Shining a Light on LAMP Assays — A Comparison of LAMP Visualization Methods Including the Novel use of Berberine

Abstract

The need for simple and effective assays for detecting nucleic acids by isothermal amplification reactions has led to a great variety of end point and real-time monitoring methods. Here we tested direct and indirect methods to visualize the amplification of potato spindle tuber viroid (PSTVd) by loop-mediated isothermal amplification (LAMP) and compared features important for one-pot in-field applications. We compared the performance of magnesium pyrophosphate, hydroxynaphthol blue (HNB), calcein, SYBR Green I, EvaGreen, and berberine. All assays could be used to distinguish between positive and negative samples in visible or UV light. Precipitation of magnesium-pyrophosphate resulted in a turbid reaction solution. The use of HNB resulted in a color change from violet to blue, whereas calcein induced a change from orange to yellow-green. We also investigated berberine as a nucleic acid-specific dye that emits a fluorescence signal under UV light after a positive LAMP reaction. It has a comparable sensitivity to SYBR Green I and EvaGreen. Based on our results, an optimal detection method can be chosen easily for isothermal real-time or end point screening applications.

Sensitivity Evaluation of SYBR Green I, SYBR Safe and Calcein Dyes for Detection of Human Papillomavirus 16 by Loop-Mediated Isothermal Amplification

“…However, the use of pre-added calcein dye before the amplification process increased in Tt (22 mins) ( Figure 5). Our findings are consistent with a previous study which reported the increased of Tt in real-time fluorescence detection by 10 min when calcein and manganese were used (Fischbach et al 2015). The addition of SYBR Green I and SYBR Safe dyes after the amplification reaction gave positive results in 12 mins at the detection limit 10 3 copies/ μl.…”

A one-step rapid screening test ofListeria monocytogenesin food samples using a real-time loop-mediated isothermal amplification turbidity assay

“…A color change is observed aer adding calcein and SYBR Green I to the reaction mixture. 19,20 Hence, these two methods are employed in analysis to avoid the use of carcinogenic ethidium bromide. However, these dyes require equipment for observing illumination which may be an inconvenience for the user.…”

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

“…To name a few, calcein has been used in detection of Trypanosoma cruzi (Besuschio et al 2017), avian influenza H5 virus (Liu et al 2013), hepatitis C virus (Zhao et al 2017), human papillomavirus , human coronavirus (Geng et al 2016) and Flavobacterium columnare in tilapia fish (Suebsing et al 2015). Calcein-based end-point detection is most commonly used due to its high sensitivity and notable colour change for naked eye observation (Fischbach et al 2015).…”