Development of a Real-Time Microchip PCR System for Portable Plant Disease Diagnosis

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

Diffractive Spectral-Splitting Optical Element Designed by Adjoint-Based Electromagnetic Optimization and Fabricated by Femtosecond 3D Direct Laser Writing

The greatest source of loss in conventional single-junction photovoltaic cells is their inefficient utilization of the energy contained in the full spectrum of sunlight. To overcome this deficiency, we propose a multijunction system that laterally splits the solar spectrum onto a planar array of single-junction cells with different band gaps. As a first demonstration, we designed, fabricated, and characterized dispersive diffractive optics that spatially separated the visible (360–760 nm) and near-infrared (760–1100 nm) bands of sunlight in the far field. Inverse electromagnetic design was used to optimize the surface texture of the thin diffractive phase element. An optimized thin film fabricated by femtosecond two-photon absorption 3D direct laser writing shows an average splitting ratio of 69.5% between the visible and near-infrared light over the 380–970 nm range at normal incidence. The splitting efficiency is predicted to be 80.4% assuming a structure without fabrication errors. Spectral-splitting action is observed within an angular range of ±1° from normal incidence. Further design optimization and fabrication improvements can increase the splitting efficiency under direct sunlight, increase the tolerance to angular errors, allow for a more compact geometry, and ultimately incorporate a greater number of photovoltaic band gaps

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

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

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

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

Pathogens tested with the real-time microchip PCR system.

<p>Pathogens tested with 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

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