Visual Detection of Isothermal Nucleic Acid Amplification Using pH-Sensitive Dyes

Abstract

Nucleic acid amplification is the basis for many molecular diagnostic assays. In these cases, the amplification product must be detected and analyzed, typically requiring extended workflow time, sophisticated equipment, or both. Here we present a novel method of amplification detection that harnesses the pH change resulting from amplification reactions performed with minimal buffering capacity. In loop-mediated isothermal amplification (LAMP) reactions, we achieved rapid (<30 min) and sensitive (<10 copies) visual detection using pH-sensitive dyes. Additionally, the detection can be performed in real time, enabling high-throughput or quantitative applications. We also demonstrate this visual detection for another isothermal amplification method (strand-displacement amplification), PCR, and reverse transcription LAMP (RT-LAMP) detection of RNA. The colorimetric detection of amplification presented here represents a generally applicable approach for visual detection of nucleic acid amplification, enabling molecular diagnostic tests to be analyzed immediately without the need for specialized and expensive instrumentation.