Abstract
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 25×16×8 cm3 in size and 843 g in weight. The disposable microchip requires only 8-µl 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 µl sample.
Highlights
Crop production worldwide is highly vulnerable to emerging and variable pathogens that can cause devastating economic losses and threaten global food security [1]
Federal and state regulators recently responded to the introduction of Phytophthora ramorum and Ralstonia solanacearum race 3 biovar 2 (R3B2) on ornamentals to quarantine and eradicate infected plants
We developed a portable and standalone real-time microchip polymerase chain reaction (PCR) system equipped with a microcontroller for controlling the entire PCR operation and with a compact fluorescence detector for acquiring fluorescence emission intensity for on-field plant disease diagnostics
Summary
Crop production worldwide is highly vulnerable to emerging and variable pathogens that can cause devastating economic losses and threaten global food security [1]. Federal and state regulators recently responded to the introduction of Phytophthora ramorum and Ralstonia solanacearum race 3 biovar 2 (R3B2) on ornamentals to quarantine and eradicate infected plants. To meet these new and growing challenges caused by plant pathogenic microbes, there is a critical need for the development of innovative technologies for rapid, accurate, and cost-effective detection and identification of such etiologic agents in field settings. A variety of commercial kits and methods are currently available for microbial pathogen detection and diagnosis such as ELISA kit (Invitrogen, Grand Island, NY, USA) and ViaLightTM kit (Lonza, Basel, Switzerland) The majority of these test kits on the market is antigen/antibody-based immunoassays and lack high-throughput capability or high sensitivity. While conventional PCR and real-time PCR systems are both widely accepted and popular in plant disease diagnostics, due to their need for sample preparation, power supply, and logistics, they are not considered for on-field operation
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