Kk Sriram, Lin Yii-Lih, Sewunet Tsegaye, Wrande Marie, Sandegren Linus, Giske Christian G, Westerlund Fredrik
Division of Chemical Biology, Department of Biology and Biological Engineering, Chalmers University of Technology, 412 96 Gothenburg, Sweden.
Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, 141 52 Stockholm, Sweden.
Micromachines (Basel). 2021 Oct 11;12(10):1234. doi: 10.3390/mi12101234.
Optical DNA mapping (ODM) has developed into an important technique for DNA analysis, where single DNA molecules are sequence-specifically labeled and stretched, for example, in nanofluidic channels. We have developed an ODM assay to analyze bacterial plasmids-circular extrachromosomal DNA that often carry genes that make bacteria resistant to antibiotics. As for most techniques, the next important step is to increase throughput and automation. In this work, we designed and fabricated a nanofluidic device that, together with a simple automation routine, allows parallel analysis of up to 10 samples at the same time. Using plasmids encoding extended-spectrum beta-lactamases (ESBL), isolated from and , we demonstrate the multiplexing capabilities of the device when it comes to both many samples in parallel and different resistance genes. As a final example, we combined the device with a novel protocol for rapid cultivation and extraction of plasmids from fecal samples collected from patients. This combined protocol will make it possible to analyze many patient samples in one device already on the day the sample is collected, which is an important step forward for the ODM analysis of plasmids in clinical diagnostics.
光学DNA图谱分析（ODM）已发展成为一种重要的DNA分析技术，在该技术中，单个DNA分子会被进行序列特异性标记并拉伸，例如在纳米流体通道中。我们开发了一种ODM检测方法来分析细菌质粒——环状的染色体外DNA，其通常携带使细菌对抗生素产生抗性的基因。与大多数技术一样，下一个重要步骤是提高通量和自动化程度。在这项工作中，我们设计并制造了一种纳米流体装置，该装置与一个简单的自动化流程相结合，可以同时对多达10个样本进行平行分析。使用从[具体来源1]和[具体来源2]分离出的编码超广谱β-内酰胺酶（ESBL）的质粒，我们展示了该装置在平行处理多个样本以及处理不同抗性基因方面的多重分析能力。作为最后一个例子，我们将该装置与一种用于从患者采集的粪便样本中快速培养和提取质粒的新方案相结合。这种组合方案将使得在样本采集当天就能在一个装置中分析多个患者样本成为可能，这是临床诊断中质粒ODM分析向前迈出的重要一步。
