Preventing Chagas disease: A new RT-qPCR method for rapid and specific quantification of viable Trypanosoma cruzi for food safety.

Without standardized methods for rapidly detecting in food matrices viable T. cruzi, foodborne outbreaks remain neglected. In this work, a reverse-transcriptase real-time PCR (RT-qPCR) mRNA-based technique was developed for the rapid and specific detection and quantification of viable Trypanosoma cruzi in açai fruits and juice. The method uses specific primer targeting region on the cyt b gene. The maximum recovery rate of T. cruzi from inoculated açai juice was 82.50%. The limit of detection and quantification in açai juice was 10 parasites/mL for RT-qPCR (mRNA-based) and qPCR (DNA-based). The RT-qPCR efficiency was estimated at 97.27% with an R2 of 0.994. The RT-qPCR was shown to be able to discriminate between viable and nonviable cells. This method provides a useful tool for rapid assessment of low concentrations of viable T. cruzi in naturally contaminated food samples, and can be applied industrially as a quality and security method.

Prevention methods of foodborne Chagas disease: Disinfection, heat treatment and quality control by RT-PCR.

The most important mode of transmission causing outbreaks of Chagas disease in the Amazon region is the oral route due to the ingestion of contaminated food. Herein, prevention methods for foodborne diseases caused by Trypanosoma cruzi, namely, sanitization, thermal treatment were investigated and the use of reverse transcription PCR (RT-PCR) amplification for the mRNA-based detection of viable T. cruzi in açai, was developed. Three T. cruzi strains (T. cruzi I, T. cruzi III and Y) were used in the present study. The Amazonian strains T. cruzi I (425) and T. cruzi III (370) showed higher resistance to sodium hypochlorite treatment and heat treatment than the reference strain Y. The blanching of fruits (70 ±â€¯1 °C for 10 s) and pasteurization of juice (82.5 °C for 1 min) efficiently eliminated T. cruzi in food matrices. Additionally, a method that uses RT-PCR amplification of mRNA was developed for the detection of viable T. cruzi in açai, which could play a role in examining food samples, ensuring consumer health, and reducing this foodborne disease.

qPCR for the detection of foodborne Trypanosoma cruzi.

Here we presented a potential real-time PCR (qPCR) method with public health importance and relevance for detection of Trypanosoma cruzi in açai pulp. There is not a current process to identify T. cruzi in açai, that ensures innocuity of this food concerning oral transmission. First, six new primers were designed using the DNA sequences of T. cruzi y152 and Emerald strains obtained from GenBank. For primers evaluation and titration they were validated regarding the amplification and not with the fluorophore chosen 1ngµL-1 of the T. cruzi DNA as target. For determination of the ideal concentration the titration of the primers drawn in this study showed T. cruzi DNA amplification in five primer pairs at concentrations 100, 200 and 300nM and DNA fixed concentrations at 1ngµL-1. For standardization all reactions were performed in triplicate with 5.0µL and positives and negatives controls were included in every run. As positive control DNA from two genotypes TcI and TcII were used. As negative control the reaction product without DNA of the parasite was used. The best primer concentration, for the expected fragments, was 300nM. From six primers improved the Ep1F/Ep1R primer detected 1×10-4ngµL-1 for both genotype of the parasite. The Bp1F/Bp1R showed amplification for 1.70.10-7ngµL-1 for TcI and 4.31.10-8ngµL-1 for TcII, based on the standard curve. The last step we tested the selected primers in qPCR for monitoring T. cruzi in açai pulp experimentally contaminated. The recovery rate for the TcII was 71%, whereas in açai samples contaminated with TcI it was 76%.