Research Institute for Health Sciences, Chiang Mai University, Chiangmai, Thailand.
Research Institute for Health Sciences, Chiang Mai University, Chiangmai, Thailand.
Int J Infect Dis. 2021 Jan;102:440-445. doi: 10.1016/j.ijid.2020.10.082. Epub 2020 Oct 29.
The aim was to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of hepatitis C virus (HCV) in a single closed tube.
Plasma samples were collected from 200 HCV-infected patients. HCV-RNA was detected by one-step RT-LAMP processed at 65 °C for 60 min. The amplified products were detected by hydroxynaphthol blue (HNB)-dependent visual method and gel electrophoresis. Specificity was tested against other viruses. Sensitivity was determined using serial dilutions of extracted RNA.
The RT-LAMP assay detected 97.5% of HCV-RNA genotype 1, 91.1% of genotype 3, and 100% of genotype 6. The color change was evidenced with the naked eye. The assay demonstrated a clinical sensitivity of 95.5% and specificity of 100%, as well as no cross-reactivity with other viruses (i.e., hepatitis B virus, HIV). The limit of detection was as low as 10 ng per reaction for HCV genotypes 1a and 6, while it was 100 ng for genotype 3a. The assay showed a 100% detection threshold at a viral load of 5.00 log IU/mL in the clinical samples tested.
This study demonstrated the use of an RT-LAMP assay for the detection of HCV in a simple, rapid, and cost-effective manner, which will be useful in resource-limited settings to allow the identification of individuals in need of HCV treatment.
旨在开发一种逆转录环介导等温扩增（RT-LAMP）检测方法，用于在单个封闭管中检测丙型肝炎病毒（HCV）。
收集 200 例 HCV 感染患者的血浆样本。采用一步法 RT-LAMP 在 65°C 下处理 60 分钟，检测 HCV-RNA。通过羟萘酚蓝（HNB）依赖的目视法和凝胶电泳检测扩增产物。用其他病毒检测特异性。用提取 RNA 的系列稀释液测定敏感性。
RT-LAMP 检测法检测到 97.5%的 HCV 基因型 1、91.1%的基因型 3 和 100%的基因型 6。肉眼可见颜色变化。该检测法具有 95.5%的临床灵敏度和 100%的特异性，与其他病毒（即乙型肝炎病毒、HIV）无交叉反应。HCV 基因型 1a 和 6 的检测限低至每个反应 10ng，而基因型 3a 的检测限为 100ng。在检测的临床样本中，该检测法在病毒载量为 5.00 log IU/mL 时具有 100%的检测阈值。
本研究证明了 RT-LAMP 检测法可用于简单、快速、经济高效地检测 HCV，这将有助于在资源有限的环境中识别需要 HCV 治疗的个体。
