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目的利用分子生物学检测技术,对2012年5月以来发生在广东省粤西地区罗定市的一起病毒性脑炎疫情进行快速的病原体检测和分子型别的鉴定。方法利用人肠道病毒通用荧光定量RT-PCR对病例的32份脑脊液(CSF)标本和7份粪便标本进行人肠道病毒的核酸快速筛查,再利用半巢式聚合酶链反应(Sn-PCR)对人肠道病毒阳性标本的部分VP1区进行扩增,并对扩增产物进行核苷酸序列测定和分析,使用分子定型方法对人肠道病毒进行型别鉴定。结果人肠道病毒通用荧光定量RT-PCR筛查结果显示32份CSF标本中有26份阳性,7份粪便标本全部阳性;阳性率分别为81.3%和100.0%。对33份阳性标本进行Sn-PCR扩增,22份显示有目的条带,测序结果显示15份CSF标本中13份为埃可病毒(ECV)30、2份为ECV9;7份粪便标本中6份为ECV30、1份为ECV9。结论发生在2012年广东省粤西地区罗定市的一起病毒性脑炎疫情是由以ECV30为主的人肠道病毒引起的;荧光定量RT-PCR法与Sn-PCR法结合核苷酸序列测定和分析可以对人肠道病毒脑炎进行快速检测和病原型别鉴定。
Objective To detect rapid pathogen detection and molecular type identification of a viral encephalitis epidemic in Luoding, Guangdong Province, western Guangdong since May 2012, using molecular biological detection techniques. Methods 32 samples of cerebrospinal fluid (CSF) and 7 samples of stool were collected for rapid detection of human enteroviruses by RT-PCR with a universal real-time fluorescent quantitative RT-PCR. Semi-nested polymerase chain reaction (Sn- PCR) to amplify a portion of the VP1 region of human enterovirus positive samples. The nucleotide sequences of the amplified products were determined and analyzed. The molecular typing method was used to identify the human enteroviruses. Results The RT-PCR results of human enterovirus showed that 26 of 32 CSF samples were positive, and 7 of them were all positive. The positive rates were 81.3% and 100.0% respectively. Sn-PCR amplification was performed on 33 positive samples, 22 of which showed objective bands. Sequencing results showed that 13 out of 15 CSF samples were ECV 30 and 2 ECV9; 1 for ECV30 and 1 for ECV9. Conclusions A viral encephalitis epidemic occurred in Luoding City, Guangdong Province, Guangdong Province, in 2012 was caused by human enterovirus (ECV30) -based human enterovirus. Fluorescent quantitative RT-PCR combined with Sn-PCR method combined with nucleotide sequence Determination and analysis of human enterovirus encephalitis rapid detection and pathogen type identification.