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

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• 作者：Keyi Liu ; Xing Yan ; Biyao Mao ; Sheng Wang ; Le Deng
• 关键词：FAM ; SYBR green I ; Graphene oxide ; Foodborne pathogen ; 16S rRNA
• 刊名：Microchimica Acta
• 出版年：2016
• 出版时间：February 2016
• 年：2016
• 卷：183
• 期：2
• 页码：643-649
• 全文大小：843 KB
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• 作者单位：Keyi Liu (1)
  Xing Yan (1)
  Biyao Mao (1)
  Sheng Wang (1)
  Le Deng (1)
  
  1. The Co-construction Laboratory of Microbial Molecular Biology of Province and Ministry of Science and Technology, College of Life Science, Hunan Normal University, Changsha, Hunan, 410081, China
  
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Analytical Chemistry
  Inorganic Chemistry
  Physical Chemistry
  Characterization and Evaluation Materials
  Monitoring, Environmental Analysis and Environmental Ecotoxicology
  
• 出版者：Springer Wien
• ISSN：1436-5073

文摘

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 π-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 102 to 105 cfu⋅mL‾^{1, with a detection limit of 300 cfu⋅mL‾1. This assay displays high sensitivity and selectivity and may possess wide applications in pathogen detection.}