Development of a direct reverse-transcription quantitative PCR (dirRT-qPCR) assay for clinical Zika diagnosis
Development of a direct reverse-transcription quantitative PCR (dirRT-qPCR) assay for clinical Zika diagnosis
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DOI：
10.1016/j.ijid.2019.06.007
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发表时间：
2019-08-01
影响因子：
8.4
通讯作者：
Gu Dayong
中科院分区：
文献类型：
--
作者：
Li, Lang;He, Jian-an;Gu Dayong
目的：基于核酸的聚合酶链反应（PCR）检测通常用于检测寨卡病毒（ZIKV）感染。然而，去除导致临床样品假阴性结果的抑制剂所需的时间和劳动力密集型样品预处理对于资源有限的地区来说是不切实际的。目的是开发一种直接逆转录定量 PCR (dirRT-qPCR) 检测方法，用于直接从临床样本诊断 ZIKV。 方法：针对包括血液和血清在内的各种临床样本，优化抑制剂耐受聚合酶、聚合酶增强剂和 dirRT-qPCR 条件的组合。通过在模拟样品中掺入标准 DNA 来评估灵敏度。使用登革热病毒和基孔肯雅病毒等其他感染的临床标本评估特异性。结果：实现了高特异性和灵敏度，检测限 (LOD) 为 9.5 x 10(1) ZIKV RNA 拷贝/反应。 ZIKV 的现场临床诊断需要 5 μl 样本，可在 2 小时内完成诊断。结论：这种强大的 dirRT-qPCR 检测显示出床边诊断的巨大潜力，引物-探针组合也可以扩展到其他病毒检测。实现了大规模现场病毒感染筛查的目标，可用于病毒爆发的早期诊断和防控。 (C) 2019 作者。由爱思唯尔有限公司代表国际传染病学会出版
Objective: The nucleic acid-based polymerase chain reaction (PCR) assay is commonly applied to detect infection with Zika virus (ZIKV). However, the time-and labor-intensive sample pretreatment required to remove inhibitors that cause false-negative results in clinical samples is impractical for use in resource-limited areas. The aim was to develop a direct reverse-transcription quantitative PCR (dirRT-qPCR) assay for ZIKV diagnosis directly from clinical samples.Methods: The combination of inhibitor-tolerant polymerases, polymerase enhancers, and dirRT-qPCR conditions was optimized for various clinical samples including blood and serum. Sensitivity was evaluated with standard DNA spiked in simulated samples. Specificity was evaluated using clinical specimens of other infections such as dengue virus and chikungunya virus.Results: High specificity and sensitivity were achieved, and the limit of detection (LOD) of the assay was 9.5 x 10(1) ZIKV RNA copies/reaction. The on-site clinical diagnosis of ZIKV required a 5 mu l sample and the diagnosis could be completed within 2 h.Conclusions: This robust dirRT-qPCR assay shows a high potential for point-of-care diagnosis, and the primer-probe combinations can also be extended for other viral detection. It realizes the goal of large-scale on-site screening for viral infections and could be used for early diagnosis and the prevention and control of viral outbreaks. (C) 2019 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases