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目的了解2007年5—6月北京市儿童中流行的手足口病的病原。方法于2007年5、6月间采集来首都儿科研究所附属儿童医院门诊就诊的手足口病患儿咽拭子标本6份和1份病情危重的手足口病并发神经系统症状而急诊收住院患儿(编号F4243,年龄9岁8个月,女性)的经气管插管呼吸道抽吸液、脑脊液及血清标本。将咽拭子和气管插管抽吸液标本接种Hep-2、MDCK和Vero细胞进行病毒分离。同时设计位于肠道病毒5’非编码区(UTR)的通用引物2对(巢式PCR)、肠道病毒71型(EV71)VP1基因不同位置的特异性引物2对半(PCR和半巢式PCR)和柯萨奇病毒A组16型(CA16)的特异性引物1对检测标本中的病毒核酸。首先用随机引物将标本中的RNA逆转录成cDNA,然后分别用上述引物进行PCR扩增肠道病毒5’UTR和EV71/CA16的VP1基因片段;对EV71阳性的标本还扩增了VP1全基因,扩增产物直接进行序列测定和分析。结果6例门诊就诊的普通手足口病患儿和F4243的呼吸道标本中均扩增到肠道病毒5’UTR基因片段,有2例门诊患儿和F4243的标本中还扩增到EV71的VP1基因片段,提示该组手足口病患儿均为肠道病毒感染,其中3例为EV71感染。序列测定和分析表明有1例普通手足口病患儿(F4211)和F4243标本确定为EV71。所有标本接种Hep-2和MDCK细胞均未出现病变,说明常见的呼吸道病毒为阴性。除1份咽拭子标本(F4283)外,其余6份接种Vero细胞的标本均出现细胞病变。用肠道病毒通用引物和EV71特异性引物对分离到的病毒株核酸的扩增结果显示这6株病毒均为肠道病毒,其中F4211和F4243为EV71,与直接从呼吸道标本中扩增的结果相符;其余4株为CA16。结论北京市儿童中流行的手足口病与肠道病毒EV71和CA16感染有关;EV71可以在5岁以上儿童中引起严重的神经系统并发症,应引起注意。
Objective To understand the pathogen of HFMD among children in Beijing from May to June in 2007. Methods In June and June 2007, 6 throat swab specimens of HFMD patients attending the Children’s Hospital Clinic affiliated to Capital Institute of Pediatrics were enrolled in this study, and 1 critically ill HFMD complicated by neurological symptoms were admitted to hospital (Intrathecal F4243, age 9 years and 8 months, females) with endotracheal intubation aspirate fluid, cerebrospinal fluid and serum samples. Throat swabs and endotracheal aspirates were inoculated into Hep-2, MDCK and Vero cells for virus isolation. At the same time, two pairs of universal primer pairs (nested PCR) and VP2 gene of enterovirus 71 (EV71) at different positions of the enterovirus 5 ’non-coding region (UTR) PCR) and coxsackievirus A group 16 (CA16) specific primer pair 1 of the detection of viral nucleic acid samples. Firstly, the RNA in samples was reverse transcribed into cDNA using random primers, and then the VP1 gene fragments of 5 ’UTR and EV71 / CA16 of enterovirus were amplified by PCR. The EV71 positive samples also amplified VP1 gene , Amplified products were directly sequenced and analyzed. Results The sequences of 5’UTR gene of enterovirus were amplified from 6 outpatients with common hand-foot-mouth disease and respiratory tract specimens from F4243. The VP1 gene of EV71 was amplified from 2 outpatients and F4243 Fragment, suggesting that the group of hand, foot and mouth disease in children are enterovirus infection, of which 3 cases of EV71 infection. Sequence analysis and analysis showed that 1 case of normal hand-foot-mouth disease (F4211) and F4243 specimens were identified as EV71. All specimens were inoculated with Hep-2 and MDCK cells without lesions, indicating that the common respiratory virus is negative. Except for 1 throat swab specimen (F4283), the remaining 6 samples of Vero cells were cytopathic. Amplification of the nucleic acid of the virus strains isolated using universal primers for enterovirus and EV71-specific primers revealed that all of the 6 viruses were enteroviruses, of which F4211 and F4243 were EV71, which was directly associated with the amplification from respiratory specimens Consistent; the remaining 4 strains were CA16. Conclusion Hand-foot-mouth disease prevalence among children in Beijing is related to enterovirus EV71 and CA16 infection. EV71 may cause serious neurological complications in children over 5 years old, and should be noted.