RESUMO
A loop-mediated isothermal amplification (LAMP)-mediated screening detection method for genetically modified (GM) papaya was developed targeting the 35S promoter (P35S) of the cauliflower mosaic virus. LAMP products were detected using a Genie II real-time fluorometer. The limit of detection (LOD) was evaluated and found to be ≤0.05% for papaya seeds. We also designed a primer set for the detection of the papaya endogenous reference sequence, chymopapain, and the species-specificity was confirmed. To improve cost-effectiveness, single-stranded tag hybridization (STH) on a chromatography printed-array strip (C-PAS) system, which is a lateral flow DNA chromatography technology, was applied. LAMP amplification was clearly detected by the system at the LOD level, and a duplex detection of P35S and chymopapain was successfully applied. This simple and quick method for the screening of GM papaya will be useful for the prevention of environmental contamination of unauthorized GM crops.
Assuntos
Carica , Quimopapaína , Carica/genética , Plantas Geneticamente Modificadas/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Verduras , Sensibilidade e EspecificidadeRESUMO
Species-specific endogenous reference sequences are indispensable in the development of methods to detect genetically modified (GM) crops for food and feed. We analyzed a partial sequence derived from the ß-fructosidase gene among several solanaceous species and developed a new eggplant specific detection method using loop-mediated isothermal amplification (LAMP). LAMP is a rapid, specific, and cost-effective technique. The species-specificity and stability of the developed method were evaluated using 18 eggplant cultivars and other crops including solanaceous plants. The limit of detection was also evaluated. The developed method showed high specificity for eggplants and stability among the eggplant cultivars tested. These results suggested that the developed method would be useful as a positive control for the detection of GM eggplants with LAMP.
Assuntos
Solanum melongena , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Plantas Geneticamente Modificadas/genética , Solanum melongena/genética , Especificidade da EspécieRESUMO
We developed a novel loop-mediated isothermal amplification (LAMP)-based detection method using lateral flow dipstick chromatography for genetically modified (GM) soybean and maize events. The single-stranded tag hybridization (STH) for the chromatography printed-array strip (C-PAS) system was used for detections targeting the cauliflower mosaic virus 35S promoter, mannose-6-phosphate isomerase gene, Pisum sativum ribulose 1, 5-bisphosphate carboxylase terminator, a common sequence between the Cry1Ab and Cry1Ac genes, and a GA21-specific sequence. The STH C-PAS system was applicable for multiplex analyses to perform simultaneous detections. The limit of detection was 0.5% or less for each target. By using the developed method, the LAMP amplification was visually detected. Moreover, the detection could be carried out without any expensive instruments, even for the DNA amplification steps, by virtue of the isothermal reaction. We demonstrated that the rapid and useful method developed here would be applicable for screening GM crops.
Assuntos
Glycine max/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Plantas Geneticamente Modificadas/genética , Zea mays/genética , Produtos Agrícolas/química , Produtos Agrícolas/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/química , Glycine max/química , Zea mays/química , Zea mays/metabolismoRESUMO
We developed new loop-mediated isothermal amplification (LAMP)-based detection methods for the screening of genetically modified (GM) maize and soybean events. The LAMP methods developed targeted seven sequences: cauliflower mosaic virus 35S promoter; 5-enolpyruvylshikimate-3-phosphate synthase gene from Agrobacterium tumefaciens strain CP4 (cp4epsps); phosphinothricin acetyltransferase (pat) gene; mannose-6-phosphate isomerase gene; Pisum sativum ribulose 1, 5-bisphosphate carboxylase terminator; a common sequence between Cry1Ab and Cry1Ac genes; and a GA21 construct-specific sequence. We designed new specific primer sets for each target, and the limit of detection (LOD) was evaluated using authorized GM maize and soybean events. LODs for each target wereâ¯≤â¯0.5%. To make the DNA extraction process simple and rapid, we also developed a direct LAMP detection scheme using crude cell lysates. The entire process, including pretreatments and detection, could be completed within 1â¯h.
Assuntos
Glycine max/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Plantas Geneticamente Modificadas/genética , Zea mays/genética , Caulimovirus/genética , Produtos Agrícolas/genética , Primers do DNA/genética , Limite de DetecçãoRESUMO
To evaluate the digestibility of rice allergenic and nonallergenic proteins under the influence of the rice grain matrix, rice powder was subjected to in vitro digestion by simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Rice proteins were extracted from the liquid and the solid phases and analysed by SDS-PAGE, and rice allergenic proteins were detected by a multiplex immunodetection method. The digestion of soluble proteins was carried out in both liquid and solid phases, while that of insoluble proteins only occurred in the solid phase. In SGF digestion, rice proteins were more quickly digested at pH 1.2 than at pH 2.0 or 2.5. Moreover, the digestibility of five kinds of rice allergenic proteins was influenced by pH level, heat processing, starch matrix, solubility, and protein properties, on a case-by-case basis. On the other hand, all detected rice allergenic proteins and non-allergenic proteins were rapidly digested in SIF.
Assuntos
Alérgenos/metabolismo , Oryza/metabolismo , Extratos Vegetais/metabolismo , Proteínas de Plantas/imunologia , Alérgenos/análise , Digestão , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Immunoblotting , Proteínas de Plantas/análiseRESUMO
The influence of different extraction solutions on the proteins extracted from rice grains was investigated. The largest amounts of salt-soluble proteins were extracted with solutions supplemented with Tris-HCl at pH 8.0. Rice allergens were analyzed by multiplex immunodetection. Except for α-globulin extracted with the solutions at pH 8.0, which showed a low-molecular-weight band besides the main band, no significant solution-dependent difference among the allergens was found. Total proteins were extracted with four kinds of solution. The extraction of the basic subunit of glutelin was found to be SDS-dependent, and more protein was obtained with extraction solutions supplemented with SDS. The contents of α-globulin and α-amylase/trypsin inhibitors were higher in the extracts without SDS than with SDS. We conclude from the present data that, in order to obtain comparable data from rice grain salt-soluble and total protein analyses, differences in the protein extraction efficiency of solutions used should be taken into consideration.
