title: Rapid and amplification-free detection of fish pathogens by utilizing a molecular beacon-based microfluidic system.
abstract: Nervous necrosis virus (NNV) and iridovirus are highly infectious pathogens that can cause lethal diseases in various species of fish. These infectious diseases have no effective treatments and the mortality rate is over 80%, which could cause dramatic economic losses in the aquaculture industry. Conventional diagnostic methods of NNV or iridovirus infected fishes, such as virus culture, enzyme-linked immunosorbent assays and nucleic acid assays usually require time-consuming and complex procedures performed by specialized technicians with delicate laboratory facilities. Rapid, simple, accurate and on-site detection of NNV and iridovirus infections would enable timely preventive measures such as immediate sacrifice of infected fishes, and is therefore critically needed for the aquaculture industry. In this study, a microfluidic-based assay that employ magnetic beads conjugated with viral deoxyribonucleic acid (DNA) capturing probes and fluorescent DNA molecular beacons were developed to rapidly detect NNV and iridovirus. Importantly, this new assay was realized in an integrated microfluidic system with a custom-made control system. With this approach, direct and automated NNV and iridovirus detection from infected fishes can be achieved in less than 30 min. Therefore, this molecular-beacon based microfluidic system presents a potentially promising tool for rapid diagnosis of fish pathogens in the field in the future.
authors.affiliation: Department of Power Mechanical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan.
authors.forename: Yi-Chih
authors.initials: YC
authors.lastname: Su
authors.affiliation: Department of Power Mechanical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan.
authors.forename: Chih-Hung
authors.initials: CH
authors.lastname: Wang
authors.affiliation: Department of Power Mechanical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan.
authors.forename: Wen-Hsin
authors.initials: WH
authors.lastname: Chang
authors.affiliation: Institute of Biotechnology, National Cheng Kung University, Tainan 70101, Taiwan. Electronic address: ibcty@mail.ncku.edu.tw.
authors.forename: Tzong-Yueh
authors.initials: TY
authors.lastname: Chen
authors.affiliation: Department of Power Mechanical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan; Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan; Institute of NanoEngineering and Microsystems, National Tsing Hua University, Hsinchu 30013, Taiwan. Electronic address: gwobin@pme.nthu.edu.tw.
authors.forename: Gwo-Bin
authors.initials: GB
authors.lastname: Lee
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation*; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005399:Fishes / Q000821:virology*; D008832:Microchemistry / Q000295:instrumentation; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D015336:Molecular Probe Techniques / Q000295:instrumentation*; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation; D018511:Systems Integration; D014780:Viruses / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D004279:DNA, Viral
keywords: Iridovirus; Microfluidics; Molecular beacon; Nervous necrosis virus; Probe conjugated magnetic beads