Protocol optimization for the detection of Trypanosoma cruzi DNA in açai (Euterpe oleraceae) pulp

CARDOSO, Gabrielle Virgínia Ferreira; OLIVEIRA, Andrey Carlos do Sacramento de; SILVA, Andréia Silva da; SILVA, Marcos Clécio de Lemos; LIMA, Joelson Sousa; ROOS, Talita Bandeira; MORAES, Carina Martins de

doi:10.1590/1809-4392201903426

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Health Sciences • Acta Amaz. 51 (1) • Jan-Mar 2021 • https://doi.org/10.1590/1809-4392201903426 copy

Protocol optimization for the detection of Trypanosoma cruzi DNA in açai (Euterpe oleraceae) pulp

Otimização de protocolo para detecção de DNA de Trypanosoma cruzi em polpa de açaí (Euterpe oleracea)

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT

Chagas disease, caused by the protozoan Trypanosoma cruzi, has often been linked to oral transmission through açai consumption. Molecular methods that allow fast and accurate identification of the pathogen are important for the detection of the presence of the parasite in this food. This study aimed to optimize polymerase chain reaction (PCR)-based detection of T. cruzi DNA in açai pulp. Several dilutions of T. cruzi DTU TcI trypomastigote forms were cultured in liver infusion tryptose (LIT) medium. Trypanosoma cruzi DNA was extracted from the cells and subjected to PCR. Subsequently, culture dilutions were added to açai pulp to evaluate the detection threshold of the optimized PCR assay. We demonstrate that our assay can detect T. cruzi DNA in açai pulp at a concentration of 1.08 × 10^-10 ng µL^-1. We conclude that our optimized protocol is effective and can be used as an important tool for the detection of T. cruzi contamination in açaí.

KEYWORDS:
detection method; foodborne disease; food pathogen; molecular detection; Chagas disease

Authorship

Gabrielle Virgínia Ferreira CARDOSO ^**Corresponding author: gabi.virginia.mv@gmail.com

Universidade Federal do Pará, Instituto de Medicina Veterinária, Laboratório de Higiene e Qualidade de Alimentos, BR 316, Km 62, Saudade II , Castanhal - PA, BrazilUniversidade Federal do ParáBrazilCastanhal, PA, BrazilUniversidade Federal do Pará, Instituto de Medicina Veterinária, Laboratório de Higiene e Qualidade de Alimentos, BR 316, Km 62, Saudade II , Castanhal - PA, Brazil

http://orcid.org/0000-0002-6407-2068

Andrey Carlos do Sacramento de OLIVEIRA

Universidade Maurício de Nassau, Av. Gentil Bitencourt, 98, Batista Campos, Belém - PA, BrazilUniversidade Maurício de NassauBrazilBelém, PA, Brazil Universidade Maurício de Nassau, Av. Gentil Bitencourt, 98, Batista Campos, Belém - PA, Brazil

Andréia Silva da SILVA

Marcos Clécio de Lemos SILVA

Universidade Norte do Paraná, Av. Máximino Porpino Silva, 2002, Centro, Castanhal - PA, BrazilUniversidade Norte do ParanáBrazilCastanhal, PA, Brazil Universidade Norte do Paraná, Av. Máximino Porpino Silva, 2002, Centro, Castanhal - PA, Brazil

Joelson Sousa LIMA

Talita Bandeira ROOS

Carina Martins de MORAES

^*Corresponding author: gabi.virginia.mv@gmail.com

ASSOCIATE EDITOR:

Izeni P. Farias

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (3)

Thumbnail

Figure 1
A 2% agarose gel showing T. cruzi DNA fragments (approximately 149 bp). L: 50-bp marker; lane 1: dilution of DNA 10⁰ (equivalent to a concentration of 133.6 ng µL^-1); lane 2: DNA 10^-1 (1.336 x 10^-3 ng µL^-1); lane 3: DNA 10^-2 (1.336 x 10^-4 ng µL^-1); lane 4: DNA 10^-3 (1.336 x 10^-5 ng µL^-1); lane 5: DNA 10^-4 (1.336 x 10^-6 ng µL^-1); lane 6: DNA 10^-5 (1.336 x 10^-7 ng µL^-1); lane 7: DNA 10^-6 (1.336 x 10^-8 ng µL^-1); lane 8: DNA 10^-7 (1.336 x 10^-9 ng µL^-1); lane 9: DNA 10^-8 (1.336 x 10^-10 ng µL^-1); lane 10: DNA 10^-9 (1.336 x 10^-11 ng µL^-1); lane 11: DNA 10^-10 (1.336 x 10^-12 ng µL^-1); lane 12: DNA 10^-11 (1.336 x 10^-13 ng µL^-1); lane 13: DNA 10^-12 (1.336 x 10^-14 ng µL^-1); lane 14: DNA 10^-13 (1.336 x 10^-15 ng µL^-1); lane 15: DNA 10^-14 (1.336 x 10^-16 ng µL^-1); lane 16: DNA 10^-15 (1.336 x 10^-17 ng µL^-1); lane 17: DNA 10^-16 (1.336 x 10^-18 ng µL^-1) - detection threshold; lane 18: DNA 10^-17 (1.336 x 10^-19 ng µL^-1); C-: negative control.

Thumbnail

Figure 2
A 2% agarose gel showing PCR-amplified T. cruzi DNA fragments (149 bp) at different dilutions of T. cruzi inoculated in açai pulp. L: 50 bp marker; lane 1: diluition of DNA 10^-0 (equivalent to a concentration 10.8 ng µL^-1); lane 2: DNA 10^-1 (1.08 x 10^-2 ng µL^-1); lane 3: DNA 10^-2 (1.08 x 10^-3 ng µL^-1); lane 4: DNA 10^-3 (1.08 x 10^-4 ng µL^-1); lane 5: DNA 10^-4 (1.08 x 10^-5 ng µL^-1); lane 6: DNA10^-5 (1.08 x 10^-6 ng µL^-1); lane 7: DNA 10^-6 (1.08 x 10^-7 ng µL^-1); lane 8: DNA 10^-7 (1.08 x 10^-8 ng µL^-1); lane 9: DNA 10^-8 (1.08 x 10^-9 ng µL^-1); lane 10: DNA 10^-9 (1.08 x 10^-10 ng µL^-1) - detection threshold; lane 11: DNA 10^-10 (1.08 x 10^-11 ng µL^-1); C-: negative control.

Thumbnail

Figure 3
Phylogenetic tree showing the relationship of alignment between sequence pairs presented by the NCBI database query produced from the Blast Tree View® program and containing the name of similar paired species and their identification code. “Unknown (Query_39595)” corresponds to the sample used in this study (Trypanosoma cruzi TcIV). This figure is in color in the electronic version.

Figure 1 A 2% agarose gel showing T. cruzi DNA fragments (approximately 149 bp). L: 50-bp marker; lane 1: dilution of DNA 10⁰ (equivalent to a concentration of 133.6 ng µL^-1); lane 2: DNA 10^-1 (1.336 x 10^-3 ng µL^-1); lane 3: DNA 10^-2 (1.336 x 10^-4 ng µL^-1); lane 4: DNA 10^-3 (1.336 x 10^-5 ng µL^-1); lane 5: DNA 10^-4 (1.336 x 10^-6 ng µL^-1); lane 6: DNA 10^-5 (1.336 x 10^-7 ng µL^-1); lane 7: DNA 10^-6 (1.336 x 10^-8 ng µL^-1); lane 8: DNA 10^-7 (1.336 x 10^-9 ng µL^-1); lane 9: DNA 10^-8 (1.336 x 10^-10 ng µL^-1); lane 10: DNA 10^-9 (1.336 x 10^-11 ng µL^-1); lane 11: DNA 10^-10 (1.336 x 10^-12 ng µL^-1); lane 12: DNA 10^-11 (1.336 x 10^-13 ng µL^-1); lane 13: DNA 10^-12 (1.336 x 10^-14 ng µL^-1); lane 14: DNA 10^-13 (1.336 x 10^-15 ng µL^-1); lane 15: DNA 10^-14 (1.336 x 10^-16 ng µL^-1); lane 16: DNA 10^-15 (1.336 x 10^-17 ng µL^-1); lane 17: DNA 10^-16 (1.336 x 10^-18 ng µL^-1) - detection threshold; lane 18: DNA 10^-17 (1.336 x 10^-19 ng µL^-1); C-: negative control.

Figure 2 A 2% agarose gel showing PCR-amplified T. cruzi DNA fragments (149 bp) at different dilutions of T. cruzi inoculated in açai pulp. L: 50 bp marker; lane 1: diluition of DNA 10^-0 (equivalent to a concentration 10.8 ng µL^-1); lane 2: DNA 10^-1 (1.08 x 10^-2 ng µL^-1); lane 3: DNA 10^-2 (1.08 x 10^-3 ng µL^-1); lane 4: DNA 10^-3 (1.08 x 10^-4 ng µL^-1); lane 5: DNA 10^-4 (1.08 x 10^-5 ng µL^-1); lane 6: DNA10^-5 (1.08 x 10^-6 ng µL^-1); lane 7: DNA 10^-6 (1.08 x 10^-7 ng µL^-1); lane 8: DNA 10^-7 (1.08 x 10^-8 ng µL^-1); lane 9: DNA 10^-8 (1.08 x 10^-9 ng µL^-1); lane 10: DNA 10^-9 (1.08 x 10^-10 ng µL^-1) - detection threshold; lane 11: DNA 10^-10 (1.08 x 10^-11 ng µL^-1); C-: negative control.

Figure 3 Phylogenetic tree showing the relationship of alignment between sequence pairs presented by the NCBI database query produced from the Blast Tree View® program and containing the name of similar paired species and their identification code. “Unknown (Query_39595)” corresponds to the sample used in this study (Trypanosoma cruzi TcIV). This figure is in color in the electronic version.

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Protocol optimization for the detection of Trypanosoma cruzi DNA in açai (Euterpe oleraceae) pulp

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[1] ^*Corresponding author: gabi.virginia.mv@gmail.com