An integrated pH "Pen" for visual nucleic acid detection based on colorimetrical LAMP at point-of-care testing.

Abstract

Nucleic acid detection serves as a critical tool employed for controlling the spread of infectious diseases. However, the majority of current nucleic acid detection methods still rely on large and complex equipment, rendering them unsuitable for point-of-care testing, particularly in remote or resource-limited areas. Therefore, the development of a simple, portable, and visual nucleic acid amplification and detection device is imperative. Based on the pH change during amplification in a non-buffered loop-mediated isothermal amplification (LAMP) system, we proposed an integrated pH "Pen" loaded with a pH test strip for endpoint detection of nucleic acid by naked eyes. The pH "Pen" was characterized by minimal instrumentation requirements, necessitating only a portable metal block for nucleic acid amplification. The device features a sealed chamber that ensures an accurate colorimetric response by preventing the pH test strip from contacting mineral oil, which is used to minimize reagent evaporation. Additionally, the image analysis method for assisted visual detection mitigates the impact of individual visual differences on color interpretation. By calculating the G/R value of the pH test strip image, this method enables the detection threshold to accommodate different imaging devices and lighting conditions. The non-viral OTU2m gene (a conserved gene derived from the Chlamydomonas reinhardtii strain) was chosen for proof of concept in our study. The "Pen" enabled the amplification and detection of the OTU2m gene within 28 min, with a limit of detection as low as 2.0 × 10² copies/mL, comparable to the sensitivity of the real-time fluorescence method. Given its simplicity, affordability, portability, and capacity for visual result analysis, the integrated pH "Pen" provided a promising method for nucleic acid detection at point-of-care testing, particularly in the condition with limited access to advanced laboratory infrastructure.