Scalable DNA-Based Magnetic Nanoparticle Agglutination Assay for Bacterial Detection in Patient Samples
Scalable DNA-Based Magnetic Nanoparticle Agglutination Assay for Bacterial Detection in Patient Samples
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DOI：
10.1021/acsnano.5b02379
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发表时间：
2015-07-01
期刊：
影响因子：
17.1
通讯作者：
Donolato, Marco
中科院分区：
文献类型：
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作者：
Mezger, Anja;Fock, Jeppe;Donolato, Marco
我们展示了一种基于纳米颗粒的检测方法，用于检测患者样本中引起尿路感染的细菌，总检测时间为4小时。这一时间明显短于目前的金标准，即平板培养，根据病原体的不同，可能需要几天的时间。该检测基于挂锁探针识别，然后进行两个循环滚动圈扩增（RCA），形成与目标细菌DNA对应的DNA线圈。RCA产品的读数是基于使用来自蓝光驱动器的低成本光电元件对dna结合磁性纳米颗粒（MNPs）特异性凝集的光磁测量。我们实施了一种检测方法，该方法依赖于RCA产品的单体化，使用单体连接和凝集两个具有通用非目标特异性检测探针功能化的MNPs群体，并引入磁培养方案。这使得大肠杆菌、奇异变形杆菌和铜绿假单胞菌在临床相关浓度下的多重检测成为可能，与以前基于mnp的检测方案相比，灵敏度提高了30倍。由于通用探针，相同的一组功能化MNPs可用于从众多RCA靶标中读取产物，使该方法真正可扩展，可在未来的综合护理点分子诊断系统中并行检测多种细菌。
We demonstrate a nanoparticle-based assay for the detection of bacteria causing urinary tract infections in patient samples with a total assay time of 4 h. This time is significantly shorter than the current gold standard, plate culture, which can take several days depending on the pathogen. The assay is based on padlock probe recognition followed by two cycles of rolling circle amplification (RCA) to form DNA coils corresponding to the target bacterial DNA. The readout of the RCA products is based on optomagnetic measurements of the specific agglutination of DNA-bound magnetic nanoparticles (MNPs) using low-cost optoelectronic components from Blu-ray drives. We implement a detection approach, which relies on the monomerization of the RCA products, the use of the monomers to link and agglutinate two populations of MNPs functionalized with universal nontarget specific detection probes and on the introduction of a magnetic incubation scheme. This enables multiplex detection of Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa at clinically relevant concentrations, demonstrating a factor of 30 improvement in sensitivity compared to previous MNP-based detection schemes. Thanks to the universal probes, the same set of functionalized MNPs can be used to read out products from a multitude of RCA targets, making the approach truly scalable for parallel detection of multiple bacteria in a future integrated point of care molecular diagnostics system.