One-pot isothermal DNA amplification – Hybridisation and detection by a disc-based method

• Low-cost, portable, robust biosensor based on compact disc technology is developed.
• Isothermal DNA amplification and detection on DVDs are integrated.
• Solid-phase amplification products are measured with a standard DVD drive.
• Applied to the multiplexed pathogen detection without microfluidics.

An integrated sensor comprising isothermal DNA amplification and in situ detection is presented. The method principle is based on recombinase polymerase amplification (RPA) and detection in the microarray format by compact disc technology as a high-throughput sensing platform. Primers were immobilised on the polycarbonate surface of digital versatile discs (DVD) and, after hemi-nested amplification, multiplexing identification of each tethered product was achieved by optical scanning with a 650 nm-laser of the DVD drive. The efficiency of one-pot hybridisation/elongation/detection depended strongly on probe density and other factors such as the concentration of the unbound primers present in solution. The optimised conditions provided equivalent amplification factors (7.3 × 108–8.9 × 108 fold) to those obtained by conventional reactions performed in vials. The proposed method was applied to Salmonella detection (generic by hns and oriC genes, and specific for subspecies I by STM4507 gene). A triplex assay was satisfactorily compared to the non-integrated protocols. Food and vaccine samples were analysed in a shorter time with less handling. The results indicate that the multiplex DVD assay is a simple, competitive, isothermal, portable system that is particularly useful for microbiological routine analysis.

Figure optionsDownload as PowerPoint slide