USE OF A TAGUCHI DESIGN FOR THE STUDY OF DIFFERENT EXPERIMENTAL CONDITIONS IN LOOP-ME- DIATED ISOTHERMAL AMPLIFICATION REACTIONS (LAMP)

MORERO, MARIANA; PARADISO LANGHOFF, FRANCO; SERENO BRUNO, ESTEFANIA; RAMIREZ, ROSANA; OYHENART, JORGE

virtual

Congreso; REUNIÓN DE SOCIEDADES DE BIOCIENCIAS 2020; 2020

SAIC-SAI-SAFIS

Loop-mediated isothermal DNA amplification (LAMP) has beenused for the diagnosis of a wide variety of diseases. This techniquehas great advantages over other molecular techniques such asPCR. It can be carried out with little equipment, with minimal trainingof personnel and achieve high sensitivity and specificity. However,it is not without its difficulties. The occurrence of false positives orfalse negatives is not unusual. Using a suitable experimental designit is possible to identify and optimize the components of the reactionthat most influence the result of the amplification. A Taguchi designallows this objective to be achieved quickly, minimizing the numberof experiments and efforts. For the reasons stated, the objective ofthis work was to evaluate the performance of a LAMP reaction underdifferent experimental conditions using a Taguchi design. For this,the work methodology consisted in the amplification of a fragment ofthe ef1a1 gene of Tritrichomonas foetus. This was carried out vary-ing the surfactant (Tween20 or Triton X-100) and the concentrationof dNTPs (0.06-0.24mM), betaine (0.4-1.8M) and MgSO4 (2-9mM).The data were analyzed by means of ANOVA, using the program,Graph Pad Prism version 5.01 software. Preliminary results showedthat the surfactant had a notable influence on the kinetics of thereactions, those containing Tween 20 being faster. The influence onthe kinetics of magnesium and betaine ions was greater in reactionswith Tween 20. The occurrence of false positives was minimized inreactions containing Tween 20 as a surfactant. In conclusion, it canbe suggested that the use of an appropriate experimental design allows not only optimizing a reaction but also studying the influenceof each of the factors on amplification