Comparison of three DNA extraction methods for the detection and quantification of GMO in Ecuadorian manufactured food

Pacheco, Ricardo; Justo, Jorge Pestana; Mendoza, Andrés Factos; Santos-Ordóñez, Efrén

doi:10.1186/s13104-017-3083-x

Cited by 29 publications

(15 citation statements)

References 14 publications

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“…Leaves from collected samples from one specimen were ground using liquid nitrogen in the grinder MM400 (Retsch, Haan, Germany) and stored at −80 °C upon DNA extraction. Approximately, 100 mg of leaf was used for DNA extraction using a CTAB protocol with some modifications (Pacheco Coello et al, 2017). PCR was performed using the 2× GoTaq ® master mix (Cat.…”

Section: Methodsmentioning

confidence: 99%

Morphological and molecular barcode analysis of the medicinal tree Mimusops coriacea (A.DC.) Miq. collected in Ecuador

Bustamante

Santos-Ordóñez

Martínez

et al. 2019

PeerJ

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Background Mimusops coriacea (A.DC.) Miq., (Sapotaceae), originated from Africa, were introduced to coastal areas in Ecuador where it is not extensively used as a traditional medicine to treat various human diseases. Different therapeutically uses of the species include: analgesic, antimicrobial, hypoglycemic, inflammation and pain relieve associated with bone and articulation-related diseases. Furthermore, Mimusops coriacea could be used as anti-oxidant agent. However, botanical, chemical or molecular barcode information related to this much used species is not available from Ecuador. In this study, morphological characterization was performed from leaves, stem and seeds. Furthermore, genetic characterization was performed using molecular barcodes for rbcL, matk, ITS1 and ITS2 using DNA extracted from leaves. Methods Macro-morphological description was performed on fresh leaves, stem and seeds. For anatomical evaluation, tissues were embedded in paraffin and transversal dissections were done following incubation with sodium hypochlorite and safranin for coloration and fixated later in glycerinated gelatin. DNA extraction was performed using a modified CTAB protocol from leaf tissues, while amplification by PCR was accomplished for the molecular barcodes rbcL, matK, ITS1 and ITS2. Sequence analysis was performed using blast in the GenBank. Phylogenetic analysis was performed with accessions queried in the GenBank belonging to the subfamily Sapotoideae. Results Leaf size was 13.56 ± 1.46 × 7.49 ± 0.65 cm; where is a macro-morphological description of the stem (see Methods). The peel of the seeds is dark brown. Sequence analysis revealed that amplicons were generated using the four barcodes selected. Phylogenetic analysis indicated that the barcodes rbcL and matK, were not discriminated between species within the same genus of the subfamily Sapotoideae. On the other hand, the ITS1 and ITS2 were discriminative at the level of genus and species of the Sapotoideae.

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Section: Methodsmentioning

confidence: 99%

Morphological and molecular barcode analysis of the medicinal tree Mimusops coriacea (A.DC.) Miq. collected in Ecuador

Bustamante

Santos-Ordóñez

Martínez

et al. 2019

PeerJ

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“…En esta investigación se analizaron 36 alimentos de consumo masivo, el 86 % de estos resultaron positivos para la presencia del promotor 35S, 72 % para el terminador NOS y 40 % para eventos específicos de maíz y soya. En Ecuador se realizó un monitoreo con 35 muestras de alimentos procesados, de los cuales 26 (74 %) resultaron positivas para la presencia de p-35S, y 8 (23 %) mostraron trazas de la secuencia del t-NOS (Coello et al, 2017). Otro estudio realizado en México con muestras (n=370) de alimentos procesados de consumo masivo y derivados del maíz, provenientes de varios países, mostró que el 82 % del total de las muestras analizadas contenía secuencias de ADN transgénico (González-Ortega et al, 2017).…”

Section: Discussionunclassified

Detección del promotor 35S mediante PCR tiempo-real: indicador de transgenicidad en alimentos y Gossypium sp.

Oviedo-Bolaños¹,

García-González

Solano-González³

et al. 2020

Agron. Mesoam.

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Introducción. Los cultivos genéticamente modificados (CGM) son de particular interés, debido al impacto en la economía global. Por lo tanto, como preocupación general, muchos países han establecido regulaciones con respecto a estos organismos. En Costa Rica, el cultivo de OGM se realiza desde 1991; sin embargo, existe un faltante de estudios que monitoreen la ejecución y el cumplimiento de las regulaciones de bioseguridad. Objetivo. El objetivo del presente estudio fue identificar la presencia o ausencia de transgenicidad en alimentos procesados para consumo humano y animal, así como en semillas provenientes de algodón. Materiales y métodos. Se utilizó la técnica de PCR tiempo real dirigida a la secuencia promotora 35S, derivada del virus del mosaico de la coliflor (CaMV, siglas en inglés), como marcador para detectar presencia de transgenes en alimentos procesados para consumo humano y animal y en semillas de algodón, silvestre o cultivado, recolectadas en áreas aledañas a una finca de algodón GM en mayo de 2017. Resultados. En las muestras analizadas hubo una alta incidencia de fragmentos de 82 pb, correspondientes a la secuencia promotora 35S, que estuvo ausente solo en cultivos de maíz orgánico y sus derivados (tortillas y harina de maíz). Los resultados sugieren la presencia de trazas de OGM en el mercado alimentario costarricense; adicionalmente, revela la urgencia de implementar un adecuado etiquetado para trazabilidad alimentaria. Se identificó, además, la presencia de algodón transgénico en las cercanías de una finca de algodón GM, lo que sugiere la pertinencia de vigilancia en aspectos de bioseguridad y manipulación genética de cultivos. Conclusiones. La presencia de trazas de OGM en alimentos procesados muestra la importancia de continuar este monitoreo para proveer elementos de discusión en cuanto a trazabilidad alimentaria y potencial flujo de transgenes en material vegetal silvestre.

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“…Leaf samples from the three S. purhampuy Ruiz plants (with codes CIBE-010, CIBE-011, CIBE-012) were ground with MM400 (Retsch, Haan, Germany) and liquid nitrogen and were stored at −80 • C. DNA extraction was performed for each Smilax plant independently. A modified CTAB protocol was used for total DNA extraction according to Pacheco Coello et al (2017). The master mix GoTaq R 2x (Cat# M7123, Promega) was used for PCR analysis according to the manufacturer's instructions using 0.5 µM for each primer according to the barcode used (Table S1) in a 50 µL PCR reaction.…”

Section: Dna Extraction and Pcrmentioning

confidence: 99%

Molecular barcode and morphological analysis of Smilax purhampuy Ruiz, Ecuador

Soledispa

Santos-Ordóñez

Miranda

et al. 2021

PeerJ

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Smilax plants are distributed in tropical, subtropical, and temperate regions in both hemispheres of the world. They are used extensively in traditional medicines in a number of countries. However, morphological and molecular barcodes analysis, which may assist in the taxonomic identification of species, are lacking in Ecuador. In order to evaluate the micromorphological characteristics of these plants, cross sections of Smilax purhampuy leaves were obtained manually. The rhizome powder, which is typically used in traditional medicines, was analyzed for micromorphological characteristics. All samples were clarified with 1% sodium hypochlorite. Tissues were colored with 1% safranin in water and were fixed with glycerinated gelatin. DNA was extracted from the leaves using a modified CTAB method for molecular barcode characterization and PCR was performed using primers to amplify the different loci including the plastid genome regions atpF-atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK–psbI spacer, and trnH–psbA spacer; and the nuclear DNA sequence ITS2. A DNA sequence similarity search was performed using BLAST in the GenBank nr database and phylogenetic analysis was performed using the maximum likelihood method according to the best model identified by MEGAX using a bootstrap test with 1,000 replicates. Results showed that the micromorphological evaluation of a leaf cross section depicted a concave arrangement of the central vein, which was more pronounced in the lower section and had a slight protuberance. The micromorphological analysis of the rhizome powder allowed the visualization of a group of cells with variable sizes in the parenchyma and revealed thickened xylematic vessels associated with other elements of the vascular system. Specific amplicons were detected in DNA barcoding for all the barcodes tested except for the trnH–psbA spacer. BLAST analysis revealed that the Smilax species was predominant in all the samples for each barcode; therefore, the genus Smilax was confirmed through DNA barcode analysis. The barcode sequences psbK-psbI, atpF-atpH, and ITS2 had a better resolution at the species level in phylogenetic analysis than the other barcodes we tested.

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Comparison of three DNA extraction methods for the detection and quantification of GMO in Ecuadorian manufactured food

Cited by 29 publications

References 14 publications

Morphological and molecular barcode analysis of the medicinal tree Mimusops coriacea (A.DC.) Miq. collected in Ecuador

Morphological and molecular barcode analysis of the medicinal tree Mimusops coriacea (A.DC.) Miq. collected in Ecuador

Detección del promotor 35S mediante PCR tiempo-real: indicador de transgenicidad en alimentos y Gossypium sp.

Molecular barcode and morphological analysis of Smilax purhampuy Ruiz, Ecuador

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