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首页> 外文期刊>Biosensors & Bioelectronics: The International Journal for the Professional Involved with Research, Technology and Applications of Biosensers and Related Devices >Isothermal and rapid detection of pathogenic microorganisms using a nano-rolling circle amplification-surface plasmon resonance biosensor
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Isothermal and rapid detection of pathogenic microorganisms using a nano-rolling circle amplification-surface plasmon resonance biosensor

机译：等温和快速检测病原微生物使用纳米滚动圆放大-表面等离子体共振生物传感器

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摘要

Rolling circle amplification (RCA) of DNA is a sensitive and cost effective method for the rapid identification of pathogens without the need for sequencing. In this study, a surface plasmon resonance DNA biosensor based on RCA with a gold (Au) nanoparticle surface was established for isothermal identification of DNA. The probes included a specific padlock probe, a capture probe (CP), which is bound to biotin, and an Au nanoparticle-modified probe, which hybridizes with the RCA products. The CP was assembled on gold nanoparticles to increase its ability to bind and hybridize. The linear padlock probe, which was designed to circularize by ligation upon recognition of the bacterial pathogen-specific sequence in 16 S rDNA, hybridizes to fully complementary sequences within the CP. Upon recognition, each target gene DNA is distinguished by localization onto the corresponding channel on the chip surface. Then, the immobilized CPs act as primers to begin the in situ solid-phase RCA reaction, which produces long single-stranded DNA. The RCA products fixed on the chip surface cause significant surface plasmon resonance angle changes. We demonstrated that six different bacterial pathogens can be identified simultaneously and that 0.5 pM of synthetic oligonucleotides and 0.5 pg μl~(-1) of genomic DNA from clinical samples can be detected by this method with low background signals. Therefore, the multiplex diagnostic method provides a highly sensitive and specific approach for the rapid identification of positive samples.
机译：DNA的滚环扩增（RCA）是一种灵敏且经济高效的方法，可快速鉴定病原体而无需测序。在这项研究中，建立了具有金（Au）纳米粒子表面的基于RCA的表面等离子体共振DNA生物传感器，用于DNA的等温鉴定。探针包括特异性的挂锁探针，与生物素结合的捕获探针（CP）和与RCA产物杂交的金纳米颗粒修饰探针。 CP被组装在金纳米颗粒上，以增加其结合和杂交的能力。线性挂锁探针旨在识别16 S rDNA中的细菌病原体特异性序列，通过连接使其环化，并与CP内的完全互补序列杂交。一旦被识别，每个靶基因DNA通过定位在芯片表面上的相应通道上而被区分。然后，固定的CP充当引物，开始原位固相RCA反应，从而产生长的单链DNA。固定在芯片表面的RCA产品会导致表面等离振子共振角发生明显变化。我们证明了可以同时鉴定出六个不同的细菌病原体，并且该方法可以在低背景信号下检测出临床样品中的0.5 pM合成寡核苷酸和0.5 pgμl〜（-1）基因组DNA。因此，多重诊断方法为快速鉴定阳性样品提供了一种高度灵敏和特异的方法。

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