The Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 8 Lavrentiev Avenue, Novosibirsk 630090, Russia.
Department of Natural Sciences, Novosibirsk State University, Novosibirsk 630090, Russia.
Int J Mol Sci. 2024 Jun 29;25(13):7215. doi: 10.3390/ijms25137215.
Human adenoviruses (HAdVs) are common pathogens that are associated with a variety of diseases, including respiratory tract infections (RTIs). Without reliable, fast, and cost-effective detection methods for HAdVs, patients may be misdiagnosed and inappropriately treated. To address this problem, we have developed a multiplex loop-mediated isothermal amplification (LAMP) assay for the detection of the species (HAdV-B), (HAdV-C) and (HAdV-E) that cause RTIs. This multiplexing approach is based on the melting curve analysis of the amplicons with a specific melting temperature for each HAdV species. Without the need for typing of HAdVs, the LAMP results can be visually detected using colorimetric analysis. The assay reliably detects at least 375 copies of HAdV-B and -C and 750 copies of HAdV-E DNA per reaction in less than 35 min at 60 °C. The designed primers have no in silico cross-reactivity with other human respiratory pathogens. Validation on 331 nasal swab samples taken from patients with RTIs showed a 90-94% agreement rate with our in-house multiplex quantitative polymerase chain reaction (qPCR) method. Concordance between the quantitative and visual LAMP was 99%. The novel multiplexed LAMP could be an alternative to PCR for diagnostic purposes, saving personnel and equipment time, or could be used for point-of-care testing.
人腺病毒（HAdVs）是常见的病原体，与多种疾病相关，包括呼吸道感染（RTIs）。由于缺乏可靠、快速且具有成本效益的 HAdV 检测方法，患者可能会被误诊和不当治疗。为了解决这个问题，我们开发了一种用于检测引起 RTIs 的 （HAdV-B）、 （HAdV-C）和 （HAdV-E）的多重环介导等温扩增（LAMP）检测方法。这种多重方法基于每个 HAdV 物种的特定熔点的扩增子的熔解曲线分析。无需对 HAdV 进行分型，LAMP 结果可以通过比色分析进行目视检测。该检测方法在 60°C 下不到 35 分钟即可可靠地检测到每个反应中至少 375 个 HAdV-B 和 -C 拷贝和 750 个 HAdV-E DNA 拷贝。设计的引物与其他人类呼吸道病原体无计算机模拟交叉反应性。对 331 份来自 RTIs 患者的鼻拭子样本进行验证，与我们内部的多重定量聚合酶链反应（qPCR）方法的一致性率为 90-94%。定量和目视 LAMP 的一致性为 99%。新型多重 LAMP 可替代 PCR 用于诊断目的，节省人员和设备时间，或可用于即时检测。
