An integrated nucleic acid detection method based on a microfluidic chip for collection and culture of rice false smut spores

Abstract

Rice false smut spores (RFSS), which are airborne spores caused by Ustilaginoidea virens (U. virens), not only cause severe yield loss and grain quality reduction, but also produce toxins that are harmful to humans and animals. Nucleic acid detection has become the main method for RFSS monitoring due to its high specificity and sensitivity. However, nucleic acid detection requires multiple steps of spore collection, DNA extraction, nucleic acid amplification and detection, which has a high demand for personnel and is hard to link with other intelligent equipment to achieve automation. Microfluidic chip has become an important approach for integrated detection of pathogens owning to miniaturization and integration in recent years. Yet there is a lack of portable methods that integrate the collection of airborne fungal spores and nucleic acid detection. Because RFSS have thick cell walls and require liquid nitrogen grinding to extract DNA, breaking the walls on-chip is difficult. Therefore, the realization of RFSS wall breaking on-chip is a major difficulty and also a very meaningful study. This study uses RFSS as the research object and provides a novel method of culturing RFSS on-chip to solve the problem of hard wall breaking, realizing the integrated detection of RFSS. The mycelium grown by RFSS germination could be easily broken to release DNA for on-chip detection, which eliminates the need for manual DNA extraction and resolves the issue of difficult wall breaking. This chip can collect RFSS based on the aerodynamic theory and achieve gas–liquid coupling through a simple microvalve structure. A micromixer is constructed to mix the liquid, and then accomplish detection quickly by recombinase polymerase amplification and lateral flow dipsticks (RPA-LFD). The detection sensitivity of this method is 1 × 10²–1 × 10⁵ CFU ml⁻¹. It can realize the “sample in and answer out” detection of RFSS due to its simple operation, independence from precision instruments, high sensitivity and specificity. The result shows that it can be used for the early detection of RFSS, has great application prospects and is expected to promote the development of on-site instant detection equipment.