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1.
Food Chem ; 305: 125426, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31522124

RESUMO

Genetically modified (GM) Atlantic salmon, AquAdvantage (AquAd), was the first GM animal approved officially for human consumption. Many countries monitor the use of this product under their GM regulations, but a pragmatic system for AquAd-specific detection is needed. Here, we developed a real-time polymerase chain reaction method with high sensitivity for detection of AquAd in foods. This method showed high specificity for the AquAd transgene and the detection limit was 12.5-25 targeted DNA copies per test reaction. An inter-laboratory study using the method developed demonstrated reproducibility at >0.1% (w/w) AquAd content.


Assuntos
Alimentos Geneticamente Modificados , Reação em Cadeia da Polimerase em Tempo Real/métodos , Salmo salar/genética , Alimentos Marinhos/análise , Animais , Animais Geneticamente Modificados , Reprodutibilidade dos Testes
2.
Biosci Biotechnol Biochem ; 84(4): 670-677, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31842715

RESUMO

Rapid DNA template preparation directly from a single rice (Oryza sativa) grain or rice flour of its equivalent weight was developed for loop-mediated isothermal amplification (LAMP). LAMP efficiency using DNA extract obtained from consecutive addition of alkaline lysis reagent (25 mM NaOH, 0.2 mM EDTA) and neutralizing reagent (40 mM Tris-HCl [pH 5]) was comparable to that using an equivalent amount of purified DNA as template. The stability of the prepared DNA extract was confirmed for up to six-day storage at room temperature. Without using any special laboratory devices, the developed method enabled a rapid, simple, and low-cost DNA template preparation method for reliable LAMP testing to detect rice genes.

3.
Data Brief ; 27: 104695, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31720342

RESUMO

This article is referred to the research article entitled "Development of a novel method for specific detection of genetically modified Atlantic salmon, AquAdvantage, using real-time polymerase chain reaction" by Soga et al. (2020). Applicability of the developed growth hormone 1 (GH1) and 18S ribosomal DNA (18S rDNA) detection methods using real-time polymerase chain reaction (PCR) for detecting Atlantic salmon (Salmo salar) to processed food commodities was examined. DNAs extracted and purified from 24 commodities labelled to include salmon as an ingredient were used as template. Yield and purity of DNAs obtained and Cq values from real-time PCR analyses were provided.

4.
Anal Chem ; 91(20): 12733-12740, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31482708

RESUMO

Nucleic acid amplification methods, such as polymerase chain reaction (PCR), are extensively used in many applications to detect target DNA because of their high sensitivity, good reproducibility, and wide dynamic range of quantification. However, analytical quality control when detecting low copy number target DNA is often missing because of a lack of appropriate reference materials. Recent advances in analytical sciences require a method to accurately quantify DNA at the single molecule level. Herein, we have developed a novel method to produce reference material containing a defined copy number of target DNA (referred to as "cell number-based DNA reference material"). In this method, a suspension of cells carrying a single target DNA sequence was ejected by an inkjet head, and the number of cells in each droplet was counted using highly sensitive cameras. The resulting solutions contained a defined copy number of target DNA and could be used as reference materials. The use of the newly developed reference material was compared with that of diluted solutions of target DNA to evaluate the performance of qualitative real-time PCR in terms of the limit of detection (LOD). Our results demonstrated that cell number-based DNA reference material provides more accurate information regarding performance quality. The reference material produced by this method is a promising tool to evaluate assay performance.

5.
Shokuhin Eiseigaku Zasshi ; 59(3): 151-156, 2018.
Artigo em Japonês | MEDLINE | ID: mdl-30033993

RESUMO

Highly processed foods, including soy sauce, cornflakes, starch sugar, beet sugar and vegetable oil, are not currently subject to genetically modified (GM) food labeling, because DNA could not be detected in these food products. Here we re-examined the method of DNA extraction from starch syrup, beet sugar and vegetable oil using commercially available DNA extraction kits. We found that DNA was not stably detected by PCR targeting a species-specific endogenous plant gene. The reason for this may have been that the DNA yield was below the detection limit, because PCR inhibition was not observed.


Assuntos
DNA de Plantas/análise , Análise de Alimentos/métodos , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase
6.
J Agric Food Chem ; 66(29): 7839-7845, 2018 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-29949351

RESUMO

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
Técnicas de Amplificação de Ácido Nucleico/métodos , Plantas Geneticamente Modificadas/genética , Soja/genética , Zea mays/genética , Produtos Agrícolas/química , Produtos Agrícolas/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/química , Soja/química , Zea mays/química , Zea mays/metabolismo
7.
Food Chem ; 252: 390-396, 2018 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-29478558

RESUMO

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 were ≤ 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 1 h.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Plantas Geneticamente Modificadas/genética , Soja/genética , Zea mays/genética , Caulimovirus/genética , Produtos Agrícolas/genética , Primers do DNA/genética , Limite de Detecção
8.
J AOAC Int ; 101(2): 507-514, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-28847345

RESUMO

Current genetically modified organism (GMO) detection methods allow for sensitive detection. However, a further increase in sensitivity will enable more efficient testing for large grain samples and reliable testing for processed foods. In this study, we investigated real-time PCR-based GMO detection methods using a large amount of DNA template. We selected target sequences that are commonly introduced into many kinds of GM crops, i.e., 35S promoter and nopaline synthase (NOS) terminator. This makes the newly developed method applicable to a wide range of GMOs, including some unauthorized ones. The estimated LOD of the new method was 0.005% of GM maize events; to the best of our knowledge, this method is the most sensitive among the GM maize detection methods for which the LOD was evaluated in terms of GMO content. A 10-fold increase in the DNA amount as compared with the amount used under common testing conditions gave an approximately 10-fold reduction in the LOD without PCR inhibition. Our method is applicable to various analytical samples, including processed foods. The use of other primers and fluorescence probes would permit highly sensitive detection of various recombinant DNA sequences besides the 35S promoter and NOS terminator.


Assuntos
DNA de Plantas/química , Grão Comestível/genética , Alimentos Geneticamente Modificados , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Zea mays/genética , Primers do DNA/genética , Limite de Detecção
9.
Food Chem ; 226: 149-155, 2017 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-28254006

RESUMO

DNA analysis of processed foods is performed widely to detect various targets, such as genetically modified organisms (GMOs). Food processing often causes DNA fragmentation, which consequently affects the results of PCR analysis. In order to assess the effects of DNA fragmentation on the reliability of PCR analysis, we investigated a novel methodology to quantify the degree of DNA fragmentation. We designed four real-time PCR assays that amplified 18S ribosomal RNA gene sequences common to various plants at lengths of approximately 100, 200, 400, and 800 base pairs (bp). Then, we created an indicator value, "DNA fragmentation index (DFI)", which is calculated from the Cq values derived from the real-time PCR assays. Finally, we demonstrated the efficacy of this method for the quality control of GMO detection in processed foods by evaluating the relationship between the DFI and the limit of detection.


Assuntos
Fragmentação do DNA , DNA de Plantas/genética , Alimentos Geneticamente Modificados , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos
10.
Breed Sci ; 67(5): 544-547, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29398950

RESUMO

Simple sequence repeat (SSR) is a popular tool for individual fingerprinting. The long-core motif (e.g. tetra-, penta-, and hexa-nucleotide) simple sequence repeats (SSRs) are preferred because they make it easier to separate and distinguish neighbor alleles. In the present study, a new set of 8 tetra-nucleotide SSRs in potato (Solanum tuberosum) is reported. By using these 8 markers, 72 out of 76 cultivars obtained from Japan and the United States were clearly discriminated, while two pairs, both of which arose from natural variation, showed identical profiles. The combined probability of identity between two random cultivars for the set of 8 SSR markers was estimated to be 1.10 × 10-8, confirming the usefulness of the proposed SSR markers for fingerprinting analyses of potato.

11.
Shokuhin Eiseigaku Zasshi ; 57(6): 187-192, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28025452

RESUMO

A real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) soybean event, MON87701. First, a standard plasmid for MON87701 quantification was constructed. The conversion factor (Cf) required to calculate the amount of genetically modified organism (GMO) was experimentally determined for a real-time PCR instrument. The determined Cf for the real-time PCR instrument was 1.24. For the evaluation of the developed method, a blind test was carried out in an inter-laboratory trial. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSDr), respectively. The determined biases and the RSDr values were less than 30 and 13%, respectively, at all evaluated concentrations. The limit of quantitation of the method was 0.5%, and the developed method would thus be applicable for practical analyses for the detection and quantification of MON87701.


Assuntos
Análise de Alimentos/métodos , Alimentos Geneticamente Modificados , Organismos Geneticamente Modificados , Reação em Cadeia da Polimerase/métodos , Soja , Sequência de Bases , Reprodutibilidade dos Testes , Soja/genética
12.
Data Brief ; 7: 1165-70, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27408919

RESUMO

This article is referred to research article entitled "Whole genome sequence analysis of unidentified genetically modified papaya for development of a specific detection method" (Nakamura et al., 2016) [1]. Real-time polymerase chain reaction (PCR) detection method for unauthorized genetically modified (GM) papaya (Carica papaya L.) line PRSV-YK (PRSV-YK detection method) was developed using whole genome sequence data (DDBJ Sequenced Read Archive under accession No. PRJDB3976). Interlaboratory validation datasets for PRSV-YK detection method were provided. Data indicating homogeneity of samples prepared for interlaboratory validation were included. Specificity and sensitivity test data for PRSV-YK detection method were also provided.

13.
Biosci Biotechnol Biochem ; 80(11): 2198-2207, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27399872

RESUMO

In rice, several allergens have been identified such as the non-specific lipid transfer protein-1, the α-amylase/trypsin-inhibitors, the α-globulin, the 33 kDa glyoxalase I (Gly I), the 52-63 kDa globulin, and the granule-bound starch synthetase. The goal of the present study was to define optimal rice extraction and detection methods that would allow a sensitive and reproducible measure of several classes of known rice allergens. In a three-laboratory ring-trial experiment, several protein extraction methods were first compared and analyzed by 1D multiplexed SDS-PAGE. In a second phase, an inter-laboratory validation of 2D-DIGE analysis was conducted in five independent laboratories, focusing on three rice allergens (52 kDa globulin, 33 kDa glyoxalase I, and 14-16 kDa α-amylase/trypsin inhibitor family members). The results of the present study indicate that a combination of 1D multiplexed SDS-PAGE and 2D-DIGE methods would be recommended to quantify the various rice allergens.

14.
Shokuhin Eiseigaku Zasshi ; 57(1): 1-6, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26936302

RESUMO

A novel real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) maize, 3272. We first attempted to obtain genome DNA from this maize using a DNeasy Plant Maxi kit and a DNeasy Plant Mini kit, which have been widely utilized in our previous studies, but DNA extraction yields from 3272 were markedly lower than those from non-GM maize seeds. However, lowering of DNA extraction yields was not observed with GM quicker or Genomic-tip 20/G. We chose GM quicker for evaluation of the quantitative method. We prepared a standard plasmid for 3272 quantification. The conversion factor (Cf), which is required to calculate the amount of a genetically modified organism (GMO), was experimentally determined for two real-time PCR instruments, the Applied Biosystems 7900HT (the ABI 7900) and the Applied Biosystems 7500 (the ABI7500). The determined Cf values were 0.60 and 0.59 for the ABI 7900 and the ABI 7500, respectively. To evaluate the developed method, a blind test was conducted as part of an interlaboratory study. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSDr). The determined values were similar to those in our previous validation studies. The limit of quantitation for the method was estimated to be 0.5% or less, and we concluded that the developed method would be suitable and practical for detection and quantification of 3272.


Assuntos
DNA de Plantas/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Zea mays/química , Zea mays/genética , Genoma de Planta/genética , Plantas Geneticamente Modificadas , Reprodutibilidade dos Testes , Sementes/genética
15.
Food Chem ; 205: 272-9, 2016 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-27006240

RESUMO

Identification of transgenic sequences in an unknown genetically modified (GM) papaya (Carica papaya L.) by whole genome sequence analysis was demonstrated. Whole genome sequence data were generated for a GM-positive fresh papaya fruit commodity detected in monitoring using real-time polymerase chain reaction (PCR). The sequences obtained were mapped against an open database for papaya genome sequence. Transgenic construct- and event-specific sequences were identified as a GM papaya developed to resist infection from a Papaya ringspot virus. Based on the transgenic sequences, a specific real-time PCR detection method for GM papaya applicable to various food commodities was developed. Whole genome sequence analysis enabled identifying unknown transgenic construct- and event-specific sequences in GM papaya and development of a reliable method for detecting them in papaya food commodities.


Assuntos
Carica/genética , Frutas/genética , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Análise de Sequência/métodos , Carica/química , Frutas/química , Genômica
16.
Anal Chem ; 88(8): 4285-93, 2016 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-27010783

RESUMO

A number of genetically modified (GM) maize events have been developed and approved worldwide for commercial cultivation. A screening method is needed to monitor GM maize approved for commercialization in countries that mandate the labeling of foods containing a specified threshold level of GM crops. In Japan, a screening method has been implemented to monitor approved GM maize since 2001. However, the screening method currently used in Japan is time-consuming and requires generation of a calibration curve and experimental conversion factor (C(f)) value. We developed a simple screening method that avoids the need for a calibration curve and C(f) value. In this method, ΔC(q) values between the target sequences and the endogenous gene are calculated using multiplex real-time PCR, and the ΔΔC(q) value between the analytical and control samples is used as the criterion for determining analytical samples in which the GM organism content is below the threshold level for labeling of GM crops. An interlaboratory study indicated that the method is applicable independently with at least two models of PCR instruments used in this study.


Assuntos
DNA de Plantas/análise , DNA de Plantas/genética , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Zea mays/genética , Calibragem , Alimentos Geneticamente Modificados , Japão
17.
Methods Mol Biol ; 1385: 249-57, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26614294

RESUMO

This chapter describes a real-time PCR-based method for quantitation of the relative amount of genetically modified (GM) soybean line GTS 40-3-2 [Roundup Ready(®) soybean (RRS)] contained in a batch. The method targets a taxon-specific soybean gene (lectin gene, Le1) and the specific DNA construct junction region between the Petunia hybrida chloroplast transit peptide sequence and the Agrobacterium 5-enolpyruvylshikimate-3-phosphate synthase gene (epsps) sequence present in GTS 40-3-2. The method employs plasmid pMulSL2 as a reference material in order to quantify the relative amount of GTS 40-3-2 in soybean samples using a conversion factor (Cf) equal to the ratio of the RRS-specific DNA to the taxon-specific DNA in representative genuine GTS 40-3-2 seeds.


Assuntos
Lectinas de Plantas/genética , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes de Fusão/genética , Soja/genética , 3-Fosfoshikimato 1-Carboxiviniltransferase , Agrobacterium/metabolismo , Proteínas de Bactérias , Proteínas de Cloroplastos , Expressão Gênica , Petunia/metabolismo , Sinais Direcionadores de Proteínas , RNA Mensageiro , Soja/metabolismo
18.
Food Chem ; 168: 606-14, 2015 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-25172754

RESUMO

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/análise
19.
Anal Chem ; 86(17): 8621-7, 2014 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-25061686

RESUMO

We developed a reference material of a single DNA molecule with a specific nucleotide sequence. The double-strand linear DNA which has PCR target sequences at the both ends was prepared as a reference DNA molecule, and we named the PCR targets on each side as confirmation sequence and standard sequence. The highly diluted solution of the reference molecule was dispensed into 96 wells of a plastic PCR plate to make the average number of molecules in a well below one. Subsequently, the presence or absence of the reference molecule in each well was checked by real-time PCR targeting for the confirmation sequence. After an enzymatic treatment of the reaction mixture in the positive wells for the digestion of PCR products, the resultant solution was used as the reference material of a single DNA molecule with the standard sequence. PCR analyses revealed that the prepared samples included only one reference molecule with high probability. The single-molecule reference material developed in this study will be useful for the absolute evaluation of a detection limit of PCR-based testing methods, the quality control of PCR analyses, performance evaluations of PCR reagents and instruments, and the preparation of an accurate calibration curve for real-time PCR quantitation.


Assuntos
DNA/análise , DNA/normas , Plantas Geneticamente Modificadas/genética , Controle de Qualidade , Reação em Cadeia da Polimerase em Tempo Real/normas
20.
Shokuhin Eiseigaku Zasshi ; 55(1): 25-33, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24598224

RESUMO

To improve the efficiency of DNA analysis of foods and agricultural products, we investigated a direct real-time PCR based on the real-time monitoring of DNA amplification directly from crude cell lysates of analytical samples. We established a direct real-time PCR system comprising sample pretreatment with a specified lysis buffer and real-time PCR using the developed master mix reagent. No PCR inhibition was observed in the analysis of crude cell lysates from 50 types of samples, indicating that the direct real-time PCR system is applicable to a wide range of materials. The specificity of the direct real-time PCR was evaluated by means of a model assay system for single nucleotide discrimination. Even when crude cell lysates coexisted in the reaction mixtures, the primer selectivity was not affected, suggesting that the sequence specificity of the direct real-time PCR was equivalent to that of PCR from purified DNA templates. We evaluated the sensitivity and quantitative performance of the direct real-time PCR using soybean flour samples including various amounts of genetically modified organisms. The results clearly showed that the direct real-time PCR system provides sensitive detection and precise quantitation.


Assuntos
Produtos Agrícolas/química , Análise de Alimentos/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Alimentos Geneticamente Modificados , Soja/química
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