Three-Dimensional-Printed Instrument for Isothermal Nucleic Acid Amplification with Real-Time Colorimetric Imaging

Abstract

Isothermal amplification methods have become popular in research due to the simplicity of the technology needed to run the reactions. Specifically, loop-mediated isothermal amplification (LAMP) has been widely used for various applications since first reported in 2000. LAMP reactions are commonly monitored with the use of colorimetry. Although color changes associated with positive amplification are apparent to the naked eye, this detection method is subjective due to inherent differences in visual perception from person to person. The objectivity of the colorimetric detection method may be improved by programmed image capture over time with simultaneous heating. As such, the development of a novel, one-step, automated, and integrated analysis system capable of performing these tasks in parallel is detailed herein. The device is adaptable to multiple colorimetric dyes, cost-effective, 3D-printed for single-temperature convective heating, and features an easy-to-use LabVIEW software program developed for automated image analysis. The device was optimized and subsequently validated using four messenger-RNA targets and mock forensic samples. The performance of our device was determined to be comparable to that of a conventional thermal cycler and smartphone image analysis, respectively. Moreover, the outlined system is capable of objective colorimetric analysis, with exceptional throughput of up to 96 samples at once.

Keywords

• 3D printing
• isothermal
• loop-mediated isothermal amplification
• image analysis
• forensic body fluid identification
• Raspberry Pi