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

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

Mar del Plata

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

Trichomonosisis a sexually transmitted disease of cattle caused by the parasiteTritrichomonasfoetus(Rhyan et al., 1988). The control of the disease is limited to theidentification and exclusion of the herd of infected breeding males.The diagnosis is based on microbiological culture, low cost but lowsensitivity and specificity, or quantitative PCR, more sensitive andspecific 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 itrequires an adequate optimization process so that it can be carriedout. The objective of this work was to optimize the conditions foramplification by LAMP of a fragment of the T. foetus ef1a generecently developed using a Taguchi scheme (Cobb & Ciarkson,1994). The optimization results were carried out by varying theconcentration of dNTPs (0.06-0.5mM), betaine (0-2M) and MgSO4(2-18mM), the results obtained were classified into 3 categories: 1positive, 2 false positive and 3 negative. The grouping of theresults was mainly due to the concentration of Mg + 2 ions. Incategories 2 and 3, regardless of the amount of betaine or dNTPsadded, the result was a false positive or negative respectively.Within category 1, the optimized final reaction contained 9mM Mg + 2ions, 1.7M betaine and 0.125mM dNTPs. Based on these data, we canconclude that LAMP could be used as an alternative tool to expensivemolecular diagnostic techniques for the detection of T.foetus.p { margin-bottom: 2.47mm; direction: ltr; line-height: 115%; text-align: left; orphans: 2; widows: 2 }