Pathogen Detection from Phalaenopsis Orchids by Using an Integrated Microfluidic System

Early detection of pathogens in high-value agricultural species is crucial. Therefore, many methods which can detect agricultural pathogens to prevent economic loss have been developed. Among them, immunoassays, nucleic acid hybridization and polymerase chain reaction have been demonstrated to detect pathogens in Phalaenopsis orchid successfully with satisfactory sensitivity and specificity. However, above-mentioned methods all have some disadvantages including lengthy process or requiring specialized laboratory facilities and well-trained technicians. The current study therefore presents an integrated microfluidic system for rapid and automatic detection of pathogens in agricultural species. The entire procedure, including pathogen-specific ribonucleic acid (RNA) purification, nucleic acid amplification using reverse transcription loop-mediated-isothermal-amplification (RT-LAMP) and optical detection, can be automatically performed on a single chip within 65 minutes. Moreover, the detection module can be interchanged between a fluorescent detector and a turbidity detector, depending on the need of the operator. Furthermore, the developed system can detect pathogens directly from fresh agriculture tissues such as leaves and flowers of the Phalaneopsis orchids. This is the first time that an interchangeable integrated microfluidic system for the detection of Phalaneopsis orchids has been demonstrated. Some of the most prevalent Phalaenopsis orchid pathogens, such as Cymbidium mosaic virus (CymMV) and Tomato spotted wilt virus (TSWV) for Phalaneopsis orchids were used in the current study to demonstrate the capabilities of the developed system. It is concluded that this system with dual detection units can directly detect pathogens from crude agricultural materials successfully.