The Experts below are selected from a list of 18192 Experts worldwide ranked by ideXlab platform
Dabing Zhang - One of the best experts on this subject based on the ideXlab platform.
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one novel multiple target plasmid Reference Molecule targeting eight genetically modified canola events for genetically modified canola detection
Journal of Agricultural and Food Chemistry, 2017Co-Authors: Canhua Wang, Dabing Zhang, Guiwen Song, Lan Zheng, Litao YangAbstract:Multiple-target plasmid DNA Reference materials have been generated and utilized as good substitutes of matrix-based Reference materials in the analysis of genetically modified organisms (GMOs). Herein, we report the construction of one multiple-target plasmid Reference Molecule, pCAN, which harbors eight GM canola event-specific sequences (RF1, RF2, MS1, MS8, Topas 19/2, Oxy235, RT73, and T45) and a partial sequence of the canola endogenous Reference gene PEP. The applicability of this plasmid Reference material in qualitative and quantitative PCR assays of the eight GM canola events was evaluated, including the analysis of specificity, limit of detection (LOD), limit of quantification (LOQ), and performance of pCAN in the analysis of various canola samples, etc. The LODs are 15 copies for RF2, MS1, and RT73 assays using pCAN as the calibrator and 10 genome copies for the other events. The LOQ in each event-specific real-time PCR assay is 20 copies. In quantitative real-time PCR analysis, the PCR efficie...
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event specific qualitative and quantitative detection of three genetically modified papaya events using a single standard Reference Molecule
Food Control, 2015Co-Authors: Jaehwan Kim, Dabing Zhang, Saetbyul Park, Hyojeong Roh, Hongbae Woo, Minki Shin, Gui Im Moon, Jinhwan Hong, Haeyeong KimAbstract:Abstract A novel standard Reference plasmid (pGEM-PAPAYA3) was constructed as a positive control and Reference standard for event-specific qualitative and quantitative detection of genetically modified (GM) papaya (55-1, 16-0-1, and Huanong No.1). The plasmid pGEM-PAPAYA3 contained the specific fragments of papaya endogenous Reference gene chymopapain and the 3 GM papaya events. The qualitative PCR assay using pGEM-PAPAYA3 was established with a limit of detection correlating to approximately 5–50 copies of papaya haploid genomes. In the quantitative PCR assay, the square regression coefficients (R2) ranged from 0.993 to 0.997. The standard deviation and relative standard deviation values for repeatability ranged from 0.04 to 0.25 and 0.05%–0.86%, respectively. The method was used to test 11 papaya products purchased from Thailand, China, the Philippines and the USA, and the results revealed 2 varieties of GM papaya in 5 of the 11 samples tested. These results indicate the developed detection methods with the standard Reference plasmid are applicable for identifying 3 GM papaya events.
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applicability of a novel Reference Molecule suitable for event specific detections of maize nk603 based on both 5 and 3 flanking sequences
Food Control, 2010Co-Authors: Kailin Shen, Shu Wang, Litao Yang, Liangwen Pan, Dabing ZhangAbstract:Abstract Plasmid Molecule based Reference material (RM) has been shown to be a good alternative as the calibrator for genetically modified organisms (GMOs) identification and quantification, while most of the currently developed plasmid RM can only be used for one specific target detection. In this study, a flexible plasmid RM pNK containing three DNA fragments, i.e. 5′ and 3′ event-specific sequences of maize NK603 and endogenous gene zSSIIb, was developed. We have proved that pNK is suitable for using as a calibrator in both 5′ and 3′ event-specific detection of maize NK603, compared with that of genuine genomic DNA. The limit of detection (LOD) was 10 copies of pNK DNA in conventional PCR assays. The absolute LOD and limit of quantification (LOQ) in quantitative PCR assays were 5 and 25 copies. The standard curves targeting to zSSIIb, 5′ and 3′ event-specific sequences based on pNK DNA showed high reaction efficiency and good linearity. Also, low bias and variations were obtained in practical samples quantification using pNK as the calibrator. These results demonstrated that the developed pNK is flexible and suitable for identification and quantification of maize NK603, as a preferable substitute of RM from the plant raw material.
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development and in house validation of a Reference Molecule pmir604 for simplex and duplex event specific identification and quantification of gm maize mir604
European Food Research and Technology, 2009Co-Authors: Shu Wang, Litao Yang, Kailin Shen, Dabing ZhangAbstract:Effective analysis methods for genetically modified organisms (GMOs) using reliable Reference Molecules as calibrators are necessary for the implementation of labeling policies. While no available detection systems based on the Reference Molecule have been reported for GM maize MIR604. Here we established the simplex and duplex qualitative and quantitative PCR systems for maize MIR604 employing a reliable new Reference Molecule pMIR604 as a calibrator, which contains fragments of the revealed 5′ flanking sequence of maize MIR604 and taxon-specific sequence zSSIIb. The limits of detection (LODs) were both 10 copies. The absolute LOD and limit of quantification were confirmed to be as low as 10 and 25 copies of pMIR604 through in-house validation both in simplex and duplex real-time PCR assays. For quantification of practical samples, results from five operators indicated that the biases of the data derived from each participant ranged from 2.00 to 23.00% and from 3.00 to 24.00% in simplex and duplex PCR systems, respectively. The relative standard deviations of the mean values for different GM content maize samples were all within 13.60% both in simplex and duplex analyses. This study showed that the detection systems of GM maize MIR604 using a new Reference Molecule pMIR604 as a calibrator are applicable for analysis of GM maize MIR604 and suitable for use as a preferable substitute of the Reference material derived from plant raw materials.
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simplex and duplex polymerase chain reaction analysis of herculex rw 59122 maize based on one Reference Molecule including separated fragments of 5 integration site and endogenous gene
Journal of AOAC International, 2009Co-Authors: Litao Yang, Shu Wang, Kailin Shen, Jianzhong Zhang, Liangwen Pan, Dabing ZhangAbstract:Reference Molecules, as positive controls and calibrators, have been recently developed in genetically modified organism analysis as a potential substitute for Reference materials derived from plant raw materials. In this study, a novel Reference Molecule p59122, including the revealed 5' integration sequence of maize Herculex ® RW (59122), was constructed that was suitable for simplex and duplex event-specific qualitative and quantitative PCR detections. The LOD values were 10 copies both for simplex and duplex qualitative PCR when p59122 was used as the calibrator. These values were comparable to those of using genomic DNA samples with 0.01 and 0.05%, approximately 5 and 25 hyploid genomic DNA copies, respectively. The absolute LOD and LOQ values were confirmed to be as low as 10 and 25 copies of p59122 DNA both in simplex and duplex quantitative systems. Furthermore, ideal quantification data with low bias, SD and RSD values were obtained from the practical samples analyses in simplex and duplex real-time PCR systems using the Reference Molecule p59122 as a calibrator. All these results suggested that the developed Reference Molecule p59122 and the qualitative and quantitative PCR detection methods are suitable for identification and quantification of GM maize 59122 and its derived products.
Litao Yang - One of the best experts on this subject based on the ideXlab platform.
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one novel multiple target plasmid Reference Molecule targeting eight genetically modified canola events for genetically modified canola detection
Journal of Agricultural and Food Chemistry, 2017Co-Authors: Canhua Wang, Dabing Zhang, Guiwen Song, Lan Zheng, Litao YangAbstract:Multiple-target plasmid DNA Reference materials have been generated and utilized as good substitutes of matrix-based Reference materials in the analysis of genetically modified organisms (GMOs). Herein, we report the construction of one multiple-target plasmid Reference Molecule, pCAN, which harbors eight GM canola event-specific sequences (RF1, RF2, MS1, MS8, Topas 19/2, Oxy235, RT73, and T45) and a partial sequence of the canola endogenous Reference gene PEP. The applicability of this plasmid Reference material in qualitative and quantitative PCR assays of the eight GM canola events was evaluated, including the analysis of specificity, limit of detection (LOD), limit of quantification (LOQ), and performance of pCAN in the analysis of various canola samples, etc. The LODs are 15 copies for RF2, MS1, and RT73 assays using pCAN as the calibrator and 10 genome copies for the other events. The LOQ in each event-specific real-time PCR assay is 20 copies. In quantitative real-time PCR analysis, the PCR efficie...
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applicability of a novel Reference Molecule suitable for event specific detections of maize nk603 based on both 5 and 3 flanking sequences
Food Control, 2010Co-Authors: Kailin Shen, Shu Wang, Litao Yang, Liangwen Pan, Dabing ZhangAbstract:Abstract Plasmid Molecule based Reference material (RM) has been shown to be a good alternative as the calibrator for genetically modified organisms (GMOs) identification and quantification, while most of the currently developed plasmid RM can only be used for one specific target detection. In this study, a flexible plasmid RM pNK containing three DNA fragments, i.e. 5′ and 3′ event-specific sequences of maize NK603 and endogenous gene zSSIIb, was developed. We have proved that pNK is suitable for using as a calibrator in both 5′ and 3′ event-specific detection of maize NK603, compared with that of genuine genomic DNA. The limit of detection (LOD) was 10 copies of pNK DNA in conventional PCR assays. The absolute LOD and limit of quantification (LOQ) in quantitative PCR assays were 5 and 25 copies. The standard curves targeting to zSSIIb, 5′ and 3′ event-specific sequences based on pNK DNA showed high reaction efficiency and good linearity. Also, low bias and variations were obtained in practical samples quantification using pNK as the calibrator. These results demonstrated that the developed pNK is flexible and suitable for identification and quantification of maize NK603, as a preferable substitute of RM from the plant raw material.
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establishment and in house validation of simplex and duplex pcr methods for event specific detection of maize syn e3272 5 using a new Reference Molecule
Journal of AOAC International, 2010Co-Authors: Kailin Shen, Shu Wang, Yingjie Pan, Zhiyi Shi, Yongning Sun, Litao YangAbstract:Despite rapid developments in the detection techniques for genetically modified organisms (GMOs), the event-specific PCR method with high specificity is still the most used technique. In this study, event-specific simplex and duplex qualitative and quantitative detection systems were developed targeting the 3' insertion site of GM maize SYN-E3272-5 (3272) construct. A Reference Molecule p3272 was constructed to act as positive control and as calibrator for quantitative analysis. The LOD for simplex and duplex qualitative PCR assays was 10 copies of p3272 control DNA. LOD and the LOQ for simplex and duplex quantitative PCR assays were 10 and 25 copies of p3272 DNA, respectively. Furthermore, four practical GM maize samples were quantified using the established simplex and duplex quantitative PCR systems by in-house validation. Results from five operators showed that the bias ranged from 3.44 to 17.24% in the simplex system and from 0.42 to 16.06% in the duplex system, respectively. These results demonstrated that the established event-specific simplex and duplex qualitative and quantitative PCR systems combined with the Reference Molecule p3272 are suitable for the detection of GM maize 3272 and its derived products.
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development and in house validation of a Reference Molecule pmir604 for simplex and duplex event specific identification and quantification of gm maize mir604
European Food Research and Technology, 2009Co-Authors: Shu Wang, Litao Yang, Kailin Shen, Dabing ZhangAbstract:Effective analysis methods for genetically modified organisms (GMOs) using reliable Reference Molecules as calibrators are necessary for the implementation of labeling policies. While no available detection systems based on the Reference Molecule have been reported for GM maize MIR604. Here we established the simplex and duplex qualitative and quantitative PCR systems for maize MIR604 employing a reliable new Reference Molecule pMIR604 as a calibrator, which contains fragments of the revealed 5′ flanking sequence of maize MIR604 and taxon-specific sequence zSSIIb. The limits of detection (LODs) were both 10 copies. The absolute LOD and limit of quantification were confirmed to be as low as 10 and 25 copies of pMIR604 through in-house validation both in simplex and duplex real-time PCR assays. For quantification of practical samples, results from five operators indicated that the biases of the data derived from each participant ranged from 2.00 to 23.00% and from 3.00 to 24.00% in simplex and duplex PCR systems, respectively. The relative standard deviations of the mean values for different GM content maize samples were all within 13.60% both in simplex and duplex analyses. This study showed that the detection systems of GM maize MIR604 using a new Reference Molecule pMIR604 as a calibrator are applicable for analysis of GM maize MIR604 and suitable for use as a preferable substitute of the Reference material derived from plant raw materials.
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simplex and duplex polymerase chain reaction analysis of herculex rw 59122 maize based on one Reference Molecule including separated fragments of 5 integration site and endogenous gene
Journal of AOAC International, 2009Co-Authors: Litao Yang, Shu Wang, Kailin Shen, Jianzhong Zhang, Liangwen Pan, Dabing ZhangAbstract:Reference Molecules, as positive controls and calibrators, have been recently developed in genetically modified organism analysis as a potential substitute for Reference materials derived from plant raw materials. In this study, a novel Reference Molecule p59122, including the revealed 5' integration sequence of maize Herculex ® RW (59122), was constructed that was suitable for simplex and duplex event-specific qualitative and quantitative PCR detections. The LOD values were 10 copies both for simplex and duplex qualitative PCR when p59122 was used as the calibrator. These values were comparable to those of using genomic DNA samples with 0.01 and 0.05%, approximately 5 and 25 hyploid genomic DNA copies, respectively. The absolute LOD and LOQ values were confirmed to be as low as 10 and 25 copies of p59122 DNA both in simplex and duplex quantitative systems. Furthermore, ideal quantification data with low bias, SD and RSD values were obtained from the practical samples analyses in simplex and duplex real-time PCR systems using the Reference Molecule p59122 as a calibrator. All these results suggested that the developed Reference Molecule p59122 and the qualitative and quantitative PCR detection methods are suitable for identification and quantification of GM maize 59122 and its derived products.
Ignacy Cukrowski - One of the best experts on this subject based on the ideXlab platform.
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density functional theory and isodesmic reaction based prediction of four stepwise protonation constants as log kh n for nitrilotriacetic acid the importance of a kind and protonated form of a Reference Molecule used
Journal of Physical Chemistry A, 2010Co-Authors: Krishna Kuben Govender, Ignacy CukrowskiAbstract:An explicit application of isodesmic reaction (a proton exchange between the studied and structurally similar Reference Molecule), where the free energy change of the protonation reaction in water was obtained using the free energies in solution from a single continuum model, was used to predict stepwise protonation constants of nitrilotriacetic acid. Calculations were performed at the RB3LYP/6-311+G(d,p) level of theory in conjunction with the PCM-UA0 solvation model. Five Reference Molecules were investigated. It has been established that one must pay special attention to structural similarities between the studied and Reference Molecules and selection of a protonated form of the Reference Molecule. The protonation reactions in which the studied and Reference Molecule are involved in must be (if possible) of the same order; e.g., the first (or generally nth) protonation reaction of the Reference Molecule must be used to compute the first (or nth) protonation constant of the studied Molecule. The lowest energy conformer must always be used. The first, second, third, and fourth computed protonation constants differed, on average, from experimental values by 3.3, 0.8, 0.2, and 0.2 log units, respectively. It appears that the charge on the Reference Molecule has more decisive influence on the accuracy of computed protonation constants than its structural differences when compared with the studied Molecule. Results reported can be used as a guide in constructing isodesmic reactions useful for the theoretical prediction of protonation constants by use of methodology described in this work.
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dft and isodesmic reaction based prediction of four stepwise protonation constants as log kh n for nitrilotriacetic acid nta the importance of a kind and protonated form of a Reference Molecule used
2009Co-Authors: Krishna Kuben Govender, Ignacy CukrowskiAbstract:An explicit application of isodesmic reaction (a proton exchange between the studied and similar in structure Reference Molecule), where the free energy change of the protonation reaction in water was obtained using the free energies in solution from a single continuum model, was used to predict stepwise protonation constants of nitrilotriacetic acid. Calculations were performed at the RB3LYP/6-311+G(d,p) level of theory in conjunction with PCM-UA0 solvation model. Five Reference Molecules were investigated. It has been established that one must pay a special attention to structural similarities between the studied and Reference Molecules and selection of a protonated form of the Reference Molecule. The protonation reactions in which the studied and Reference Molecule are involved in must be (if possible) of the same order; e.g. first (or generally nth) protonation reaction of the Reference Molecule must be used to compute the first (or nth) protonation constant of studied Molecule. The lowest energy conformer must always be used. The first, second, third and fourth computed protonation constants differed, on average, from experimental values by 3.3, 0.8, 0.2 and 0.2 log units, respectively. It appears that the charge on the Reference Molecule has more decisive influence on accuracy of computed protonation constants than its structural differences when compared with the studied Molecule. Results reported can be used as guide in constructing isodesmic reactions useful for the theoretical prediction of protonation constants by use of methodology described in this work.
Haeyeong Kim - One of the best experts on this subject based on the ideXlab platform.
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event specific qualitative and quantitative detection of five genetically modified rice events using a single standard Reference Molecule
Food Chemistry, 2017Co-Authors: Jaehwan Kim, Saetbyul Park, Hyojeong Roh, Minki Shin, Gui Im Moon, Jinhwan Hong, Haeyeong KimAbstract:Abstract One novel standard Reference plasmid, namely pUC-RICE5, was constructed as a positive control and calibrator for event-specific qualitative and quantitative detection of genetically modified (GM) rice (Bt63, Kemingdao1, Kefeng6, Kefeng8, and LLRice62). pUC-RICE5 contained fragments of a rice-specific endogenous Reference gene (sucrose phosphate synthase) as well as the five GM rice events. An existing qualitative PCR assay approach was modified using pUC-RICE5 to create a quantitative method with limits of detection correlating to approximately 1–10 copies of rice haploid genomes. In this quantitative PCR assay, the square regression coefficients ranged from 0.993 to 1.000. The standard deviation and relative standard deviation values for repeatability ranged from 0.02 to 0.22 and 0.10% to 0.67%, respectively. The Ministry of Food and Drug Safety (Korea) validated the method and the results suggest it could be used routinely to identify five GM rice events.
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event specific qualitative and quantitative detection of three genetically modified papaya events using a single standard Reference Molecule
Food Control, 2015Co-Authors: Jaehwan Kim, Dabing Zhang, Saetbyul Park, Hyojeong Roh, Hongbae Woo, Minki Shin, Gui Im Moon, Jinhwan Hong, Haeyeong KimAbstract:Abstract A novel standard Reference plasmid (pGEM-PAPAYA3) was constructed as a positive control and Reference standard for event-specific qualitative and quantitative detection of genetically modified (GM) papaya (55-1, 16-0-1, and Huanong No.1). The plasmid pGEM-PAPAYA3 contained the specific fragments of papaya endogenous Reference gene chymopapain and the 3 GM papaya events. The qualitative PCR assay using pGEM-PAPAYA3 was established with a limit of detection correlating to approximately 5–50 copies of papaya haploid genomes. In the quantitative PCR assay, the square regression coefficients (R2) ranged from 0.993 to 0.997. The standard deviation and relative standard deviation values for repeatability ranged from 0.04 to 0.25 and 0.05%–0.86%, respectively. The method was used to test 11 papaya products purchased from Thailand, China, the Philippines and the USA, and the results revealed 2 varieties of GM papaya in 5 of the 11 samples tested. These results indicate the developed detection methods with the standard Reference plasmid are applicable for identifying 3 GM papaya events.
Krishna Kuben Govender - One of the best experts on this subject based on the ideXlab platform.
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density functional theory and isodesmic reaction based prediction of four stepwise protonation constants as log kh n for nitrilotriacetic acid the importance of a kind and protonated form of a Reference Molecule used
Journal of Physical Chemistry A, 2010Co-Authors: Krishna Kuben Govender, Ignacy CukrowskiAbstract:An explicit application of isodesmic reaction (a proton exchange between the studied and structurally similar Reference Molecule), where the free energy change of the protonation reaction in water was obtained using the free energies in solution from a single continuum model, was used to predict stepwise protonation constants of nitrilotriacetic acid. Calculations were performed at the RB3LYP/6-311+G(d,p) level of theory in conjunction with the PCM-UA0 solvation model. Five Reference Molecules were investigated. It has been established that one must pay special attention to structural similarities between the studied and Reference Molecules and selection of a protonated form of the Reference Molecule. The protonation reactions in which the studied and Reference Molecule are involved in must be (if possible) of the same order; e.g., the first (or generally nth) protonation reaction of the Reference Molecule must be used to compute the first (or nth) protonation constant of the studied Molecule. The lowest energy conformer must always be used. The first, second, third, and fourth computed protonation constants differed, on average, from experimental values by 3.3, 0.8, 0.2, and 0.2 log units, respectively. It appears that the charge on the Reference Molecule has more decisive influence on the accuracy of computed protonation constants than its structural differences when compared with the studied Molecule. Results reported can be used as a guide in constructing isodesmic reactions useful for the theoretical prediction of protonation constants by use of methodology described in this work.
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dft and isodesmic reaction based prediction of four stepwise protonation constants as log kh n for nitrilotriacetic acid nta the importance of a kind and protonated form of a Reference Molecule used
2009Co-Authors: Krishna Kuben Govender, Ignacy CukrowskiAbstract:An explicit application of isodesmic reaction (a proton exchange between the studied and similar in structure Reference Molecule), where the free energy change of the protonation reaction in water was obtained using the free energies in solution from a single continuum model, was used to predict stepwise protonation constants of nitrilotriacetic acid. Calculations were performed at the RB3LYP/6-311+G(d,p) level of theory in conjunction with PCM-UA0 solvation model. Five Reference Molecules were investigated. It has been established that one must pay a special attention to structural similarities between the studied and Reference Molecules and selection of a protonated form of the Reference Molecule. The protonation reactions in which the studied and Reference Molecule are involved in must be (if possible) of the same order; e.g. first (or generally nth) protonation reaction of the Reference Molecule must be used to compute the first (or nth) protonation constant of studied Molecule. The lowest energy conformer must always be used. The first, second, third and fourth computed protonation constants differed, on average, from experimental values by 3.3, 0.8, 0.2 and 0.2 log units, respectively. It appears that the charge on the Reference Molecule has more decisive influence on accuracy of computed protonation constants than its structural differences when compared with the studied Molecule. Results reported can be used as guide in constructing isodesmic reactions useful for the theoretical prediction of protonation constants by use of methodology described in this work.
