OPTIMIZATION OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) FOR THE DETECTION OF TRITRICHOMONAS FOETUS

PARADISO LANGHOFF, FRANCO; MORERO, MARIANA; RAMIREZ, ROSANA; OYHENART, JORGE ANIBAL

Mar del Plata

Congreso; Reunión Anual de Sociedades de Biociencia 2019; 2019

SAIC

Abstract/Resumen: Trichomonosis is a sexually transmitteddisease of cattle caused by the parasite Tritrichomonas foetus(Rhyan et al., 1988). The control of the disease is limited to theidentification and exclusion of the herd of infected breedingmales. The diagnosis is based on microbiological culture, low costbut low sensitivity and specificity, or quantitative PCR, moresensitive and specific but more expensive (Parker et al., 2003;BonDurant et al., 2003; Cobo et al., 2007). Isothermal loopmediated DNA amplification (LAMP) has great advantages overthese diagnostic techniques (rapid, requires no equipment, moresensitive and specific), although it requires an adequateoptimization process so that it can be carried out. The objectiveof this work was to optimize the conditions for amplification byLAMP of a fragment of the T. foetus ef1a gene recentlydeveloped using a Taguchi scheme (Cobb & Ciarkson, 1994). Theoptimization results were carried out by varying theconcentration of dNTPs (0.06 - 0.5 mM), betaine (0 ? 2 M) andMgSO4 (2 ? 18 mM), the results obtained were classified into 3categories: 1 positive, 2 false positive and 3 negative. Thegrouping of the results was mainly due to the concentration ofMg2+ ions. In categories 2 and 3, regardless of the amount ofbetaine or dNTPs added, the result was a false positive ornegative respectively. Within category 1, the optimized finalreaction contained 9 mM Mg2+ ions, 1.7 M betaine and 0.125 mMdNTPs. Based on these data, we can conclude that LAMP could beused as an alternative tool to expensive molecular diagnostictechniques for the detection of T. foetus.