Lermo A, Zacco E, Barak J, Delwiche M, Campoy S, Barbé J, Alegret S, Pividori M I
Departament de Química, Grup de Sensors i Biosensors, Universitat Autònoma de Barcelona, 08193 Bellaterra, Catalonia, Spain.
Biosens Bioelectron. 2008 Jul 15;23(12):1805-11. doi: 10.1016/j.bios.2008.02.020. Epub 2008 Feb 29.
A very sensitive assay for the rapid detection of pathogenic bacteria based on electrochemical genosensing has been designed. The assay was performed by the PCR specific amplification of the eaeA gene, related with the pathogenic activity of Escherichia coli O157:H7. The efficiency and selectivity of the selected primers were firstly studied by using standard Quantitative PCR (Q-PCR) based on TaqMan fluorescent strategy. The bacteria amplicon was detected by using two different electrochemical genosensing strategies, a highly selective biosensor based on a bulk-modified avidin biocomposite (Av-GEB) and a highly sensitive magneto sensor (m-GEC). The electrochemical detection was achieved in both cases by the enzyme marker HRP. The assay showed to be very sensitive, being able to detect 4.5 ng microl(-1) and 0.45 ng microl(-1) of the original bacterial genome after only 10 cycles of PCR amplification, when the first and the second strategies were used, respectively. Moreover, the electrochemical strategies for the detection of the amplicon showed to be more sensitive compared with Q-PCR strategies based on fluorescent labels such as TaqMan probes.
设计了一种基于电化学基因传感的用于快速检测病原菌的高灵敏度检测方法。该检测方法通过对与大肠杆菌O157:H7致病活性相关的eaeA基因进行PCR特异性扩增来进行。首先基于TaqMan荧光策略，通过标准定量PCR（Q-PCR）研究所选引物的效率和选择性。使用两种不同的电化学基因传感策略检测细菌扩增子，一种是基于本体修饰抗生物素蛋白生物复合材料的高选择性生物传感器（Av-GEB），另一种是高灵敏度磁传感器（m-GEC）。在这两种情况下，均通过酶标记物HRP实现电化学检测。该检测方法显示出非常高的灵敏度，当分别使用第一种和第二种策略时，在仅进行10轮PCR扩增后，就能检测到每微升4.5纳克和0.45纳克的原始细菌基因组。此外，与基于荧光标记（如TaqMan探针）的Q-PCR策略相比，用于检测扩增子的电化学策略显示出更高的灵敏度。
