A paper and plastic device for the combined isothermal amplification and lateral flow detection of Plasmodium DNA

Background: Isothermal amplification techniques are emerging as a promising method for malaria diagnosis since they are capable of detecting extremely low concentrations of parasite target while mitigating the need for infrastructure and training required by other nucleic acid based tests. Recombinase polymerase amplification (RPA) is promising for further development since it operates in a short time frame (<30 min) and produces a product that can be visually detected on a lateral flow dipstick. A self-sealing paper and plastic system that performs both the amplification and detection of a malaria DNA sequence is presented. Methods: Primers were designed using the NCBI nBLAST tools and screened using gel electrophoresis. Paper and plastic devices were prototyped using commercial design software and parts were cut using a laser cutter and assembled by hand. Synthetic copies of the Plasmodium 18S gene were spiked into solution and used as targets for the RPA reaction. To test the performance of the device the same samples spiked with synthetic target were run in parallel both in the paper and plastic devices and using conventional bench top methods. Results: Novel RPA primers were developed that bind to sequences present in the four species of Plasmodium which infect humans. The paper and plastic devices were found to be capable of detecting as few as 5 copies/mu L of synthetic Plasmodium DNA (50 copies total), comparable to the same reaction run on the bench top. The devices produce visual results in an hour, cost approximately $1, and are self-contained once the device is sealed. Conclusions: The device was capable of carrying out the RPA reaction and detecting meaningful amounts of synthetic Plasmodium DNA in a self-sealing and self-contained device. This device may be a step towards making nucleic acid tests more accessible for malaria detection.