title: A microfluidic system integrated with buried optical fibers for detection of Phalaenopsis orchid pathogens.
abstract: Orchids of the genus Phalaenopsis are some of the most economically important plants in Taiwan. Fast, accurate, and on-site detection of pathogens in these orchids is therefore of critical importance in order to prevent or suppress costly disease outbreaks. Traditional pathogen detection methods are time-consuming, require well-equipped laboratories with highly trained personnel, and cannot be conducted in situ. In this study, a microfluidic system integrated with buried optical fibers was developed to detect viral pathogens of Phalaenopsis spp. Briefly, virus-specific ribonucleic acid (RNA) purification was achieved by a pre-treatment incubation with magnetic beads, and reverse-transcription loop-mediated isothermal amplification (RT-LAMP) was used subsequently to amplify the viral RNA. Positive RT-LAMP reactions resulted in the precipitation of magnesium pyrophosphate, which caused a change in turbidity that could be seen by the naked eye. A buried optical fiber-based detection module and a micro-stirring device were then integrated into the microfluidic chip to detect the RT-LAMP reaction product directly on the chip itself by measuring the change in the optical signals caused by the turbidity change associated with a positive amplification. The limit of detection for this system was found to be 25 fg, which is of similar sensitivity to existing, more laborious methods. Therefore, by using the integrated microfluidic system, a sensitive, rapid, accurate, and automatic diagnosis of viral pathogens in Phalaenopsis spp. orchids could be achieved within only 65 min.
authors.affiliation: Department of Power Mechanical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan.
authors.forename: Chih-Lin
authors.initials: CL
authors.lastname: Lin
authors.affiliation: Department of Power Mechanical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan.
authors.forename: Wen-Hsin
authors.initials: WH
authors.lastname: Chang
authors.affiliation: Department of Power Mechanical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan.
authors.forename: Chih-Hung
authors.initials: CH
authors.lastname: Wang
authors.affiliation: Department of Plant Pathology, National Chung Hsing University, Taichung 402, Taiwan.
authors.forename: Chia-Hwa
authors.initials: CH
authors.lastname: Lee
authors.affiliation: Institute of Biotechnology, National Cheng Kung University, Tainan 701, Taiwan.
authors.forename: Tzong-Yueh
authors.initials: TY
authors.lastname: Chen
authors.affiliation: Department of Plant Pathology, National Chung Hsing University, Taichung 402, Taiwan. Electronic address: fjjan@nchu.edu.tw.
authors.forename: Fuh-Jyh
authors.initials: FJ
authors.lastname: Jan
authors.affiliation: Department of Power Mechanical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan; Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan; Institute of NanoEngineering and Microsystems, National Tsing Hua University, Hsinchu 300, Taiwan. Electronic address: gwobin@pme.nthu.edu.tw.
authors.forename: Gwo-Bin
authors.initials: GB
authors.lastname: Lee
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005336:Fiber Optic Technology / Q000295:instrumentation*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation*; D029595:Orchidaceae / Q000821:virology*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D018511:Systems Integration; D017865:Tospovirus / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D012367:RNA, Viral
keywords: Microfluidics; Optical fibers; Pathogen detection; Phalaenopsis orchid; Reverse transcription loop-mediated isothermal amplification