目的:分析2021年杭州地区新生儿至1岁婴儿患者肠道病毒的感染情况,了解其分子特征及流行趋势,从而为疾病的预防提供科学依据。方法:采集患者的肛拭子或咽拭子,采用实时荧光定量PCR技术检测肠道病毒(EV)通用型和柯萨奇病毒A组16型(CA16型);然后通过PCR扩增和测序验证CA16阳性样本;对于EV阳性而非CA16样本,则针对部分VP1区设计引物,进行PCR扩增和测序分析以鉴定型别;最后,在NCBI网站利用BioEdit、MEGA4生信分析软件对PCR产物序列进行比对并构建进化树以确定肠道病毒基因分型。结果:在2021年杭州地区临床初步诊断疑似肠道病毒感染的60例儿童患者中,共检出EV阳性标本29份,总阳性检出率为48%,其中CA16阳性检出率为20%。在随机抽取的10份EV阳性标本中,CA16阳性5份,EV其他分型(CA6)阳性样本1份。结论:2021年杭州地区EV CA16为主要感染型别,同时也存在EV其他型别如CA6等。因此,加强肠道病毒的监测,对解释EV的感染趋势及疾病的防控至关重要。 Objective: To analyze the infection status, molecular characteristics and epidemic trend of entero-virus in 2021 of newborns to infants within 1 year old in Hangzhou. Methods: Feces and throat swabs from patients were collected, and real-time RT-PCR was performed to determine the geno-types of enterovirus (EV) and group A 16 (CA16) of Coxsackievirus. CA16 positive samples were further analyzed with PCR amplification and sequencing. For samples of EV positive but CA16 nega-tive, primers were designed based on part of VP1 and PCR amplification and sequencing were per-formed to identify the genotypes. Finally, BioEdit and MEGA4 softwares were used to analyze the homology of the PCR products in NCBI website and phylogenetic tree was constructed to determine the EV genotyping. Results: 29 EV positive samples were found in all the 60 children with suspected EV infection in Hangzhou in 2021. The constituent ratio of EV positive was 48%, and among them, the constituent ratio of CA16 positive was 20%. Among the randomly selected 10 samples, 5 were CA16 positive and 1 was CA6 positive. Conclusion: CA16 was the major genotyping of the enterovi-rus in newborns to infants within 1 year old in Hangzhou in 2021. Meanwhile, other genotyping such as CA6 was also found. Therefore, it is important to monitor the EV genotyping for explaining the infection trend of EV and for the prevention of disease.
目的:分析2021年杭州地区新生儿至1岁婴儿患者肠道病毒的感染情况,了解其分子特征及流行趋势,从而为疾病的预防提供科学依据。方法:采集患者的肛拭子或咽拭子,采用实时荧光定量PCR技术检测肠道病毒(EV)通用型和柯萨奇病毒A组16型(CA16型);然后通过PCR扩增和测序验证CA16阳性样本;对于EV阳性而非CA16样本,则针对部分VP1区设计引物,进行PCR扩增和测序分析以鉴定型别;最后,在NCBI网站利用BioEdit、MEGA4生信分析软件对PCR产物序列进行比对并构建进化树以确定肠道病毒基因分型。结果:在2021年杭州地区临床初步诊断疑似肠道病毒感染的60例儿童患者中,共检出EV阳性标本29份,总阳性检出率为48%,其中CA16阳性检出率为20%。在随机抽取的10份EV阳性标本中,CA16阳性5份,EV其他分型(CA6)阳性样本1份。结论:2021年杭州地区EV CA16为主要感染型别,同时也存在EV其他型别如CA6等。因此,加强肠道病毒的监测,对解释EV的感染趋势及疾病的防控至关重要。
新生儿至1岁内婴儿,肠道病毒,流行病学,型别分析
Wei Liu*, Chao Chen, Shu Teng
Hangzhou Children’s Hospital, Hangzhou Zhejiang
Received: May 27th, 2022; accepted: Jun. 19th, 2022; published: Jun. 29th, 2022
Objective: To analyze the infection status, molecular characteristics and epidemic trend of enterovirus in 2021 of newborns to infants within 1 year old in Hangzhou. Methods: Feces and throat swabs from patients were collected, and real-time RT-PCR was performed to determine the genotypes of enterovirus (EV) and group A 16 (CA16) of Coxsackievirus. CA16 positive samples were further analyzed with PCR amplification and sequencing. For samples of EV positive but CA16 negative, primers were designed based on part of VP1 and PCR amplification and sequencing were performed to identify the genotypes. Finally, BioEdit and MEGA4 softwares were used to analyze the homology of the PCR products in NCBI website and phylogenetic tree was constructed to determine the EV genotyping. Results: 29 EV positive samples were found in all the 60 children with suspected EV infection in Hangzhou in 2021. The constituent ratio of EV positive was 48%, and among them, the constituent ratio of CA16 positive was 20%. Among the randomly selected 10 samples, 5 were CA16 positive and 1 was CA6 positive. Conclusion: CA16 was the major genotyping of the enterovirus in newborns to infants within 1 year old in Hangzhou in 2021. Meanwhile, other genotyping such as CA6 was also found. Therefore, it is important to monitor the EV genotyping for explaining the infection trend of EV and for the prevention of disease.
Keywords:Newborns to Infants within 1 Year Old, Enterovirus, Epidemiology, Genotype
Copyright © 2022 by author(s) and Hans Publishers Inc.
This work is licensed under the Creative Commons Attribution International License (CC BY 4.0).
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人类肠道病毒(Enteroviruses, EV)是一种具有约7500个核苷酸的单股正链无包膜的小型RNA病毒,属于小核糖核酸病毒属 [
肠道病毒感染以柯萨奇病毒A组(Coxsaekievirus group A, CA)16和人肠道病毒71型(Enterovirus 71, EV71)最为常见 [
本研究中,我们收集了2021年杭州地区新生儿至1岁婴儿患者标本,这些患者的临床初步诊断结果为手足口病、疱疹性咽峡炎、脑炎或者惊厥等。通过对临床初步诊断结果及导致其感染的主要病原体进行分型,进一步了解杭州地区肠道病毒的优势型别,有助于提高疾病诊断率,从而达到早期治疗的效果,可获得较高的社会和经济效益。
收集2021年杭州地区4~10月份临床初步诊断为手足口病、疱疹性咽峡炎、脑炎、发热性惊厥等症状的患儿肛拭子或咽拭子标本60份,患儿年龄为新生儿至1岁内,均签署知情同意书。
纳入/排除标准:纳入标准1) 符合《手足口病诊疗指南(2018年版)》的诊断标准;2) 年龄为新生儿至1岁内。排除标准1) 未征得家长同意;2) 未收集到咽拭子/肛拭子标本;3) 没有完整的病史资料;4) 入院时合并其他感染性疾病者;5) 既往有运动、精神发育较同龄儿落后者。
取140 μl样本,根据天根的病毒RNA提取试剂盒(天根DP419)提取病毒RNA;提取好的RNA用NanoDrop 2000 (美国Thermo)测定浓度和纯度,RNA样本保存于−80℃备用。
用Rever Tra Ace qPCR RT Kit试剂盒(FSQ-101, TOYOBO),把RNA逆转录成cDNA。主要步骤简述如下:取4 μl RNA溶液加入1 μl Oligo (dT),70℃变性5 min,冰上静置2 min,然后加入10 μl RNase Free H2O,4 μl 5×RT Buffer,1 μl Rever Tra Ace试剂。在PCR仪上按照如下程序进行逆转录:37℃ 45 min;98℃ 5 min。反应结束后,保存于−20℃备用。
用肠道病毒核酸通用型引物和探针,对60份经逆转录获得的cDNA样品在ABI7500仪器上进行qRT-PCR (探针法)检测筛查。其中试剂是TOYOBO公司的THUNDERBIRDTM Probe qPCR Mix (QPS-101, TOYOBO)。
筛查得到的肠道病毒阳性样本,用柯萨奇病毒A组16型(即CA16)设计的特异性引物在ABI7500仪器上进行qRT-PCR(SYBR green法)进行CA16分型检测。引物如下:CAP-F: 5’-TATGTTAATTGGGACATTGATTTGA-3’,CAP-R:5’-ACCATTGGGTTTGGCTACG-3’。使用试剂为SYBR Green Realtime PCR Master Mix (QPK-212, TOYOBO)。
分型检测到的CA16阳性样本,通过特异性引物CAP-F/CAP-R进行普通PCR扩增,PCR扩增所用试剂为HotStarTaq Master Mix (QIAGEN),反应条件为95℃,15 min;94℃,30 sec,50℃,30 sec,72℃,30 sec,45个循环;72℃,10 min。PCR产物送擎科生物公司进行测序。
在肠道病毒的VP1区设计能扩增几乎所有肠道病毒的引物:I-F: 5’-ATGTAYGT(dI)CC(dI)CC(dI)GG(dI)GG-3’;I-R:5’-GC(dI)CC(dI)GAYTG(dI)TG(dI)CCRAA-3’,进行两轮PCR扩增。PCR扩增所用试剂为HotStarTaq Master Mix (QIAGEN),反应条件为95℃,15 min;94℃,30 sec,50℃,30 sec,72℃,30 sec,45个循环;72℃,10 min。PCR产物送擎科生物公司进行测序。
测序得到的序列与NCBI数据库中标准序列进行比对,初步确定肠道病毒型别后,进一步通过BioEdit和MEGA4生物软件,采用邻近法进行多序列比对构建进化树,对初筛结果进行审核,从而确定肠道病毒基因分型。
本研究共收集杭州地区2021年度4~10月份新生儿至1岁内婴儿患者标本60份,共检出EV阳性样本29例,总阳性检出率为48%。其中在19例患者的咽拭子标本中,EV阳性有12例,占63%,是总样本数的20%,而肛拭子标本检测出EV阳性的为17个,占肛拭子标本数的41%,是总样本数的28%。具体检出情况如表1所示。
样本类型 | 检测数 | EV阳性数 | EV阴性数 | EV阳性率(%) |
---|---|---|---|---|
咽拭子 | 19 | 12 | 7 | 63% |
肛拭子 | 41 | 17 | 24 | 41% |
总数 | 60 | 29 | 31 | 48% |
表1. EV具体检出情况
针对EV阳性的29例样本,通过CA16特异性引物初步筛选出CA16阳性样本12例。CA16的阳性检出率为41.3%。
随机选取CA16阳性样本6例,测序得到序列经与NCBI标准序列Blast比对后,确定肠道病毒CA16有5例,其中1例为假阳性,Blast结果如图1所示。
图1. CA16阳性标本测序比对结果演示图
随机选取CA16阴性的EV阳性样本6例,测序得到序列经与NCBI标准序列Blast比对后,发现是肠道病毒CA6型,比对结果如图2所示。
通过测序获得CA16阳性标本5例的序列分别标记为:CAP3、CAP5、CAP8、CAP31和CAP36;EV其他分型CA6序列标记为I-primer62-1;这些序列与NCBI数据库中公布的代表序列构建进化树。结果显示,6株EV与相对应的原型株系聚在一起,该结果与NCBI比对结果一致。具体结果见图3:
图2. CA6阳性标本测序比对结果演示图
图3. 2021年杭州地区肠道病毒序列进化树
肠道病毒是人类疾病的一个主要来源,主要发生在婴幼儿身上,而成年人感染较少 [
本研究发现肠道病毒CA16阳性数居多,其他肠道病毒阳性所占比例较小,从某种程度上可以说明,2021年杭州地区CA16肠道病毒为优势型别。在本研究中,只发现了一例CA6阳性标本。CA6阳性率低的确切原因还不清楚,样本例数不够或样本采集地区的覆盖率不够广泛可能是部分原因。近年来的数据显示,CA6的感染率在升高,它在很多呼吸道疾病中发挥越来越重要的作用 [
肠道病毒感染是一个严重的全球公共卫生问题,尤其多发于婴幼儿时期,严重威胁着婴幼儿的健康及生命安全。肠道病毒主要通过粪–口途径感染(这个已多次提到,显得有些重复),并在其生命周期早期以胃肠道上皮为目标,还可通过呼吸道传播,如肠道病毒D68,不干净的卫生环境及不良的卫生习惯对肠道病毒的传播过程非常重要 [
本研究存在一定局限性:由于肠道病毒的种类很多,而且保守序列很短,所以本研究的通用引物筛查肠道病毒阳性病例有一定的假阴性可能;另外本次研究虽然收集杭州多个区的样本,但入组的样本例数还是比较少的,得到的结论可能反映不了整个肠道病毒流行季的趋势;将来有关新生儿不同型别肠道病毒感染还需要增大样本量进行深入研究,以便为临床预防和治疗肠道病毒感染提供有力依据。
浙江省医药卫生科技计划项目2019KY524;杭州市农业和社会发展科研计划20191203B122。
刘 伟,陈 超,滕 淑. 杭州地区新生儿至1岁患儿肠道病毒分型分析Analysis of Enterovirus Genotyping in Newborns to Infants within 1 Year Old in Hangzhou[J]. 临床医学进展, 2022, 12(06): 5856-5862. https://doi.org/10.12677/ACM.2022.126847