruses.Results：A total of nineteen specimens（6 throat swabs and 13 rectal swabs）were used for virus isolation and identification.Nine enterovirus strains（47.4%）were successfully isolated,of which six are CoxB5（66.7%）,two Echo6（22.2%）and only one CoxB3（11.1%）.Conclusion：Although EV71 and CoxA16 viruses are the major agents causing epidemic of HFMD,several other enteroviruses including CoxB5,CoxB3 and Echo6 can also induce the same disease.Therefore,besides EV71 and CoxA16,the enhanced surveillance of other enteroviruses should be necessary for more effective prevention and control of HFMD.