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.
- 2.Ng JK, Chong S (2011) Multiplexing Capabilities of Biosensors for Clinical Diagnostics. INTECH Open Access Publisher, RijekaGoogle Scholar
- 8.McCalla SE, Tripathi A (2011) Microfluidic reactors for diagnostics applications. Annu Rev. Biomed Eng 13:321–343Google Scholar
- 14.del Río JS, Yehia Adly N, Acero-Sánchez JL, Henry OY, O’Sullivan CK (2014) Electrochemical detection of Francisella tularensis genomic DNA using solid-phase recombinase polymerase amplification. Biosens Bioelectron 54: 674–678Google Scholar
- 22.International Organization for Standardization, Geneva, Switzerland (2005a) ISO 21569:2005. Foodstuffs-methods of analysis for the detection of genetically modified organisms and derived products-Qualitative nucleic acid based methodsGoogle Scholar
- 23.International Organization for Standardization, Geneva, Switzerland (2005b) ISO 21570:2005. Foodstuffs-methods of analysis for the detection of genetically modified organisms and derived products-Quantitative nucleic acid based methodsGoogle Scholar
- 28.Zhou QJ, Wang L, Chen J, Wang RN, Shi YH, Li CH, Zhang DM, Yan XJ, Zhang YJ (2014) Development and evaluation of a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP) for rapid and simultaneous detection of ten pathogenic bacteria in aquatic animals. J Microbiol Meth 104:26–35CrossRefGoogle Scholar