Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, Yunnan, China.
Yunnan Kecan Biotechnology Co., Ltd, Kunming, Yunnan, China.
J Clin Microbiol. 2024 Sep 11;62(9):e0038324. doi: 10.1128/jcm.00383-24. Epub 2024 Aug 14.
Chikungunya fever is an acute infectious disease caused by chikungunya virus (CHIKV), which is transmitted by mosquitoes. Simple, rapid, and sensitive detection of CHIKV is critical for its prevention and spread. To address this issue, we combined one-tube, reverse transcription semi-nested, multi-enzyme isothermal rapid amplification, and lateral flow dipstick strips assay to detect CHIKV RNA. The study used a 318-bp gene fragment of CHIKV NSP4 as the target of the assay. This method of amplification takes 30 min for two-step amplification at 39°C. The dilution of amplification products was added to the LFD strip with results visible to the naked eye after 10 min. The method has a sensitivity of 1 copy/μL for the detection of CHIKV RNA, which is 100-fold higher than the conventional reverse transcription-multi-enzyme isothermal rapid amplification and 10-fold higher than the reverse transcription quantitative PCR (RT-qPCR) method. In addition, the method demonstrated good specificity and a better detection rate (85.7%, 18 of 21) than RT-qPCR (80.9%, 17 of 21) in clinically confirmed patient plasma samples. Thus, the rapid CHIKV RNA assay developed in this study will be an important tool for the rapid and accurate screening of patients for chikungunya fever.
This study presents a new one-tube, reverse transcription semi-nested, multi-enzyme isothermal rapid amplification assay combined with lateral flow dipstick strips for the detection of CHIKV. This technique significantly improves sensitivity and outperforms RT-qPCR for the detection of CHIKV, especially in samples with low viral loads. It is also significantly faster than conventional RT-qPCR and does not require special equipment or a standard PCR laboratory. The combination of the isothermal amplification technology developed in this study with point-of-care molecular testing offers the potential for rapid, on-site, low-cost molecular diagnosis of CHIKV.
基孔肯雅热是一种由基孔肯雅病毒（CHIKV）引起的急性传染病，通过蚊子传播。简单、快速、灵敏地检测 CHIKV 对于预防和控制其传播至关重要。为了解决这个问题，我们将单管、逆转录半巢式、多酶等温快速扩增与侧流纸条检测相结合，以检测 CHIKV RNA。该研究使用 CHIKV NSP4 的 318bp 基因片段作为检测靶标。该扩增方法在 39°C 下进行两步扩增，耗时 30 分钟。将扩增产物的稀释液加入 LFD 条中，10 分钟后肉眼可见结果。该方法检测 CHIKV RNA 的灵敏度为 1 拷贝/μL，比常规逆转录-多酶等温快速扩增高 100 倍，比逆转录定量 PCR（RT-qPCR）高 10 倍。此外，该方法在临床确诊的患者血浆样本中的特异性和检测率（85.7%，21 份中的 18 份）均优于 RT-qPCR（80.9%，21 份中的 17 份）。因此，本研究中开发的快速 CHIKV RNA 检测方法将成为快速准确筛选基孔肯雅热患者的重要工具。
本研究提出了一种新的单管、逆转录半巢式、多酶等温快速扩增与侧流纸条检测相结合的 CHIKV 检测方法。该技术显著提高了 CHIKV 的检测灵敏度，优于 RT-qPCR，尤其是在病毒载量较低的样本中。它也比传统的 RT-qPCR 快得多，并且不需要特殊的设备或标准的 PCR 实验室。本研究中开发的等温扩增技术与即时分子检测相结合，为快速、现场、低成本的 CHIKV 分子诊断提供了潜力。
