In-situ fixation (ISF): A rapid, reusable, and high-throughput nucleic acid extraction method for plant molecular...

Rapid, sensitive, and high-throughput nucleic acid testing (NAT) is crucial for diverse applications in plant breeding and crop protection, including genotyping, transgenic detection, and pathogen diagnosis. However, efficient plant DNA/RNA extraction methods suitable for point-of-care testing remain a significant challenge due to the complex plant cell composition. Here, we present a novel in-situ fixation (ISF) method that eliminates the need for sample grinding, water bath, centrifugation, and pipetting, enabling rapid (6 min) and high-throughput (96 samples) nucleic acid extraction from plant leaves. The ISF method fixes DNA/RNA within the cells, allowing reuse of the extraction reagents without cross-contamination. The extracted nucleic acids are suitable for various NAT techniques, including PCR, RT-PCR, qPCR, and LAMP. We demonstrate the integration of ISF with qPCR and LAMP for rapid detection of genetically modified content and plant pathogens. Furthermore, an ISF-based high-throughput device was developed for efficient genotyping of rice hybrid offspring. The ISF method's simplicity, reusability, and compatibility with field-deployable isothermal amplification offer a promising solution for on-site, rapid, and cost-effective plant molecular analysis.