Type-specific EV-D68 real-time RT-PCR assay for the detection of all extant enterovirus D68 strains.

IF 5.4 2区 医学 Q1 MICROBIOLOGY
Journal of Clinical Microbiology Pub Date : 2025-09-10 Epub Date: 2025-08-20 DOI:10.1128/jcm.01492-22
Terry Fei Fan Ng, W Allan Nix, Shannon L Rogers, Brian Emery, Shur-Wern Chern, Kiantra Butler, M Steven Oberste
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Abstract

We developed a type-specific Enterovirus D68 (EV-D68) real-time RT-PCR (rRT-PCR) assay termed CDC2022, which targets sequences encoding conserved amino acid regions of all extant EV-D68 strains. We targeted three motifs conserved among all strains in the last 60 years. The assay achieved 100% (281/281) sensitivity and 100% (344/344) specificity when tested with a collection of 625 respiratory specimens, compared to the gold-standard EV semi-nested VP1 PCR and sequencing assay (snPCR/Seq). CDC2022 gave negative results with 289/289 non-target viruses, including 104 EV A-D isolates, 165 rhinovirus (RV) isolates or clinical specimens, and 14 other common respiratory viruses. The limit of detection (LOD) of the CDC2022 assay is 361 copies per reaction, as determined using serially diluted RNA transcripts. It can detect as few as 0.28 CCID50 per reaction with titrated isolates. An in silico "phylo-primer-mismatch" analysis was performed to visualize primer/probe mismatches and to compare CDC2022 with other EV-D68 rRT-PCR assays. It showed that CDC2022 has the fewest primer/probe mismatches among all assays analyzed and is suitable for all clades. As a type-specific assay targeting conserved amino acids, the CDC2022 assay allowed detection of newer strains from 2024. The CDC 2022 assay could provide a critical tool for molecular surveillance of EV-D68.IMPORTANCEEV-D68 has caused recurring respiratory disease outbreaks in the United States since 2014. As recurrent outbreaks and continued virus evolution are expected for EV-D68, the CDC2022 rRT-PCR provides a robust test that detects known strains as well as potential emerging strains. This type-specific assay approach is critical for national EV-D68 surveillance and clinical diagnostics. An in silico "phylo-primer-mismatch" approach is invented to show EV-D68 assay robustness, but it has utility in new molecular tests for pathogen detection.

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Abstract Image

Abstract Image

建立EV-D68型实时RT-PCR检测所有现存肠道病毒D68株的方法。
我们开发了一种名为CDC2022的肠病毒D68 (EV-D68)实时RT-PCR (rRT-PCR)检测方法,该方法针对所有现存EV-D68菌株的保守氨基酸区域编码序列。我们以近60年来在所有菌株中保守的三个基序为目标。与金标准EV半巢式VP1 PCR和测序法(snPCR/Seq)相比,该方法在625份呼吸道标本中获得100%(281/281)的灵敏度和100%(344/344)的特异性。CDC2022检测结果为阴性的非靶病毒289/289种,其中EV A-D分离株104株,鼻病毒(RV)分离株或临床标本165株,其他常见呼吸道病毒14种。CDC2022检测的检测限(LOD)为每个反应361个拷贝,使用连续稀释的RNA转录物来确定。它可以在每次反应中检测到0.28个CCID50。通过计算机“门源-引物-错配”分析,将引物/探针错配可视化,并将CDC2022与其他EV-D68 rRT-PCR分析进行比较。结果表明,CDC2022在所有分析中引物/探针错配最少,适用于所有进化支。作为一种针对保守氨基酸的类型特异性检测,CDC2022检测允许检测2024年以来的新菌株。CDC 2022检测可以为EV-D68的分子监测提供关键工具。自2014年以来,ev - d68在美国引起了反复发作的呼吸道疾病暴发。由于EV-D68预计会反复暴发和持续的病毒进化,CDC2022 rRT-PCR提供了一种检测已知毒株和潜在新出现毒株的强大测试。这种类型特异性检测方法对于国家EV-D68监测和临床诊断至关重要。发明了一种硅“种-引物错配”方法来显示EV-D68检测的稳健性,但它在新的病原体检测分子测试中具有实用性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Journal of Clinical Microbiology
Journal of Clinical Microbiology 医学-微生物学
CiteScore
17.10
自引率
4.30%
发文量
347
审稿时长
3 months
期刊介绍: The Journal of Clinical Microbiology® disseminates the latest research concerning the laboratory diagnosis of human and animal infections, along with the laboratory's role in epidemiology and the management of infectious diseases.
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