Shanghai Tenth People's Hospital, School of Medicine, Tongji University, Shanghai 200092, China.
College of Life Sciences, Ningxia University, Ningxia 750021, China.
J Pharm Biomed Anal. 2022 Jul 15;216:114812. doi: 10.1016/j.jpba.2022.114812. Epub 2022 May 4.
We developed a novel approach to analyze multiple DNA targets based on microdroplet PCR coupled with denaturing gradient gel electrophoresis (MPDG) to achieve high-throughput detection of biological samples. The target DNAs were preamplified using specific primers. Subsequently, the preamplified products were separated into individual microreactors for parallel amplification with high efficiency, avoiding the interference of different primers and templates, and preventing inconsistent amplification efficiency and non-specific amplification. The final products were analyzed using denaturing gradient gel electrophoresis (DGGE). Using genetically modified (GM) maize as samples, the MPDG method could be used for the simultaneous detection of three DNA targets with an absolute limit of detection of 0.5% (w/w), with no cross reaction with other non-GM samples. The simulated sample assay of MPDG suggested that the method is suitable for practical application. The MPDG approach, with high sensitivity and specificity, could play a crucial role in the field of nucleic acid detection.
我们开发了一种新的方法，基于微滴式 PCR 结合变性梯度凝胶电泳（MPDG）来分析多个 DNA 靶标，以实现对生物样本的高通量检测。使用特定的引物对靶 DNA 进行预扩增。随后，将预扩增产物分离到单个微反应中进行高效平行扩增，避免了不同引物和模板的干扰，防止了扩增效率不一致和非特异性扩增。最终产物使用变性梯度凝胶电泳（DGGE）进行分析。使用转基因（GM）玉米作为样品，MPDG 方法可用于同时检测三个 DNA 靶标，绝对检测限为 0.5%（w/w），与其他非 GM 样品无交叉反应。MPDG 方法的模拟样品测定表明，该方法适用于实际应用。该 MPDG 方法具有高灵敏度和特异性，在核酸检测领域具有重要作用。
