Nucleic acid detection techniques have played a crucial role in identifying specific genetic indicators or species, with Polymerase Chain Reaction (PCR) being the established gold standard in this field. However, PCR's dependence on specialized equipment and skilled personnel has limited its utility in resource-limited or field settings, and its multitemperature stage protocol hinders rapid nucleic acid detection. The emergence of isothermal amplification methods, particularly recombinase polymerase amplification (RPA), has addressed some of these limitations, offering high sensitivity and efficiency. Nevertheless, the challenge of RPA amplicon detection, typically reliant on labeling methods, has persisted, potentially introducing false positives and increased costs. This study introduces an innovative approach to nucleic acid detection, harnessing hyperspectral quantitative interference for label-free, isothermal nucleic acid detection within a remarkably short 25-minute timeframe. By employing a solid-phase RPA amplification process that transforms the product into a DNA molecule layer and leveraging Fourier domain optical slice separation and spectral phase shift analysis, this method enables the semi-quantitative determination of amplification results. The integration of digital microfluidic technology further enhances the method's performance, enabling parallel, integrated, and clinical multi-indicator pathogen detection. Overall, this research presents a practical and rapid solution for label-free nucleic acid detection, addressing the current limitations associated with nucleic acid amplification techniques. This advancement holds promise for a wide range of applications, from point-of-care diagnostics to field-based pathogen detection, ultimately contributing to more accessible and efficient nucleic acid testing methodologies.
Nucleic acid detection is widely used in life science and clinical medical diagnosis. Innovative methods and platform research to improve its key performances are of great significance to ensure population health, promote precision medical technology, and even ensure social stability and development. Most of the existing nucleic acid detection technologies utilized PCR as the amplification method, relying on professional and complex scientific instruments and thus is time-consuming and laborious. Fortunately, RPA offers a feasible alternative. It has the advantages of fast amplification speed, high sensitivity, simple primer design, no temperature cycle control and complex manual operations. However, the detection of amplified products is difficult and costly, and there is a lack of low-cost real-time detection methods with parallel multiple detection abilities. In this work, a label-free and real-time RPA amplicon detection method based on hyperspectral interferometry is presented. A solid-phase biochip helps to capture the RPA product in a real-time meaner and the interference spectrum signal is used to read the solid thickness increment brought by the amplicon. A Fourier domain thickness computation method contributes to calculating the thickness increase and excluding scattering noise. The detection sensitivity reaches 6 copies/reaction and the consuming time is less than 20 min. Moreover, the detection method can also be used for single point mutation readout with the specificity of merelya1%mutation-wild type ratio. Combined with a microfluidic platform, parallel, simultaneous and multiple tests can be realized with 3 microliters.
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