Optimización de un protocolo de extracción de ADN para la detección molecular de ooquistes de Toxoplasma gondii en coles verdes (Brassica oleracea var. Capitata) procedentes del mercado Central de Ambato

Abstract

Toxoplasmosis is a foodborne zoonosis of public health relevance, particularly in pregnant women, neonates, and immunocompromised individuals. Raw vegetables may act as vehicles for Toxoplasma gondii oocysts due to contact with contaminated water, soil, and handling practices. In this context, green cabbage marketed in Ambato represents a sanitary matrix of interest, since its compact leaf structure favors the retention of parasitic particles and hinders their removal through washing. This study was framed within the Microbiology and Biotechnology research line, linked to food safety, and was developed using an experimental approach. Green cabbage samples were analyzed through recovery with PBS 1X whit Tween® 80, concentration using the Ritchie technique, microscopic observation with Lugol and safranin staining, and molecular evaluation. To optimize DNA extraction, a 2 cubed factorial design was applied, considering thermal lysis, mechanical lysis, and proteinase K digestion. The recovered DNA was quantified by spectrophotometry, assessed using A260 out of 280 and A260 out of 230 ratios, and verified by conventional PCR and electrophoresis, amplifying a 196 bp fragment. The results showed structures compatible with oocysts in 8 of 21 samples using Lugol staining and in 6 samples using safranin staining. The treatment with 10 thermal shock cycles, 2 mechanical lysis cycles, and 20 microliter of proteinase K showed the best overall performance. The scientific novelty lies in proposing a combined, cost-efficient protocol adapted to plant matrices, achieving 100 percentage PCR detection compared with 50 percentage using the GeneJET® kit.