Fraunhofer Institute for Cell Therapy and Immunology - Bioanalytics and Bioprocesses (IZI-BB), 14476, Potsdam, Germany.
Institute for Biochemistry and Biology, University of Potsdam, 14476, Potsdam, Germany.
Sci Rep. 2021 Oct 11;11(1):20137. doi: 10.1038/s41598-021-99774-z.
In this report we describe Cy5-dUTP labelling of recombinase-polymerase-amplification (RPA) products directly during the amplification process for the first time. Nucleic acid amplification techniques, especially polymerase-chain-reaction as well as various isothermal amplification methods such as RPA, becomes a promising tool in the detection of pathogens and target specific genes. Actually, RPA even provides more advantages. This isothermal method got popular in point of care diagnostics because of its speed and sensitivity but requires pre-labelled primer or probes for a following detection of the amplicons. To overcome this disadvantages, we performed an labelling of RPA-amplicons with Cy5-dUTP without the need of pre-labelled primers. The amplification results of various multiple antibiotic resistance genes indicating great potential as a flexible and promising tool with high specific and sensitive detection capabilities of the target genes. After the determination of an appropriate rate of 1% Cy5-dUTP and 99% unlabelled dTTP we were able to detect the bla gene in less than 1.6E-03 ng genomic DNA corresponding to approximately 200 cfu of Escherichia coli cells in only 40 min amplification time.
在本报告中，我们首次描述了在重组酶聚合酶扩增（RPA）产物的扩增过程中直接进行 Cy5-dUTP 标记。核酸扩增技术，特别是聚合酶链式反应以及各种等温扩增方法，如 RPA，已成为检测病原体和靶标基因的有前途的工具。实际上，RPA 甚至提供了更多的优势。由于其速度和灵敏度，这种等温方法在即时诊断中很受欢迎，但需要预标记的引物或探针来检测扩增子。为了克服这一缺点，我们使用 Cy5-dUTP 对 RPA 扩增子进行了标记，而无需预标记的引物。对各种多重抗生素耐药基因的扩增结果表明，它具有很大的潜力，是一种灵活且有前途的工具，具有高特异性和灵敏性的靶基因检测能力。在确定了 1% Cy5-dUTP 和 99% 未标记的 dTTP 的适当比例后，我们能够在不到 40 分钟的扩增时间内检测到少于 1.6E-03 ng 基因组 DNA 中的 bla 基因，相当于大约 200 个大肠杆菌细胞的 cfu。
