Pathogens tested with the real-time microchip PCR system.

<p>Pathogens tested with the real-time microchip PCR system.</p

Simulated temperature profile of the microchip.

<p>(A) COMSOL Multiphysics® simulation showing uniform temperature distribution in the PCR chamber region when heated to 94°C (White dotted line shows the position of the reaction chamber). (B) Temperature profile across A-A’ shows the uniformity of the temperature in the chamber region of the PCR microchip within 1°C variation when the PCR microchip is heated to 94°C.</p

Performance of the portable real-time microchip PCR system using different concentrations of <i>Fv</i> DNA.

<p>(A) Amplication plot of PMT voltage output over the thermocycle number. Using 300, 180, 100, 50, 12, and 5 ng/sample, the Cq was 18, 19, 20, 22, 24, and 29, respectively (enlarged graph shows the Cq). The error bar of the negative control (0 ng) is the standard deviation. (B) Correlation graph of the DNA amount and the Cq (R²>0.947).</p

Comparison of fungal and bacterial genomic DNA extracted by a variety of methods with or without the use of liquid nitrogen, phenol and chloroform.

<p><i>Fusarium oxysporum</i> f. sp. <i>lycopersici</i> (FOL) and <i>Pseudomona syringae</i> pv. <i>syringae</i> (<i>Pss</i>) genomic DNA was isolated from 50 mg of wet fungal and bacterial biomass, respectively.^a</p>b<p>Modifications as described in the materials and methods (section 2.2).</p>c<p>Values are the means of three biological replicates ± SE.</p

Analysis of pathogen DNAs using the real-time microchip PCR system.

<p>Analysis of pathogen DNAs using the real-time microchip PCR system.</p

Photographs of the compact fluorescence detector housing assembly for real-time detection of amplified DNA samples.

<p>(A) A PCR chip with a thermocouple placed on top of the optical detector housing (bottom part of the image) and a cover integrated with a cooling fan and having septa rubbers to seal the inlet and outlet of the PCR chip (top part of the image). This cover also completely encloses the PCR chip to prevent ambient light from affecting the reading of the PMT. (B) The fully assembled housing that encloses the PCR microchip. The cooling fan can be seen on top of the housing, as well as screws that provide the tight seal.</p

Gel electrophoresis result to verify the portable real-time microchip PCR system.

<p>The picture shows strong bands of <i>B. glumae</i> DNA (150 ng/sample), <i>Fv</i> DNA (50 ng/sample), and <i>Pss</i> B728a DNA (75 ng/sample) amplified using the real-time PCR microchip system (sample 1 to 10). <i>Fv</i> DNA 50 ng/samples amplified using a conventional PCR machine and water were used as control (sample 11 to 13).</p

Illustration of the portable real-time microchip PCR system.

<p>(A) An overview of the real-time microchip PCR system, including a PCR microchip, a fluorescence detector housing coupled to an LED and a PMT, a cooling fan to accelerate cooling, and a microcontroller unit (MCU) for thermocycle control and fluorescence signal acquisition. (B) The PCR microchip is composed of a heater layer and a reaction chamber layer bonded together. The widest line of the heater is located at the center of the chamber, and the widths gets bigger by 1.25 times from the outermost trace to the center trace, thus are 1280, 1600, 2000, and 2500 µm. The gap between the spiral heater lines is 630 µm. The diameter and depth of the circular reaction chamber is 7.8 mm and 80 µm, respectively. White dotted line in the image shows the location of the reaction chamber and the inlet/outlet. (C) Schematic of the compact fluorescence detector housing and optical components inside the housing.</p

Photograph of the portable real-time microchip PCR system.

<p>(A) The portable real-time microchip PCR system controlled by an MCU and powered by a battery. The entire size is 16×28×9 cm³, and the total weight is 843 g. (B) The LCD of the MCU board displays several information about the real time PCR such as the number of cycle, current PCR step, current temperature, fluorescence intensity, the increasing amount of the present cycle’s fluorescence intensity compared to the first cycle’s fluorescence intensity, and the graph showing the trace of the fluorescence intensity of each cycle.</p