A simple, robust and equipment-free DNA amplification readout in less than 30 seconds

Molecular based diagnostic methods rely on the amplification of pathogen DNA but naked eye visualization of results is still challenging. We present here a simple and highly reliable DNA amplification readout system for naked eye detection of isothermally or PCR amplified DNA in less than 30 seconds. This system utilizes spermine to precipitate DNA amplicons and initiate bridging flocculation of a mix of charcoal and diatomaceous earth particles in suspension. In the absence of amplification, the charcoal particles remain suspended resulting in a black, non-transparent colloid solution while positive samples in which DNA amplification has occurred can be identified within seconds as the particles flocculate and settle leaving a transparent liquid phase. We have coupled this method with our rapid dipstick DNA purification method and isothermal DNA amplification to create a simple four-step diagnostic system that can be preassembled to reduce unnecessary manipulation in the field. The method's simplicity, low cost, minimal equipment and clear presence/absence readout makes it ideal for rapid diagnostic testing in the laboratory and in situations where users have limited technical training or resources including high school science classes and field-based research.