RESUMO
An affordable, portable, and easy-to-build instrument was implemented to perform loop-mediated isothermal amplification (LAMP) in the field. Controlled by Arduino, CELDA heats the reaction tubes, reads the intensity of green fluorescent light and stores data in a computer. In the laboratory and in the field, CELDA was successfully used for the detection of Tritrichomonas foetus DNA in less than 60 min and helped distinguish positives with less difficulty than visual inspection.
Assuntos
Tritrichomonas foetus , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Sensibilidade e Especificidade , Tritrichomonas foetus/genéticaRESUMO
Taguchi method was used to optimize loop mediated isothermal amplification tests aimed to amplify segments of the elongation factor 1a1 (tf-ef1a1), the 5,8 ribosomal gene (tf-5,8 r) or the beta tubulin 2 (tf-btub2) from the protozoan parasite Tritrichomonas foetus. L9 orthogonal array and quadratic loss functions that penalize deviations from prediction values revealed the effect of amplification reaction components. Analysis of variance (ANOVA) decomposed the contribution of individual factors to a small Ct. Confirmation experiments established that optimum conditions were predictable, verifiable and reproducible. Primers concentration conditioned the non-specific amplification of tf-ef1a1 while betaine and magnesium concentration contributed to accelerate the time to reach a positive threshold in tf-ef1a1, tf-5,8 r and tf-btub2. The general strategy of simple and robust experimental design holds potential as a general optimization protocol for LAMP tests in every diagnostic laboratory.