Abstract
Orchids of the genus Phalaenopsis are some of the most economically important plants in Taiwan. Rapid, accurate, and on-site detection of pathogens from these orchids is therefore of critical importance for preventing or suppressing costly disease outbreaks. Traditional pathogen detection methods are relatively time-consuming, require well-equipped laboratories with well-trained personnel, and cannot be conducted in field. 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 using a pre-treatment incubation with magnetic beads surface-coated with specific nucleotide probes, 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. Alternatively, 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 in situ. With the micro-stirring device, the RT-LAMP product became uniformly distributed after the stirring step. Therefore, the situation of amplification can be detected by measuring the change of the optical signals caused by the turbidity change. The optical sensitivity and limit of detection of the optical detection system were also improved by this approach. The limit of detection for this system was found to be 25 femtograms (about 5000 target copies) for capsicum chlorosis virus and 25 picograms (about 5000000 target copies) for cymbidium mosaic virus, which is of similar sensitivity of existing, delicate laborious methods. Therefore, by using the integrated microfluidic system with buried optical fibers, a sensitive, rapid, accurate, and automatic diagnosis of viral pathogens in Phalaenopsis spp. orchids could be achieved within only 65 minutes. Furthermore, the integrated system is compact in size and is relatively easy to operate. The power and reagent consumption are greatly reduced. Therefore, it would be promising for in-field study.