Due to the extreme infectivity of Yersinia pestis it poses a serious threat as a potential biowarfare agent, which can be rapidly and facilely disseminated. A cost-effective and specific method for its rapid detection at extremely low levels is required, in order to facilitate a timely intervention for containment. Here, we report an ultrasensitive method exploiting a combination of isothermal nucleic acid amplification with a tailed forward primer and biotinylated dNTPs, which is performed in less than 30 min. The polymerase chain reaction (PCR) and enzyme linked oligonucleotide assay (ELONA) were used to optimise assay parameters for implementation on the LFA, and achieved detection limits of 45 pM and 940 fM using SA-HRP and SA-polyHRP, respectively. Replacing PCR with isothermal amplification, namely recombinase polymerase amplification, similar signals were obtained (314 fM), with just 15 min of amplification. The lateral flow detection of the isothermally amplified and labelled amplicon was then explored and detection limits of 7 fM and 0.63 fg achieved for synthetic and genomic DNA, respectively. The incorporation of biotinylated dNTPs and their exploitation for the ultrasensitive molecular detection of a nucleic acid target has been demonstrated and this generic platform can be exploited for a multitude of diverse real life applications.

中文翻译：

---

使用生物素化 dNTP 检测鼠疫耶尔森氏菌以增强横向流动测定中的信号

由于鼠疫耶尔森菌具有极强的传染性，它作为一种潜在的生物战剂构成了严重威胁，可以迅速而轻松地传播。需要一种具有成本效益和特定的方法来快速检测极低水平的物质，以促进及时干预以进行遏制。在这里，我们报告了一种利用等温核酸扩增与带尾正向引物和生物素化 dNTP 相结合的超灵敏方法，该方法在 30 分钟内完成。聚合酶链反应 (PCR) 和酶联寡核苷酸测定 (ELONA) 用于优化在 LFA 上实施的测定参数，并分别使用 SA-HRP 和 SA-polyHRP 实现了 45 pM 和 940 fM 的检测限。用等温扩增代替PCR，即重组酶聚合酶扩增，获得了类似的信号 (314 fM)，只需 15 分钟的放大。然后探索了等温扩增和标记扩增子的侧流检测，合成 DNA 和基因组 DNA 的检测限分别为 7 fM 和 0.63 fg。已经证明了生物素化 dNTP 的掺入及其对核酸靶标的超灵敏分子检测的开发，并且该通用平台可用于多种不同的现实生活应用。