肠道病毒5’-非翻译区测序分型的应用评估  

Evaluation of 5＇-untranslated region amplification and sequencing for enterovirus serotypes identification diagnosis

作　　者：唐诗欢 谢争华 刘朵朵[1] 袁颖 陈满君 范笑地 丁细霞[1] 余楠[1] Tang Shihuart;Xie Zhertghua;Liu Duoduo;Yuart Yirtg;Chert Manjun;Fan Xiaodi;Ding Xixia;Yu Nan(Department of Clinical Laboratory,Zhujiang Hospital Affiliated to Southern Medical University,Key Laboratory of Pathogenic Microorganisms of Emerging Infection Diseases,Guangzhou 510282,China)

机构地区：[1]南方医科大学珠江医院检验医学部广东省突发传染病病原微生物重点实验室,广州510282

出　　处：《中华实验和临床病毒学杂志》2018年第5期488-491,共4页Chinese Journal of Experimental and Clinical Virology

基　　金：国家重大科技专项课题（2017ZX10103011-006）

摘　　要：目的评估5’-非翻译区（5’-untranslated region，5’-UTR）扩增测序法对临床标本直接进行肠道病毒（enterovirus，EV）血清型分型的价值。方法收集实时荧光聚合酶链反应（real-time PCR）检测EV通用型核酸为阳性的肛拭518份、鼻拭148份，采用5’-UTR区逆转录PCR（reverse transcription PCR，RT—PCR）对标本中的EV进行扩增、测序、比对及血清型分型，与VP1（viral protein1）区简并引物巢式PCR测序分型方法比较，两者不一致的标本经EV血清型特异性RT-PCR验证。结果666份EV通用型核酸为阳性的标本，5’-UTR和VP1区各分型成功553例（83．0％）和318例（47．7％），P〈0．001；以VP1法为参考，对肛拭中主要检出的血清型CoxA6（217例）、CoxA16（88例）、EVA71（40例）、CoxA10（28例）和CoxA4（27例），5’-UTR法敏感度（57．1％～100％）、特异度（67．4％～98．1％）以及方法一致性（kappa值0．188～0．847），均因血清型而异；对鼻拭中主要检出的血清型EVD68（15例），5’-UTR法敏感度100％，特异度91．1％，一致性差（kappa值0．217）。分型不一致标本，经CoxA6、CoxA16、EVA71、CoxA10和EVD68型特异性RT—PCR验证，大部分得以确认。以VP1法联合型特异性RT—PCR法为参考，5’-UTR法的敏感度和特异性以及与参考方法的一致性均提高。结论5’-UTR对肠道病毒的分型的敏感度和特异度因血清型而异，应用时应根据研究目的综合考虑。Objective To evaluate an assay permitting amplification of target 5＇-untranslated region （5＇-UTR） sequences directly from clinical specimens and distinction among serotypes of enterovirus （EV）. Methods A total of 518 rectal swabs and 148 nasal swabs tested positive by pan-enterovirus real-time PCR were collected. 5＇-UTR and the viral protein 1 （VP1） gene fragments were amplified and sequenced separately for serotyping. The inconsistent samples by 5＇-UTR and VP1 serotyping were further determined by using the serotype-specific RT-PCR. Results A total of 553 （83.0%） samples were detected by 5＇- UTR serotyping and 318 （47.7%） were detected by VP1 serotyping in all 666 positive specimens, and there was significant difference in the detection rates between two methods in rectal and nasal swabs （ P 〈 0. 001 ）. For the rectal swabs, the mainly detected serotypes were CoxA6 （217）, CoxA16 （88）, EVA71 （40）, CoxA10 （28） and CoxA4 （27） by 5＇-UTR serotyping. Compared with the VP1 serotyping, the sensitivity and specificity of 5＇-UTR serotyping were 57. 1%-100% and 67.4%-98. 1% respectively, with varied consistence with serotypes （kappa value 0. 214-0. 283）. For the nasal swabs, the most frequently detected serotype was EVD68, with the sensitivity of 100%, the specificity of 91.1%, and the poor consistence （kappa value 0. 217）. CoxA6, CoxA16, EVA71, CoxA10 and EVD68 were further confirmed by serotypespecific RT-PCR. Using VP1 serotyping combined with serotype-specific RT-PCR as a reference method , the effect of performance of 5＇-UTR serotyping on diagnosis was increased. Conclusions The performances of 5＇-UTR serotyping in enterovirus vary with serotypes. The application of 5＇-UTR serotyping should be considered comprehensively according to the purpose of the study.

关 键 词：肠道病毒 5’-非翻译区 逆转录-聚合酶链式反应 测序 分型 

分 类 号：R373.2[医药卫生—病原生物学]