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日本語

In the context of increased food demand due to world population growth and decreased production due to abnormal weather, the "steady supply of safe and high quality agricultural, forest, and fishery products and food" has become a pressing issue common to all humankind in order to actualize a sustainable world (Sustainable Development Goals, SDGs). This research aims at developing a diagnostic technology to support the effective and stable production of high quality crops. By utilizing this technology, even regular agricultural producers without special knowledge or skills can easily and rapidly test for plant diseases and insect pests at their farms at the genetic level.

Loop-mediated isothermal amplification (LAMP) * is one of the methods for detecting target nucleic acids (DNA or RAN). This approach can amplify the targeted gene at a constant temperature (60–65℃ for 30 minutes to 1 hour) without expensive instrumentation for high-precision temperature control in PCR assays, which is the most commonly used genetic diagnosis technique. Therefore, the LAMP method has considerable potential for providing an easy-to-use diagnostic tool and enabling on-site diagnoses. However, with the conventional LAMP method, multiple-item viral diagnosis is complicated because of the amount of samples (target DNA or target RNA), reagents preparation, and gene amplification reactions required for the test. This process also requires specialized knowledge and skills.

Here, the research team has solved this problem by employing microfluidic chip technology. We have developed a polydimethylsiloxane (PDMS)-based microfluidic device for the multiplex genetic diagnosis of plant diseases by using semiconductor manufacturing technology. The fabricated multiplex genetic diagnostic device consists of an array of five reaction chambers (3 µL in volume) and a microchannel (200 µm in width and 80 µm in height which together form a connected network. The device is approximately 45 mm×25 mm in size (less than 1/3 the size of a standard business card). As a sample, an RNA sample containing viral RNA target extracted from diseased cucumber leaves collected at a farm was used. In the operating procedure for the multiplex LAMP assay, a mixture of sample and reagents were autonomously dispensed into the multiple reaction chambers, with just one operation for introducing the mixture into the inlet port of the device. Then, the device was heated in hot water (63℃ for 40 minutes to 1 hour), resulting in the specific amplification of targeted nucleic acids. As shown in the figure, two kinds of RNA viruses were successfully detected simultaneously on our diagnostic device. It should be noted that the device has the ability to simultaneously diagnose up to four different kinds of plant viral diseases.

The team now plans to develop a diagnostic device for enabling the simultaneous detection of a total of eight items, including four kinds of cucumber viral diseases and four kinds of insect pests, with the aim of putting the device to practical use. In principle, it is possible to freely customize the types of target viruses to meet individuals’ specific needs on our diagnostic device. Therefore, looking ahead to the "life with corona" era, we will provide a platform for the rapid multiplex diagnosis of human infectious diseases (such as the novel coronavirus and the influenza viruses). We will also realize rapid multiplex allergen testing in food production (seven specified raw material items: wheat, buckwheat, peanut, egg, milk, shrimp, and crab) as a tool for improving food safety.

This research was partially supported by "Knowledge Hub Aichi", Priority Research Project from Aichi Prefectural Government. Additionally, this work was conducted as a part of "the Cooperative Project for Innovative Research" (Microfluidic-based Genetic Diagnostic and Improving Technologies for Enhancing Food Safety), funded by Toyohashi University of Technology.

* LAMP is an isothermal gene amplification method developed by Eiken Chemical Co., Ltd. (http://loopamp.eiken.co.jp/e/lamp/). This is a technique to amplify a target DNA at a constant temperature (60 - 65℃) by using a set of four to six primers specially designed to recognize six to eight distinct regions on the target gene based on strand displacement reaction.