Optimization of loop-mediated isothermal amplification (LAMP) for the detection of Tritrichomonas foetus

MORERO M; OYHENART J; RAMIREZ MR,; PARADISO L F

Mar del Plata

Congreso; Reunion anual SAIC-SAFE-SAB-SAP 2019; 2019

Trichomonosis is a sexually transmitted disease of cattle caused by the parasite Tritrichomonas foetus (Rhyan et al., 1988). The control of the disease is limited to the identification and exclusion of the herd of infected breeding males. The diagnosis is based on microbiological culture, low cost but low sensitivity and specificity, or quantitative PCR, more sensitive and specific but more expensive (Parker et al., 2003; BonDurant et al., 2003; Cobo et al., 2007). Isothermal loop-mediated DNA amplification (LAMP) has great advantages over these diagnostic techniques (rapid, requires no equipment, more sensitive and specific), although it requires an adequate optimization process so that it can be carried out. The objective of this work was to optimize the conditions for amplification by LAMP of a fragment of the T. foetus ef1a gene recently developed using a Taguchi scheme (Cobb & Ciarkson, 1994). The optimization results were carried out by varying the concentration of dNTPs (0.06-0.5mM), betaine (0-2M) and MgSO4 (2-18mM), the results obtained were classified into 3 categories: 1 positive, 2 false positive and 3 negative. The grouping of the results was mainly due to the concentration of Mg + 2 ions. In categories 2 and 3, regardless of the amount of betaine or dNTPs added, the result was a false positive or negative respectively. Within category 1, the optimized final reaction contained 9mM Mg + 2 ions, 1.7M betaine and 0.125mM dNTPs. Based on these data, we can conclude that LAMP could be used as an alternative tool to expensive molecular diagnostic techniques for the detection of T. foetus.