Dados do Trabalho

Título

Development of an one-step RT- qPCR assay for the detection and quantification of viable forms of Trypanosoma cruzi in açaí samples from areas at risk of oral transmission of Chagas disease.

Introdução

About 70% of new cases of Chagas disease (CD) in Brazil have been attributed to oral transmission, mostly of foods based on açaí, bacaba and sugarcane juices in the north and northeast of the country, making evident the need for control of oral transmission of the disease through the improvement of tools for sanitary vigilance and diagnosis. Currently, the methods used to carry out quality control of food associated with outbreaks, and to assess the potential for oral transmission of CD by the consumption of açaí, are mostly based on the isolation of the parasite or inoculation of food in experimental animals, restricting the analyzes to the major research centers.

Objetivo(s)

To develop a molecular methodology, based on RT-qPCR, for the detection and quantification of viable T. cruzi in a sample based on açaí pulp, through a protocol that allows a stabilization and preservation of the nucleic acids in açaí, associated with a standardization exogenous quality controls.

Material e Métodos

Açaí samples were artificially contaminated with six different T. cruzi DTUs and preserved in a Guanidine-EDTA solution. The RNA extraction method was standardized using the High Pure RNA Tissue Kit (ROCHE), in a simple and reproducible way, and the RT-qPCR reactions were designed for TaqMan duplex systems (FAM and VIC), targeting T. cruzi (satDNA) and exogenous internal positive control (Luciferase gene from the Coleoptera firefly).

Resultados e Conclusão

The results for the satDNA target showed high linearity (R2=0.99, from 1,000,000 to 1 parasite equivalent/mL) and a reaction efficiency of 86.74%. In addition, the technique was able to detect parasites from the six DTUs of T. cruzi and proved to be highly sensitive when detecting up to 0.1 parasite equivalents/mL in açaí samples, differentiating viable from total parasites (when compared to DNA detection). Finally, it was possible to validate this RT-qPCR assay using açaí samples positive for T. cruzi DNA collected in a municipality with a history of oral CD outbreak (Coari-AM). 88.8% were also positive for T. cruzi RNA, suggesting that viable parasites can also be detected in these samples collected in the field.

Palavras-chave

Trypanosoma cruzi, viability, molecular diagnosis, oral Chagas disease, açaí, RT- qPCR.