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自2014年肠道病毒D组68型(Enterovirus D68,EV-D68)引起急性弛缓性脊髓炎暴发以来，一直被广泛关注。建立一种快速、高效且便捷的EV-D68特异性Real-time RT-PCR检测方法，对于及时发现潜在的传染源并迅速阻断传播至关重要。本研究参考EV-D68各个基因型VP1区代表序列设计探针和引物，通过制作标准品，对其特异性、灵敏度以及重复性进行全面评价，并用临床样本检测其可靠性。结果显示，本研究所设计的探针和引物仅能检出EV-D68，其他73种血清型肠道病毒均无检出情况，最低检测下限为2×101拷贝/μL，拷贝数与循环阈值之间具有良好的线性关系(R2=0.9933)，重复性检测的变异系数小于1.70%，稳定性良好。采用本研究设计的探针和引物对41份临床样本进行检测时，实现了零漏检，与测序的金标准结果完全吻合，对于目前流行的B3和D3基因亚型可以实现全覆盖。综上所述，本研究设计的特异性探针引物在EV-D68检测中展现出高效且准确的特性，同时对目前流行的基因型具有较强的通用性，能够为EV-D68感染的临床诊断、疫情监测以及流行病学调查工作提供有力支持，为EV-D68的快速鉴定和相关疾病防控提供关键技术支撑。
Abstract：Since the 2014 outbreaks of acute flaccid myelitis caused by Enterovirus D68 (EV-D68),the virus has attracted widespread public health attention.Establishing a rapid,efficient,and convenient EV-D68 specific real-time RT-PCR assay is critical for timely identification of potential sources of infection and effective interruption of transmission.In this study,probes and primers targeting the VP1 region were designed based on representative sequences from various EV-D68 genotypes.A reference standard was prepared to comprehensively evaluate the specificity,sensitivity,and reproducibility of the assay,and its reliability was further assessed using clinical samples.The results demonstrated that the probes and primers specifically detected EV-D68 without cross-reactivity to any of the other 73 enterovirus serotypes tested.The limit of detection was determined to be 2×101 copies/μL,with a strong linear correlation between copy number and cycle threshold (R2=0.9933).The coefficient of variation for repeatability was less than 1.70%,indicating excellent assay stability.When applied to 41 clinical samples,the assay achieved complete concordance with sequencing results,with no missed detections,and successfully covered the currently circulating B3 and D3 subgenotypes.In conclusion,the specific probes and primers developed in this study exhibit high efficiency,accuracy,and broad applicability across prevalent EV-D68 genotypes.This assay provides robust technical support for the clinical diagnosis,surveillance,and epidemiological investigation of EV-D68 infections,as well as for the rapid identification of EV-D68 and the prevention and control of associated diseases.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.250123
中图分类号:R373.2
引用信息:
[1]肖梦怡,韩振志,路环环等.肠道病毒D68型流行基因型高灵敏通用型Real-time RT-PCR检测方法建立及应用[J].病毒学报,2025,41(03):625-633.DOI:10.13242/j.cnki.bingduxuebao.250123.
基金信息:
公共卫生人才培养支持项目(项目号:GJJKJ-2024-ZY)~~