qPCR for the detection of foodborne Trypanosoma cruzi

• Published in: Parasitology international Vol. 66; no. 5; pp. 563 - 566
• Main Authors: de Souza Godoi, Poliana Alves, Piechnik, Claudio Adriano, de Oliveira, Ana Caroline, Sfeir, Michelle Zibetti, de Souza, Emanuel Maltempi, Rogez, Hervé, Thomaz Soccol, Vanete
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.10.2017
• Subjects: Animals; Açaiberries; Chagas disease; Chagas Disease - diagnosis; Chagas Disease - parasitology; Chagas Disease - transmission; DNA Primers; DNA, Protozoan - genetics; Euterpe - parasitology; Foodborne Diseases - diagnosis; Foodborne Diseases - parasitology; Gastroenterology and Hepatology; Genotype; Humans; Infectious Disease; Protozoan; Public Health; qPCR; Real-Time Polymerase Chain Reaction - methods; Trypanosoma cruzi - genetics; Trypanosoma cruzi - isolation & purification; Trypanosoma foodborne transmission; Chagas disease; Açaiberries; Protozoan; qPCR; Trypanosoma foodborne transmission
• ISSN: 1383-5769; 1873-0329
• DOI: 10.1016/j.parint.2017.06.001

Abstract 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 1 ng μL − 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 300 nM and DNA fixed concentrations at 1 ng μL − 1 . For standardization all reactions were performed in triplicate with 5.0 μL 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 300 nM. From six primers improved the Ep1F/Ep1R primer detected 1 × 10 − 4 ng μL − 1 for both genotype of the parasite. The Bp1F/Bp1R showed amplification for 1.70.10 − 7 ng μL − 1 for TcI and 4.31.10 − 8 ng μL − 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%.