应用集成盘上实验室快速分子诊断细菌感染

J. Loo , C.C.H. Leung , H.C. Kwok , S.Y. Wu , I.L.G. Law , M.L. Chin , M. Hui , S.K. Kong , H.P. Ho
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引用次数: 2

摘要

细菌感染是重症监护病房的一大灾难,死亡率很高。传染性细菌的快速分析对于正确用药是必要的。目前使用平板接种的金标准是不准确的,耗时和劳动密集。因此,我们开发了一种分子诊断方法来快速分析感染性细菌的目标标记DNA。采用微流控平台lab-on-a-disc (LOAD),因为只需一个简单的旋转动作就可以为驱动圆盘边界内任何位置的样品和试剂提供高度可控的离心驱动力。采用离心力触发阀,可以利用不同的离心力控制复杂的液体顺序流动。这将使许多反应在流体泵送和流量控制设计中以最小的复杂性同时并行执行。我们报告了用于直接样品到答案应用的集成LOAD -从患者样品输入到检测信号输出的全自动分析。与pdms制成的微流控盘集成的LOAD具有DNA提取、LAMP反应和检测三大功能。利用微流体技术,将低至10 μL的血液样品装入样品加载点,即可检测出目标细菌。细胞在加热部位裂解后释放的DNA结合在二氧化硅膜上。洗涤后,纯化的DNA洗脱液进行LAMP反应,扩增目标基因序列。环介导等温扩增(LAMP)是一种在65℃下进行反应的等温核酸扩增方法。放大后的信号由DNA结合荧光染料报道。我们的原型显示了从临床样本(如血液)中提取细菌DNA的高产量和纯度。我们展示了使用LOAD平台在临床血液样本中检测鲍曼不动杆菌,这是导致医院获得性感染的关键病原体之一。LOAD平台内的快速信号检测和主动温度控制也使实时LAMP靶向特定DNA序列作为条形码来识别受感染的细菌种类。我们发现,DNA LAMP检测灵敏度为10-15 g,而细菌浓度为102 cfu/ml。该系统能够在2小时内提供细菌DNA分析。总之,我们的集成LOAD是一种简单(从样本到答案)、特异性(特定基因序列识别)、鲁棒性(微流控盘自动检测)的细菌感染快速分子诊断方法。在快速细菌检测的一个LOAD平台方法中,从样品到答案的短周转时间和技术进步应该在解决护理点医疗诊断应用的需求方面具有很大的潜力。其简单性使临床医护人员无需技术培训即可使用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Rapid Molecular Diagnosis of Bacterial Infection Using Integrated Lab-on-a-disc

Bacterial infection is a disaster leading to high fatal rate in intensive caring unit. Rapid profiling of infectious bacteria is necessary for applying the correct medication. Current gold standard using plate inoculation is inaccurate, time-consuming and labour-intensive. Therefore we have developed a molecular diagnostic approach to target marker DNA of the infectious bacteria for rapid profiling. A micro-fluidic platform lab-on-a-disc (LOAD) has been adopted because using one simple spinning action can provide highly controllable centrifugation drive force for the actuation of samples and reagents anywhere within the boundary of the disc. When centrifugal force-triggered valve is applied, complex sequential flow of liquid can be controlled with various centrifugal force. This will enable parallel execution of many reactions simultaneously with minimal complexity in the design of fluidic pumping and flow control.

We report an integrated LOAD for direct sample-to-answer applications - fully automated assay from patient's sample input to detection of signal output. The integrated LOAD with PDMS-made microfludic disc performs three major functions, namely DNA extraction, LAMP reaction and detection. Using microfluidics technology, target bacteria can be detected using as little as 10 μL blood sample loaded into sample loading site. The DNA release after cell lysis in heating site is bound on the silica membrane. After washing, the purified DNA elution is subjected to LAMP reaction to amplify the target genetic sequence. Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification method where reaction occurs under 65 oC. The amplified signal is reported by DNA binding fluorescent dye. Our prototype shows high yield and purity of bacterial DNA from clinical samples such as blood. We demonstrated the detection of Acinetobacter baumanii, which is one of the key pathogens resulting in hospital-acquired infections, in clinical blood sample using the LOAD platform. Fast signal detection and active temperature control within the LOAD platform has also enabled real-time LAMP targeting of specific DNA sequences as barcodes to identify infected bacterial species. We found the detection sensitivity of LAMP using DNA is 10-15 g, while that of bacteria concentration is 102 cfu/ml. The system is capable of providing bacterial DNA profiling within 2 hours.

In conclusion, our integrated LOAD is a simple (sample-to-answer), specific (specific genetic sequences recognition), robust (automated assay on microfluidic disc) method for rapid molecular diagnosis of bacterial infection. The short turnaround time and the technical advancement of sample-to-answer in one LOAD platform approach for rapid bacterial detection should have much potential in addressing the needs of point-of-care medical diagnosis applications. The simplicity allows the clinical healthcare workers to utilize without needing technical training.

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