Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
Analyst. 2017 Dec 4;142(24):4661-4669. doi: 10.1039/c7an01438d.
The demand for rapid and sensitive bacterial detection is continuously increasing due to the significant requirements of various applications. In this study, a terahertz (THz) biosensor based on rolling circle amplification (RCA) was developed for the isothermal detection of bacterial DNA. The synthetic bacterium-specific sequence of 16S rDNA hybridized with a padlock probe (PLP) that contains a sequence fully complementary to the target sequence at the 5' and 3' ends. The linear PLP was circularized by ligation to form a circular PLP upon recognition of the target sequence; then the capture probe (CP) immobilized on magnetic beads (MBs) acted as a primer to initialize RCA. As DNA molecules are much less absorptive than water molecules in the THz range, the RCA products on the surface of the MBs cause a significant decrease in THz absorption, which can be sensitively probed by THz spectroscopy. Our results showed that 0.12 fmol of synthetic bacterial DNA and 0.05 ng μL of genomic DNA could be effectively detected using this assay. In addition, the specificity of this strategy was demonstrated by its low signal response to interfering bacteria. The proposed strategy not only represents a new method for the isothermal detection of the target bacterial DNA but also provides a general methodology for sensitive and specific DNA biosensing using THz spectroscopy.
由于各种应用的巨大需求，对快速和敏感的细菌检测的需求不断增加。在这项研究中，开发了一种基于滚环扩增（RCA）的太赫兹（THz）生物传感器，用于细菌 DNA 的等温检测。合成的细菌特异性序列 16S rDNA 与带有发夹探针（PLP）杂交，PLP 在 5'和 3'末端包含与靶序列完全互补的序列。线性 PLP 通过连接环化，在识别靶序列后形成环状 PLP；然后，固定在磁性珠（MBs）上的捕获探针（CP）充当引物，以初始化 RCA。由于在太赫兹范围内，DNA 分子的吸收性远低于水分子，因此 MBs 表面上的 RCA 产物会导致太赫兹吸收显着下降，这可以通过太赫兹光谱灵敏地探测到。我们的结果表明，使用该检测方法可以有效检测到 0.12 fmol 的合成细菌 DNA 和 0.05 ng μL 的基因组 DNA。此外，该策略的特异性通过其对干扰细菌的低信号响应得到了证明。该策略不仅代表了用于目标细菌 DNA 等温检测的新方法，而且还为使用太赫兹光谱进行灵敏和特异性 DNA 生物传感提供了一般方法。
