Use of a Taguchi design for the study of different experimental conditions in loop-mediated isothermal amplification reactions (LAMP)

SERENO E. ; RAMIREZ MR,; MORERO M; PARADISO F; OYHENART J.

Buenos Aires

Congreso; Reunion anual de sociedades de biociencias; 2020

SAIC

Amplification isothermal loop medianted (LAMP) has been used for the detection and diagnosis of a wide variety of diseases. In a LAMP reaction, a thermostable polymerase with strand displacement activity and a system of 4-6 primers that recognize 6-8 different sequences within the target DNA are used. This technique has great advantages over molecular culture and PCR techniques. It can be carried out with little equipment, with minimal staff training, with high sensitivity, specificity and speed. However, it is not without problems. The occurrence of false positives or, in the opposite, the absence of amplification is common in the use of this technique. Factors that can affect amplification need to be identified and optimized to improve amplification performance and quality. Most optimizations in a typical laboratory boil down to testing a limited number of components to approximate the most appropriate concentration to carry out the reaction. These optimizations do not include an adequate experimental design, which allows minimizing time, reducing costs and efforts to optimize the LAMP reaction. The main objective of this work is use a Taguchi design to evaluate the effect on the performance of a LAMP reaction under different experimental conditions.DNA. It was extracted with the CTAB (hexadecyltrimethylammonium bromide) method. Primers. A set of previously described primers based on the amplification of the ef1a gene was used. The concentration of primers used was FIP-FIP 0.8uM, B3-F3 0.1uM and 0.4uM LB-LF. LAMP. A Taguchi scheme was used for the evaluation and approximation of the adequate concentration of magnesium ions, betaine and dNTPs. LAMP reactions were performed in which the concentration of dNTPs was varied between 0.06-0.24mM, 0.4-1.8M betaine and 2.25-9mM MgSO4. The orthogonal arrangement was carried out using two different surfactants Tween 20 or Triton X-100 in a final concentration of 0.1% v/v to determine which was more suitable for amplification. For each reaction, 100ng of T. foetus DNA and 2ul of a 1/10000 dilution of SYBR GREEN I were added. The LAMP reactions were carried out for 120 minutes and the fluorescence signals of the amplification products were measured every 60s and quantified with a Heal Force X-960 Real-time PCR thermal cycler. The Ct value obtained in each reaction was used as a response variable to perform the analysis of the experimental design used. The optimal conditions were those that produced the fastest reactions while minimizing the occurrence of false positives. The change in surfactant in LAMP reactions significantly influences the kinetics of the reactions. Reactions containing Tween 20 as surfactant were faster (mean Ct = 69min) than reactions containing Triton X-100 (mean Ct = 104min). False positives appeared after 70 min in reactions with Tween 20, while in reactions with Triton they appeared very close to the amplifications that contained T. fetus DNA. The change of surfactant in the reactions not only affects the kinetics, but also influences the occurrence of false positives. The final reaction was optimized to: 2.25 mM MgSO4, 0.8M betaine, 0.12 mM dNTPs, 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 11 mM (NH4) 2SO4, 0.1% Tween 20 and 4U Bst DNA polymerase at 65 °C. for 120 min and stopped heating at 80 °C for 20 min. The application of an orthogonal Taguchi arrangement for the optimization of LAMP reactions allowed us in a quick and easy way to optimize the appropriate concentrations of each of the components, analyze their effect on the kinetics of the reactions and minimize the occurrence of false positives, greatly improved the quality of LAMP amplification