Parallel solid-phase isothermal amplification and detection of multiple DNA targets in microliter-sized wells of a digital versatile disc

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• 作者：Sara Santiago-Felipe ; Luis Antonio Tortajada-Genaro ; Rosa Puchades…
• 关键词：Multiplex PCR ; DNA ; Recombinase polymerase amplification (RPA) ; Compact disc ; High ; throughput screening ; Food safety
• 刊名：Microchimica Acta
• 出版年：2016
• 出版时间：March 2016
• 年：2016
• 卷：183
• 期：3
• 页码：1195-1202
• 全文大小：710 KB
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• 作者单位：Sara Santiago-Felipe (1)
  Luis Antonio Tortajada-Genaro (1)
  Rosa Puchades (1)
  Ángel Maquieira (1)
  
  1. Instituto Interuniversitario de Reconocimiento Molecular y Desarrollo Tecnológico (IDM) - Departamento de Química, Universitat Politècnica de València, Camino de Vera s/n, 46022, Valencia, Spain
  
• 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

An integrated method for the parallelized detection of multiple DNA target sequences is presented by using microstructures in a digital versatile disc (DVD). Samples and reagents were managed by using both the capillary and centrifugal forces induced by disc rotation. Recombinase polymerase amplification (RPA), in a bridge solid phase format, took place in separate wells, which thereby modified their optical properties. Then the DVD drive reader recorded the modifications of the transmitted laser beam. The strategy allowed tens of genetic determinations to be made simultaneously within <2 h, with small sample volumes (3 μL), low manipulation and at low cost. The method was applied to high-throughput screening of relevant safety threats (allergens, GMOs and pathogenic bacteria) in food samples. Satisfactory results were obtained in terms of sensitivity (48.7 fg of DNA) and reproducibility (below 18 %). This scheme warrants cost-effective multiplex amplification and detection and is perceived to represent a viable tool for screening of nucleic acid targets.