Aptamer-based detection of Salmonella enteritidis using double signal amplification by Klenow fragment and dual fluorescence

作者：Liu, Keyi; Yan, Xing; Mao, Biyao; Wang, Sheng; Deng, Le^*

来源：Microchimica Acta, 2016, 183(2): 643-649.

DOI：10.1007/s00604-015-1692-4

• FAM
• SYBR green I
• Graphene oxide
• Foodborne pathogen
• 16S rRNA

This article describes a sensitive and selective fluorometric method for the determination of Salmonella enteritidis by exploiting the polymerase activity of the Klenow fragment and dual fluorescence. First, one end of a target-selective aptamer was labeled with the fluorophore 6-carboxyfluorescein (FAM). Once the labeled aptamer binds to graphene oxide (GO) via pi-stacking interaction, the fluorescence of FAM is quenched. However, the addition of target (16S rRNA) leads to the restoration of fluorescence due to the binding of probe and target which shifts the FAM fluorophore away from the quenching GO. By using the Klenow fragment and by exploiting the synergistic effect of FAM and the DNA probe SYBR Green I (which is strongly fluorescent in presence of dsDNA only), fluorescence is strongly amplified and sensitivity improved. The analyte 16SrRNA can be determined by this method in the 60 pM to 100 nM concentration range, and the detection limit is 60 pM. It is also shown that Salmonella enteritidis can be determined in milk samples by this method in concentrations between 10(2) to 10(5) cfua <...mLaEuro3/4(1), with a detection limit of 300 cfua <...mLaEuro3/4(1). This assay displays high sensitivity and selectivity and may possess wide applications in pathogen detection.

• 出版日期2016-2
• 单位湖南师范大学