Polymerization-Based Amplification for Target-Specific Colorimetric Detection of Amplified <i>Mycobacterium tuberculosis</i> DNA on Cellulose

Abstract

Loop-mediated isothermal amplification (LAMP) is an appealing method for low-cost, point-of-care nucleic acid diagnostic assays due to high sensitivity, minimal equipment requirements, and compatibility with user-friendly colorimetric detection methods. The enhanced sensitivity LAMP offers comes with vulnerability to cross-contamination, where negative samples are exposed to minute amounts of nucleic acids from positive samples. These amounts are insignificant in less sensitive amplification methods, but visible when LAMP is paired with common colorimetric methods. Here, we examined the use of eosin photopolymerization, a tunable reaction, for colorimetric detection of LAMP products to reduce this false positive risk. Using eosin and biotin end-labeled primers, we successfully amplified target regions of the Mycobacterium tuberculosis (MTB) genome using PCR and LAMP, captured amplicons on streptavidin-coated cellulose, and detected DNA targets via eosin photopolymerization, producing a bright pink color only if MTB DNA was present in the sample. Consistent with previous reports, the LAMP-based method exhibited high background signal, but tuning the illumination time for the photopolymerization reaction allowed readouts from samples with no added MTB DNA to remain blank and visually distinct from pink positives. This method yielded limits of detection of 30 and 300 copies/μL for LAMP and PCR amplification, respectively. When confronted with boiled MTB culture samples, this method gave clear positive readouts, compared to negligible signal from other Mycobacterium boiled culture samples. This new method of LAMP colorimetric detection has the potential to increase the utility of LAMP as a nucleic acid assay technique by mitigating sensitivity to cross-contamination.