Department of Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand; Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Graduate Program in Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand; Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Talanta. 2022 Oct 1;248:123588. doi: 10.1016/j.talanta.2022.123588. Epub 2022 May 29.
A combination of Hybridization Chain Reaction (HCR) and Lateral Flow Immunoassay (LFIA) is an attractive strategy for a simple signal amplification DNA/RNA detection. The present study aimed to report a strategy used to solve a problem encountered when the target DNA contained folded secondary structure during HCR, enabling HCR hairpin probes to easily access the target site. The 24-nt conserved sequence within 3'-UTR, present only in dengue virus genome but not in other species, is an ideal target to use as a probe binding site for pan-dengue virus detection. Thus, the 105-nt target containing the 24-nt target sequence was chosen as a target with secondary structures. The 24-nucleotide (nt) synthetic target DNA successfully induced HCR reaction within 5 min at room temperature. However, the HCR detection of the 105-nt synthetic target DNA with secondary structures was problematic. The probe hybridization was prevented by the secondary structures of the target, resulting in a failure to generate HCR product. To solve this problem, two helper oligonucleotides (helper1 and helper2) were designed to linearize the folded structure of the 105-nt target through strand-displacement mechanism, allowing the HCR hairpin probes to easily access the target site. The HCR product with the labeled helper oligonucleotides and the labeled probes were successfully detected by LFIA. With this strategy, the combination of the helper-enhanced HCR and LFIA exhibited a limit of detection (LOD) in a nanomolar range of the 105-nt DENV synthetic target DNA. Our study demonstrated that signal amplification by the combination of HCR and LFIA could successfully detect the target DNA with secondary structure, but not target RNA with secondary structure. In summary, this work provided a proof of concept of two main issues including probe hybridization enhancement by helper oligonucleotide for the target with complicated secondary structure and the advantage of a combination of labeled helper and HCR probes design for LFIA to overcome the false positive result from HCR probe leakage. Our findings on the use of helper oligonucleotides may be beneficial for the development of other isothermal amplification, since the secondary structure of the target is one of the major obstacles among hybridization-based methods.
杂交链式反应（HCR）和侧流免疫分析（LFIA）的结合是一种用于简单信号放大的 DNA/RNA 检测的有吸引力的策略。本研究旨在报告一种策略，用于解决在 HCR 过程中目标 DNA 包含折叠二级结构时遇到的问题，使 HCR 发夹探针能够轻松访问目标位点。仅存在于登革热病毒基因组中而不存在于其他物种中的 3'UTR 内的 24-nt 保守序列是作为用于泛登革热病毒检测的探针结合位点的理想目标。因此，选择包含二级结构的 105-nt 目标作为含有二级结构的目标。24 个核苷酸（nt）合成目标 DNA 可在室温下 5 分钟内成功诱导 HCR 反应。然而，具有二级结构的 105-nt 合成靶 DNA 的 HCR 检测存在问题。目标的二级结构阻止了探针的杂交，导致 HCR 产物无法生成。为了解决这个问题，设计了两个辅助寡核苷酸（helper1 和 helper2）通过链置换机制使 105-nt 目标的折叠结构线性化，使 HCR 发夹探针能够轻松访问目标位点。带有标记的辅助寡核苷酸和标记探针的 HCR 产物通过 LFIA 成功检测到。通过这种策略，结合了辅助增强的 HCR 和 LFIA 的信号放大在纳摩尔范围内对 105-nt DENV 合成靶 DNA 显示出检测限（LOD）。我们的研究表明，通过 HCR 和 LFIA 的结合进行信号放大可以成功检测具有二级结构的目标 DNA，但不能检测具有二级结构的目标 RNA。总之，这项工作证明了两个主要问题的概念验证，包括通过辅助寡核苷酸增强目标物杂交的探针和结合标记辅助物和 HCR 探针设计的 LFIA 的优势，以克服 HCR 探针泄漏引起的假阳性结果。我们关于辅助寡核苷酸的使用的发现可能对其他等温扩增的发展有益，因为目标的二级结构是杂交方法中的主要障碍之一。
