Parallel solid-phase isothermal amplification and detection of multiple DNA targets in microliter-sized wells of a digital versatile disc
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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.
KeywordsMultiplex PCR DNA Recombinase polymerase amplification (RPA) Compact disc High-throughput screening Food safety
This research has been funded through projects FEDER PrometeoII/2014/040 (GVA), and CTQ/2013/45875-R (MINECO). The Spanish Ministry of Education and Science provided S.S.F. with a grant for PhD studies.
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