title: Development of a real-time microchip PCR system for portable plant disease diagnosis.
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 cm(3) 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.
authors.affiliation: Department of Biomedical Engineering, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Chiwan
authors.initials: C
authors.lastname: Koo
authors.affiliation: Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Martha
authors.initials: M
authors.lastname: Malapi-Wight
authors.affiliation: Department of Electrical and Computer Engineering, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Hyun Soo
authors.initials: HS
authors.lastname: Kim
authors.affiliation: Department of Electrical and Computer Engineering, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Osman S
authors.initials: OS
authors.lastname: Cifci
authors.affiliation: Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Vanessa L
authors.initials: VL
authors.lastname: Vaughn-Diaz
authors.affiliation: Department of Electrical and Computer Engineering, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Bo
authors.initials: B
authors.lastname: Ma
authors.affiliation: Department of Electrical and Computer Engineering, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Sungman
authors.initials: S
authors.lastname: Kim
authors.affiliation: Department of Electrical and Computer Engineering, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Haron
authors.initials: H
authors.lastname: Abdel-Raziq
authors.affiliation: Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Kevin
authors.initials: K
authors.lastname: Ong
authors.affiliation: Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Young-Ki
authors.initials: YK
authors.lastname: Jo
authors.affiliation: Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Dennis C
authors.initials: DC
authors.lastname: Gross
authors.affiliation: Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Won-Bo
authors.initials: WB
authors.lastname: Shim
authors.affiliation: Department of Biomedical Engineering, Texas A&M University, College Station, Texas, United States of America ; Department of Electrical and Computer Engineering, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Arum
authors.initials: A
authors.lastname: Han
mesh_terms: D046210:Microfluidic Analytical Techniques; D010935:Plant Diseases; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity