【摘要】 目的探讨肠道病毒(EV)中枢神经系统感染患儿脑脊液(CSF)中VP1序列分子分型研究的临床价值。方法2003年1月至2005年12月青岛大学附属医院和滨州医学院附属医院采用通用引物和VP1段分子分型引物分别对63例无菌性脑膜炎和脑炎患儿急性期和恢复期的CSF标本进行扩增,对VP1分型引物扩增阳性片段进行克隆、测序及分型研究。结果采用通用引物经2次PCR检测急性期患儿的CSF标本共47例(47/63,74.6%)阳性,恢复期仅3例(3/57,5.3%)阳性。采用分型引物经2次PCR检测通用引物扩增阳性的47例急性期的CSF标本共31例(62.0%)阳性,其中4例脑炎和20例脑膜炎进行了序列测定。测序结果通过BLAST软件进行同源性对比分析,发现同一型别内序列同源性在97%~99%,不同型别之间同源性只有74%~76%,其中COX B3(9例)、ECHO 12(7例)、ECHO 30(3例)、COX A7(2例)、COX A9(1例)、COX B5(1例)、COX A11(1例)。所有CSF进行病毒分离,11例(11/63,17.5%)阳性,阳性率明显低于VP1段分子分型的检测结果,除1例CSF标本病毒分离为ECHO 7的患儿,通用引物扩增阳性而VP1分型引物扩增失败外,其余血清型别与VP1段分子分型研究结果完全一致。结论EV是儿童无菌性脑膜炎和脑炎最重要的病原体;采用通用引物所建的RT-PCR是快速检测EV中枢神经系统感染的有效方法;分型引物扩增VP1部分序列并测序,与GENBANK中的EV标准毒株进行对比,根据基因序列的同源性进行EV分型,为EV的分型研究提供了新的方法和思路。更多还原

【Abstract】 Objective To investigate the methods of molecular typing of enteroviruses(EV) by VP1 sequencing from cerebrospinal fluid(CSF) of children with central nervous system infection.Methods The 63 CSF specimen of childhood with aseptic meningitis and encephalitis were detected by RT-PCR of both consensus primer and VP1 typing primer.The positive specimen by using VP1 typing primer were investigated by nucleotide sequence analysis.Results In 63 CSF specimen of aseptic meningitis and encephalitis,47 cases(47/63,74.6%)were positive by using consensus primer amplification to carry out twice RT-PCR in acute stage,3 cases(3/57,5.3%)were positive in 57 cases of convalescence.In 47 cases of amplification positive by using consensus primer,31 cases(62.0%)were positive by using typing primer,of which the EV VP1 nucleotide sequence of 4 cases of encephalitis and 20 cases of meningitis was searched.The results of RT-PCR and sequencing by BLAST software showed that the nucleotide homology was 97%~99% in identical type,while the nucleotide homology was 74%~76% in diversity type.The molecular typing of EV by VP1 sequencing were coxsackie B3(9 cases),echoviruses 12(7 cases),echoviruses 30(3 cases),coxsackie A7(2 cases),coxsackie A9(1 case),coxsackie B5(1 case),COX A11(1 case).Eleven cases(11/63,17.5%)were positive by viral isolation in CSF specimen of acute stage.The positive rate was significantly lower by viral isolation than that by RT-PCR and molecular typing.The VP1 typing was failure only in one case with meningitis,while the viral isolation was echoviruses 7.Conclusion EV is the most important cause in meningitis and encephalitis of children.The PR-PCR by consensus primer is a modus operandi in detection EV infection,while contrasting homology bath VP1 nucleotide sequencing and EV standard strain in GENBANK by BLAST provide a new approach for EV typing.更多还原