Improved visual detection of DNA amplification using pyridylazophenol metal sensing dyes

Abstract

Detection of nucleic acid amplification has typically required sophisticated laboratory instrumentation, but as the amplification techniques have moved away from the lab, complementary detection techniques have been implemented to facilitate point-of-care, field, and even at-home applications. Simple visual detection approaches have been widely used for isothermal amplification methods, but have generally displayed weak color changes or been highly sensitive to sample and atmospheric effects. Here we describe the use of pyridylazophenol dyes and binding to manganese ion to produce a strong visible color that changes in response to nucleic acid amplification. This detection approach is easily quantitated with absorbance, rapidly and clearly visible by eye, robust to sample effects, and notably compatible with both isothermal and PCR amplification. Nucleic acid amplification and molecular diagnostic methods are being used in an increasing number of novel applications and settings, and the ability to reliably and sensitively detect them without the need for additional instrumentation will enable even more access to these powerful techniques.

Vivid COVID-19 LAMP is an ultrasensitive, quadruplexed test using LNA-modified primers and a zinc ion and 5-Br-PAPS colorimetric detection system

“…A recent report suggests that Mn 2+ could be used with 5-Br-PAPS for colorimetric detection of amplification 98 , despite a previous report of this complex not forming under mild conditions 99 and Mn 2+ possessing certain theoretical disadvantages such as reduced Bst enzyme fidelity 100 and inducing mutagenesis during synthesis 101 , 102 . Intrigued, we conducted a thorough comparison of our ZBP system with the one based on Mn 2+ .…”

“…8f, g ). A more thorough comparison of these two systems is warranted as both might be advantageous under different conditions, like amplification method (e.g., PCR vs LAMP or other isothermal amplification techniques), alternative primer sets (for example, the LAMP primers used by Zhang et al 98 are optimized for the Mn 2+ -based system and would likely perform better) or sample input types. With respect to the latter, it will be crucial to identify a direct sample inactivation procedure compatible with the Mn 2+ /5-Br-PAPS system, a task made complicated by the weakly stable Mn 2+ /5-Br-PAPS complex likely being more susceptible to disruption by compounds found in direct samples and inactivation buffers/reagents 99 .…”

Metal Ion‐Mediated Optical Biosensors with Signal Amplification: Recent Advances

“…Following this principle, Tanner et al. selected two pyridylazophenol metal sensing dyes (5‐Bromo‐PAPS and 5‐Nitro‐PAPS) to improve visual detection of DNA amplification [27] . Both of them showed yellow color, but give a red color upon complexing with Mn 2+ .…”