Collection, nucleic acid release, amplification, and visualization platform for rapid field detection of rice false smut

Abstract

Rice false smut (RFS) has brought serious food safety problems to the world. Reliable diagnostic tools are needed for the field detection of RFS. Traditional polymerase chain reaction (PCR) is inefficient due to sample transport and preparation, which cannot adapt to the needs of field detection. Herein, we successfully developed a simple, portable microfluidic test platform to rapidly detect RFS. To simplify the operation, we integrated spore purification, nucleic acid release, and amplification into one chip. A micro air pump was used to separate the spores from the impurities and complete the collection of the spores through the airflow. We rapidly lysed spores and released nucleic acids by the benzyl chloride method. The loop-mediated isothermal amplification (LAMP) products could be combined with SYBR Green I to observe the results visually. On-chip sample tests showed that the spore collection efficiency was approximately 78%. By providing on-chip detection results, the chip had 100% specificity and a detection limit of 100 copies/reaction. At the same time, the stability (CV < 5%) and quantitative ability (R² = 0.989) of the chip were also guaranteed. Through the visual detection of large samples, the on-chip detection results were highly concordant with the classical RT-PCR detection results, and the detection timeliness was greatly enhanced. Compared with RT-PCR, the single-sample detection time was shortened by about twenty minutes. The proposed micro-diagnostic tool did not require any large end-point detection instruments and avoided the complicated operation of nucleic acid extraction. As a result, in the future, our microfluidic chip could be used for rapid and real-time monitoring and early warning of rice false smut spores in rice paddies.