A Microfluidic System Integrated One-step PCR and High-resolution Melting Analysis for Rapid Rice Mutant Detection

Rice is a crucial crop to meet global food demands, therefore, it is necessary to develop rapid, efficient and inexpensive methods for screening mutants of important genes in molecular breeding. In this research, a simple microfluidic chip-integrated one-step polymerase chain reaction (PCR) and high-resolution melting (HRM) analysis were designed for the rapid screening of rice mutants. In order to complete the above processes, we also installed a self-developed functional instrument integrated with a temperature control system and a fluorescence detection department. This microfluidic platform was validated by analysing genes related to insect resistance, phenotype and yield of two important varieties of rice, Oryza sativa L. ssp. japonica. and Oryza sativa L. ssp. indica (93-11). The results showed that the platform could detect single-nucleotide mutations between the wild and the mutant rice plants in 1 h and the accuracy was consistent with gene sequencing regarded as the golden standard of the mutants screening. Compared to the traditional method (requiring approximately 3 h), this platform can distinguish mutants from wild types more rapidly. This system is a simple, integrated, disposable and low-cost tool for high-throughput screening of mutants in molecular breeding. More important, this portable instrument can be used as a point-of-care platform for nucleic acid diagnostics in many scientific fields.