手足口病病毒分离方法研究  

Study on ioslation of viruses from patients with hand-foot-mouth disease patients

作　　者：罗敏[1] 杨洪[1] 张海龙[1] 冼慧霞[1] 陈惠玲[1] 吴微[1] 姚相杰[1] 何雅青[1] 

机构地区：[1]深圳市疾病预防控制中心,广东深圳518055

出　　处：《中国热带医学》2012年第4期403-405,共3页China Tropical Medicine

基　　金：广东省医学科技基金(No.B2011288)

摘　　要：目的探讨影响手足口病病毒分离培养的可能因素以及方法的改进。方法取手足口病患儿的粪便、肛拭子或脑脊液、疱疹液、咽拭子,其中以粪便为主,经Hank’s平衡缓冲液(HBSS)重悬震荡后,离心取上清液接种于横纹肌肉瘤传代细胞(RD-A细胞),二次传代后通过肠道病毒致细胞病变效应(CPE)分析及EV71、CA16及肠道病毒通用试剂盒进行荧光定量PCR的方法来鉴定病毒分离效果。结果 RD-A细胞能够有效的分离EV71、CA16和其他肠道病毒,采样时间在发病3日内,病毒分离效果达到66.7%,3日以上,病毒分离效果仅有29.1%。未添加万古霉素、阿米卡星和制菌霉素的病毒分离,污染率达到75%,分离效率为13.6%,添加三种抑制细菌和霉菌的抗生素后,污染率达到3.93%,分离效率达到54.9%。结论采样时间在发病3日内,有效的抑制细菌及霉菌的生长,合理的细胞接种密度可以明显提高病毒的分离培养效率。Objective To explore the factors influencing the isolation of virus from hand-foot-month disease patients and improve the isolation rate.Methods The specimens including cerebral spinal fluid,vesicle fluid and throat swabs,were collected and inoculated into RD-A cells for isolation of enteroviruses（EV）.The isolates were identified by cytopathic effect test（CPE） and then the real time reverse transcription polymerase reaction（realtime RT-PCR） for EV,Enterovirus71（Enterovirus71,EV71） and Coxsacki virus16（CA16）.Results Human RD-A cells were highly susceptible to EV71,CA16 and other enterovirus.If the samples were taken within 72 hours of onset of the disease,the virus isolation rate could reach 66.7%,and it was only 29.1% in case of over 72 hours.With addition of vancomycin,amikin and nystatin in culture,the contamination rate of mciroaoganisms would reduced from 75% to 3.93%,and the isolation rate increased from 13.6% to 54.9%.Conclusions The viruses isolation rate can be greatly increased if the samples were taken in time,and effective inhition of bacterium and fungus.and properly cell inoculation density could be achieved.

关 键 词：手足口病 病毒分离 细胞病变效应 

分 类 号：R512.5[医药卫生—内科学]