基于环介导等温扩增结合侧流层析检测骨条藻
Detection of Skeletonema costatum based on loop-mediated isothermal amplification combined with lateral flow dipstick.
机构信息
Key Laboratory of Applied Marine Biotechnology, Ningbo University, Ningbo 315211, China.
出版信息
Mol Cell Probes. 2017 Dec;36:36-42. doi: 10.1016/j.mcp.2017.08.003. Epub 2017 Aug 19.
We developed a new assay method, which combines loop-mediated isothermal amplification (LAMP) with a chromatographic lateral flow dipstick (LFD) for the rapid and special detection of the diatom Skeletonema costatum. Four groups of LAMP primers were derived from a conserved DNA sequence unique to S. costatum. The amplifications were carried out at 61, 63, and 65 °C for 60 min in various combinations by the quantitative PCR thermal cycler to confirm optimal primers and reaction temperature. The LAMP-LFD detection limit was 0.94 pg/μL of S. costatum genomic DNA and was 100 times more sensitive than conventional PCR. The LAMP-LFD method had high specificity and accurately identified S. costatum algal isolates, but not other algal isolates. The new LAMP-LFD assay can be used as a reliable and easy method to detect S. costatum.
我们开发了一种新的检测方法，该方法结合环介导等温扩增（LAMP）和层析侧向流试纸条（LFD），用于快速、特异检测硅藻小环藻。根据小环藻特有的保守 DNA 序列，设计了 4 组 LAMP 引物。通过定量 PCR 热循环仪，在不同组合下于 61、63 和 65°C 下进行 60min 的扩增，以确定最佳的引物和反应温度。LAMP-LFD 的检测限为 0.94pg/μL 的小环藻基因组 DNA，比常规 PCR 灵敏 100 倍。LAMP-LFD 方法具有高度特异性，能准确识别小环藻藻株，但不能识别其他藻株。该新的 LAMP-LFD 检测方法可作为一种可靠且易于使用的方法来检测小环藻。
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