病毒学报

目前肠道病毒(Enterovirus,EV)分子分型主要基于VP1区,此区不仅可以分型还能区分亚型,但不同型别的肠道病毒在VP1区的核苷酸变异很大,需要使用多对引物或高简并度的简并引物进行巢式PCR才能定型,实验过程繁琐。本研究发现肠道病毒5'UTR-VP4-VP2区两端序列高度保守能利用通用引物进行扩增、中间序列差异大于10%能区分不同型别。本研究建立并验证5'UTR-VP4-VP2区测序鉴定手足口病(Hand,foot,and mouth disease,HFMD)临床标本中肠道病毒的方法。根据ICTV公布的人肠道病毒104个型别的核苷酸全序列,在肠道病毒5'UTR-VP4-VP2区设计引物(EV543F/EV1197R)。收集702份肠道病毒核酸检测阳性的手足口病临床标本,用EV543F/EV1197R引物扩增5'UTR-VP4-VP2区基因并测序,BLAST比对定型;对407份标本同时进行RTPCR分型检测。在702份标本中鉴定出15个型别679份肠道病毒,阳性率为96.7%;407份RT-PCR分型结果和本法比较,结果显示EV-A71、CV-A16和CV-A10两法一致性很好(Kappa值0.966～0.970),本法CV-A6阳性率(38.82%)优于RT-PCR分型(34.40%),Mcnemar卡方P值=0.003。肠道病毒5'UTR-VP4-VP2区测序法具有较好的敏感性和简便、快速的特点,可用于手足口病肠道病毒感染的实验室诊断和相关研究。

The molecular typing of enteroviruses(EVs) is based mainly on the VP1 region,which can be differentiated not only into types but also into subtypes. However,the nucleotide variation of different types of enteroviruses in the VP1 region is very wide. Therefore,multiple pairs of primers or degenerate primers with high degeneracy are needed for nested polymerase chain reaction(PCR) to determine the type,which is intrinsically a complex process. We found that the sequences at the two ends of the 5'UTR-VP4-VP2 region of enteroviruses were highly conserved and could be amplified using universal primers. Intermediate sequences with a difference of >10% could be classified as different types. To establish and verify sequencing of the5'UTR-VP4-VP2 region for identification of enteroviruses in clinical specimens of hand,foot,and mouth disease(HFMD),a primer(EV543 F/EV1197 R) for the 5'UTR-VP4-VP2 region of enteroviruses was designed based on the complete nucleotide sequences of 104 human enterovirus types published by the International Committee on Taxonomy of Viruses. A total of 702 clinical specimens of enterovirus nucleic acidpositive HFMD were collected. The 5′UTR-VP4-VP2 region was amplified and sequenced using primer EV543 F/EV1197 R,and alignment of the genotyping were conducted by BLAST. Simultaneous reverse transcription-polymerase chain reaction(RT-PCR)typing was carried out for 407 specimens. Among the 702 specimens,679 enteroviruses of 15 types were identified(96.7%). The results of RT-PCR typing for 407 specimens were compared with those in this method,demonstrating that EV-A71,CV-A16 and CV-A10 in the two methods had high consistency(kappa= 0.966 – 0.970). The positive rate of CV-A6(38.82%)in this method was higher than that of RT-PCR typing(34.40%)(P=0.003;McNemar chi-square test). Sequencing of the 5'UTR-VP4-VP2 region of enteroviruses is characterized by high sensitivity,simplicity and rapidity,and can be used for laboratory diagnosis and related research of enterovirus infection in HFMD.