肠道病毒VP1基因定型巢式RT-PCR反应体系建立及评价  

Establishment and evaluation of nested RT-PCR assay for detection of enterovirus VP1 genotyping

作　　者：陈燕[1] 杨爱平[1] 汪敏[1] 胡萍芳[1] 杨文娟[1] CHEN Yan;YANG Ai-ping;WANG Min;HU Ping-fang;YANG Wen-juan(Zhejiang Xiaoshan Hospital,Hangzhou,Zhejiang 310000,China)

机构地区：[1]浙江萧山医院,浙江杭州310000

出　　处：《中国卫生检验杂志》2021年第2期162-164,190,共4页Chinese Journal of Health Laboratory Technology

基　　金：杭州市科技计划引导项目(20181228Y86);萧山区科技计划项目(2017216)。

摘　　要：目的建立一种快速、灵敏、特异的肠道病毒VP1基因分型巢式RT-PCR检测方法。方法根据Gen Bank发表的肠道病毒全基因组序列,收集国内外文献,将文献中用于测序分型的引物进行总结分析,在其VP1区设计并合成巢式RT-PCR引物,优化PCR反应条件,建立检测肠道病毒基因分型的巢式RT-PCR方法。将200株肠道病毒通用阳性,EV71、CA16阴性的临床标本及阴性对照、EB病毒、登革热病毒、流感病毒、腺病毒用该方法进行巢式RT-PCR扩增,随机选择30例PCR产物进行测序,明确本区肠道病毒流行基因型。结果 200份临床病例样本巢式RT-PCR扩增,124例有准确PCR扩增条带,阳性率为62%;阴性对照、EB病毒、登革热病毒、流感病毒、腺病毒均未扩增出条带;23例测序成功,7例比对失败,其中检出16份CA6阳性,阳性率为69.5%,6份CA12阳性,阳性率为26.1%,1份CA10阳性,阳性率为4.3%。结论肠道病毒VP1基因分型巢式RT-PCR检测方法具有良好的灵敏度和特异度;本院就诊患儿肠道病毒基因型主要以CA6和CA12型为主。Objective To establish a rapid,sensitive,and specific nested RT-PCR method for the detection of human enterovirus(HEV) VP1 genotype. Methods Nested RT-PCR primers were designed according to the VP1 section of HEV isolated from whole gene sequences published in Gen Bank. Reaction conditions were optimized and a nested RT-PCR method for the detection of enterovirus genotypes was established. 200 clinical specimens with enterovirus universal primer positive,EV71 and CA16 negative. EB virus,dengue virus,influenza virus,adenovirus and negative control were carried out by this method. 30 samples of PCR products were randomly selected for sequencing and comparison to confirm the epidemic genotype of enterovirus in our region. 30 cases of PCR products were randomly selected and sequenced to verify the epidemic genotype of enterovirus.Results Among 200 clinical cases,124 cases had correct PCR amplification bands,the positive rate was 62%;negative control,EB virus,dengue virus,influenza virus,adenovirus did not amplify the bands;23 cases were successfully sequenced,7 cases failed to compare,16 cases were CA6,the positive rate was 69. 5%,6 cases were CA12,the positive rate was 26. 1%,and 1 case was CA10,the positive rate was 4. 3%. Conclusion The nested RT-PCR method for detecting VP1 genotype of enterovirus has good sensitivity and specificity,and CA6 and CA12 are the main types of enteroviruses.

关 键 词：肠道病毒 巢式RT-PCR 基因分型 检测 

分 类 号：R446.1[医药卫生—诊断学]