Development of a Software Application for Loop-Mediated Isothermal Amplification (LAMP) Primer Design

Loop-mediated isothermal AMPlification (LAMP) is an innovative gene amplification technique alternative to PCR, whose main features consist of constant temperature (isothermal reaction), the employment of a strand-displacement polymerase and the use of a set of 6 primers, that target 8 separate regions of a nucleic acid sequence. An accurate selection of the primers, their concentrations and their melting temperatures (Tm) is essential in order that the annealing of the numerous primers on the target can be finely orchestrated at the proper reaction temperature: therefore the phase of primers’ design is crucial in order to obtain successful LAMP reactions. Although some software applications specific for LAMP primer design exist, they often have several limitations: as a consequence, the selection of the candidate primer set is time-consuming and needs to be done by numerous trial-and-error laboratory experimentations. The aim of this project was the development of a primer designing software application tailored to DiaSorin LAMP requisites. As a first step, we have created a database of thermodynamic parameters that has been integrated with a hybridization algorithm: this tool has enabled us to obtain primers Tm calculation and prediction of secondary structures formation at different temperatures and salt concentrations. Once created the engine of our prediction system, we have developed an assisted and an automatic primer design modes. In the first case, after selecting the target sequence, the experimental conditions and the design criteria, the user is helped during the manual selection of primers along the target thanks to a conditional formatting highlighting data that differ from the design criteria previously set. In the automatic design mode, the software generates a number of primer sets based on the acceptance criteria inserted into the system and identifies the best sets by assigning them a score. In addition, the software has been implemented through the development of a tool for the evaluation of primers dimers in multiplex assays, characterized by the simultaneous amplification of more transcripts in one single reaction. This tool predicts the formation of possible intermolecular secondary structures between the numerous primers present in the solution, displays them graphically and provides information about their extensibility. Finally, we have developed a tool for the graphical representation of dumbbells, which are the key structures of the exponential amplification characteristic of a LAMP reaction. In the future we will assess if our primer designing software provides accurate predictions of the real experimental conditions of a LAMP reaction: at this purpose we will perform explorative studies on the software through dedicated tests. In addition we will evaluate the possibility to add further improvements to the software, based on DiaSorin experience with LAMP technique. The creation of such a software guarantees the existence of a DiaSorin primer designing system, which ensures intellectual property, can make a significant contribution to an easier and more efficient development of LAMP assays and can be further implemented according to our needs.