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    Item type:Publication,
    An RT-RPA-Cas12a platform for rapid and sensitive detection of tilapia lake virus
    (2022-11-15)
    Sukonta, Thanwarat
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    Senapin, Saengchan
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    Taengphu, Suwimon
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    ;
    Kitthamarat, Mintra
    Tilapia lake virus (TiLV) or Tilapia tilapinevirus is a highly contagious pathogen that causes severe symptoms and massive mortalities in tilapia, affecting numerous tilapia-farming communities across the continents. Though TiLV has grown into a serious threat that could undermine the aquaculture-based economy and global food security, effective therapeutics and vaccines are not yet commercially available, rendering prompt detection and control the most practical strategy. Current TiLV diagnostic techniques, however, face a number of constraints that restrict their utility, including low throughput and high resource demands imposed upon users. To fill in this critical gap, we developed a novel CRISPR-Cas12a method coupled with reverse-transcriptase recombinase polymerase amplification (RT-RPA-Cas12a) for TiLV detection, using Segment 9 as the target. The platform could detect as few as 200 copies of RNA and produced no false positive results when tested with other fish pathogens. The assay could be carried out at a constant temperature between 37 and 42 °C in less than an hour, allowing naked-eye interpretation of the results through lateral flow and smartphone-based readouts. Taken together, our RT-RPA-Cas12a platform is accurate, streamlined, and user-friendly, and thus has the potential to be a valuable asset in combating the growing threat posed by TiLV.
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    Item type:Publication,
    A Cost-Effective Digital Microfluidic System for CRISPR Diagnostic Automation
    (2024-01-01)
    Vunkong, Jirachaya
    ;
    CRISPR diagnostics offer precise detection of genetic material with high specificity and accuracy while adaptable for field deployment. However, the labor-intensive steps required can reduce usability and increase the risk of human error. To overcome these limitations, we developed a low-cost digital microfluidic (DMF) platform integrated with a custom fluorescence imaging module for the automation of CRISPR diagnostics. Our DMF platform was built from a basic PCB array coated with a layer of parafilm and silicone oil. The fluorescence module was custom designed and fabricated through 3D printing. Our integrated platform effectively facilitated movement, mixing, and fluorescence imaging of water and CRISPR-Cas reagents. This affordable DMF system demonstrates its initial feasibility for automating CRISPR diagnostics, enhancing accessibility and user- friendliness.