Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) - A New Versatile Isothermal Amplification Method.

The growing demand for cost-effective nucleic acid detection assays leads to an increasing number of different isothermal amplification reaction methods. However, all of the most efficient methods suffer from highly complex assay conditions due to the use of complicated primer sets and/or auxiliary enzymes. The present study describes the application of a new linker moiety that can be incorporated between a primer and a secondary target binding site which can act both as a block to polymerase extension as well as a hinge for refolding. This novel "hinge-primer" approach results in an efficient regeneration of the primer binding site and thus improves the strand-displacement and amplification process under isothermal conditions. Our investigations revealed that the reaction with forward and reverse hinge-primer including an abasic site is very efficient. The assay complexity can be reduced by combining the hinge-primer with a corresponding linear primer. Furthermore, the reaction speed can be increased by reducing the length of the amplified target sequence. We tested the sensitivity down to 104 copies and found a linear correlation between reaction time and input copy number. Our approach overcomes the usually cumbersome primer-design and extends the range of isothermal amplification methods using a polymerase with strand-displacement activity.

Alizarin Red S for Online Pyrophosphate Detection Identified by a Rapid Screening Method.

We identified Alizarin Red S and other well known fluorescent dyes useful for the online detection of pyrophosphate in enzymatic assays, including the loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assays. An iterative screening was used for a selected set of compounds to first secure enzyme compatibility, evaluate inorganic pyrophosphate sensitivity in the presence of manganese as quencher and optimize conditions for an online detection. Of the selected dyes, the inexpensive alizarin red S was found to selectively detect pyrophosphate under LAMP and PCR conditions and is superior with respect to its defined red-shifted spectrum, long shelf life and low toxicity. In addition, the newly identified properties may also be useful in other enzymatic assays which do not generate nucleic acids but are based on inorganic pyrophosphate. Finally, we propose that our screening method may provide a blueprint for rapid screening of compounds for detecting inorganic pyrophosphate.

Shining a light on LAMP assays--a comparison of LAMP visualization methods including the novel use of berberine.

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.