Development of Novel PCR Assays for Improved Detection of Enterovirus D68
Development of Novel PCR Assays for Improved Detection of Enterovirus D68
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DOI：
10.1128/jcm.01151-21
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发表时间：
2021-11-01
影响因子：
9.4
通讯作者：
Saitoh, Akihiko
中科院分区：
文献类型：
--
作者：
Ikuse, Tatsuki;Aizawa, Yuta;Saitoh, Akihiko
肠病毒D68 （EV-D68）引起一系列临床表现，包括哮喘样疾病、严重呼吸系统疾病和急性弛缓性脊髓炎。EV-D68自2014年以来在世界范围内引起暴发，现在在许多国家被认为是一种重新出现的感染。EV-D68特异性PCR检测被广泛用于EV-D68感染的诊断；然而，由于最近流行的EV-D68的遗传变化，检测敏感性值得关注。为了解决这个问题，我们在GenBank上总结了2014年至2020年之前报道的全球暴发的EV-D68序列，并在现有EV-D68特异性PCR检测的引物和探针结合位点发现了几个突变。随后，我们设计了两种与最近报道的EV-D68序列相对应的新检测方法：EV-D68特异性实时PCR和插入式PCR。在对日本最近一次EV-D68暴发期间22例EV-D68确诊病例的分析中，新的实时PCR比现有的检测方法灵敏度更高（100%对45%，P < 0.01）， CT值中位数更低（27.8对32.8,P = 0.005）。新的非巢式PCR的灵敏度（91%）高于现有的半巢式PCR （50%, P < 0.01）。使用非ev - d68感染病例（n = 135）的样本，新的实时PCR的特异性为100%。总之，我们的新检测方法比现有的检测方法具有更高的灵敏度，并且可能更准确地诊断最近流行的EV-D68。为应对未来EV-D68疫情，必须持续监测和更新EV-D68特异性检测方法。
Enterovirus D68 (EV-D68) causes a range of clinical manifestations, including asthma-like illness, severe respiratory disease, and acute flaccid myelitis. EV-D68 has caused worldwide outbreaks since 2014 and is now recognized as a re-emerging infection in many countries. EV-D68-specific PCR assays are widely used for the diagnosis of EV-D68 infection; however, assay sensitivity is a concern because of genetic changes in recently circulated EV-D68. To address this, we summarized EV-D68 sequences from previously reported world outbreaks from 2014 through 2020 on GenBank, and found several mutations at the primer and probe binding sites of the existing EV-D68-specific PCR assays. Subsequently, we designed two novel assays corresponding to the recently reported EV-D68 sequences: an EV-D68-specific realtime and seminested PCR. In an analysis of 22 EV-D68 confirmed cases during a recent EV-D68 outbreak in Japan, the new real-time PCR had higher sensitivity than the existing assay (100% versus 45%, P < 0.01) and a lower median CT value (27.8 versus 32.8, P = 0.005). Sensitivity was higher for the new nonnested PCR (91%) than for the existing seminested PCR assay (50%, P < 0.01). The specificity of the new real-time PCR was 100% using samples from non-EV-D68-infected cases (n = 135). In conclusion, our novel assays had higher sensitivity than the existing assay and might lead to more accurate diagnosis of recently circulating EV-D68. To prepare for future EV-D68 outbreaks, EV-D68-specific assays must be continuously monitored and updated.