Medical School of Southeast University, Nanjing, 210009, People's Republic of China.
Center of Clinical Laboratory Medicine, Zhongda Hospital, Southeast University, Nanjing, 210009, People's Republic of China.
Talanta. 2022 Mar 1;239:123122. doi: 10.1016/j.talanta.2021.123122. Epub 2021 Dec 5.
Currently, PCR is the gold standard for the detection of hepatitis C virus (HCV). However, the PCR technique is complicated and time-consuming, which prevents its application and, clinical point-of-care testing (POCT). Herein, we report a POCT method with simplicity, high sensitivity and specificity, which consists of a catalytic hairpin assembly (CHA) signal amplification system coupled with a lateral flow immunochromatographic (LFIA) test strip for the detection of HCV. Two ingeniously designed hairpin probes were hybridized to form the H1-H2 duplex in the presence of the target DNA. The catalytic hairpin assembly which was characterized of isothermal and enzyme-free, was accomplished within 40 min and the reaction was then applied to a LFIA test strip. Only the H1-H2 duplex labeled with both digoxin and biotin could be captured by the test strip, and the fluorescence value was determined. In addition, we evaluated the application potential for the detection of clinical samples. The reported method demonstrated high sensitivity with a detectable minimum concentration at 1 fM and showed a good linear range from 10 nM to 10pM, and high specificity for various mismatched sequences. The results demonstrated that clinically positive samples could be successfully detected. In conclusion, the reported method is simple, rapid, and free of large-scale equipment. POCT is expected to be useful for HCV detection in clinic.
目前，PCR 是检测丙型肝炎病毒 (HCV) 的金标准。然而，PCR 技术复杂且耗时，这阻碍了其在临床上的即时检测（POCT）中的应用。在此，我们报告了一种具有简单、高灵敏度和特异性的 POCT 方法，该方法包括催化发夹组装（CHA）信号放大系统和侧向流动免疫层析（LFIA）测试条，用于检测 HCV。两个巧妙设计的发夹探针在存在目标 DNA 的情况下杂交形成 H1-H2 双链。催化发夹组装具有等温且无酶的特点，可在 40 分钟内完成，然后将反应应用于 LFIA 测试条。只有标记有地高辛和生物素的 H1-H2 双链才能被测试条捕获，并测定荧光值。此外，我们评估了该方法用于检测临床样本的应用潜力。所报道的方法具有高灵敏度，最低检测浓度为 1 fM，从 10 nM 到 10 pM 具有良好的线性范围，对各种错配序列具有高特异性。结果表明，临床上阳性的样本可以被成功检测到。总之，该方法简单、快速，无需大型设备。POCT 有望在 HCV 的临床检测中发挥作用。
