Rapid and accurate detection of plant pathogens in the field is crucial to prevent the proliferation of infected crops.
Polymerase chain reaction (PCR) process is the most reliable and accepted method for plant pathogen diagnosis, however
current conventional PCR machines are not portable and require additional post-processing steps to detect the amplified
DNA (amplicon) of pathogens. Real-time PCR can directly quantify the amplicon during the DNA amplification without the
need for post processing, thus more suitable for field operations, however still takes time and require large instruments that
are costly and not portable. Microchip PCR systems have emerged in the past decade to miniaturize conventional PCR
systems and to reduce operation time and cost. Real-time microchip PCR systems have also emerged, but unfortunately all
reported portable real-time microchip PCR systems require various auxiliary instruments. Here we present a stand-alone
real-time microchip PCR system composed of a PCR reaction chamber microchip with integrated thin-film heater, a compact
fluorescence detector to detect amplified DNA, a microcontroller to control the entire thermocycling operation with data
acquisition capability, and a battery. The entire system is 2561668 cm3 in size and 843 g in weight. The disposable
microchip requires only 8-ml sample volume and a single PCR run consumes 110 mAh of power. A DNA extraction protocol,
notably without the use of liquid nitrogen, chemicals, and other large lab equipment, was developed for field operations.
The developed real-time microchip PCR system and the DNA extraction protocol were used to successfully detect six
different fungal and bacterial plant pathogens with 100% success rate to a detection limit of 5 ng/8 ml sample.