Collaborative trial for the validation of event-specific PCR detection methods of genetically modified papaya Huanong No.1

Wei, Jiaojun; Han, Le; Pan, Aihu; Li, Feiwu; Li, Xiang; Quan, Sheng; Guo, Jinchao; Yang, Litao

doi:10.1016/j.foodchem.2015.07.010

Cited by 12 publications

(3 citation statements)

References 21 publications

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“…The development of RMs for the MON810 event is very important to ensure the legalized planting and production processing. During the development of a plasmid-DNA-based CRM, it is critical to validate its characteristic values in addition to its homogeneity and stability [ 24 , 25 ]. In this study, we constructed a plasmid DNA calibrant, pMON810, for quantitative qPCR analysis of GM maize MON810, and we organized a collaborative ring trial for inter-laboratory validation of the applicability and viability of pMON810 as a plasmid DNA calibrant.…”

Section: Introductionmentioning

confidence: 99%

Collaborative Ring Trial of the Applicability of a Reference Plasmid DNA Calibrant in the Quantitative Analysis of GM Maize Event MON810

Meng

Wang

Guo

et al. 2022

Foods

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Certified reference materials (CRMs) is one of the critical requirements in a quantitative analytical method, such as in the quantification of genetically modified (GM) contents in food/feed products. Plasmid-DNA-based CRMs are becoming essential in GM content quantification. Herein, we report the construction of one plasmid DNA calibrant, pMON810, for the quantification of the GM maize event MON810 which is commercially planted and used for food/feeds worldwide, and the collaborative ring trial was used to validate its applicability. pMON10 was proven to have high specificity for the MON810 event. The limit of detection (LOD) and limit of quantification (LOQ) of real-time PCR assays of MON810 event and maize endogenous gene using pMON810 as calibrant was 2 copies/μL and 5 copies/μL, respectively. A total of eight laboratories participated in the ring trial and returned valid test results. Each sample was performed with three repeats and three parallels in each repeat. Statistical analysis of the ring trial results showed that pMON810 as a calibrant had high PCR efficiency (ranging from 0.885 to 1.008) and good linearity (ranging from 0.9933 to 0.9997) in MON810 and endogenous gene real-time PCR assays. The bias between the test values and true values ranged from 4.60 to 20.00% in the quantification of five blind samples. These results indicate that pMON810 is suitable for use as a calibrant for the quantification of MON810 events in routine lab analysis or to evaluate detection methods for MON810, as well as being used as a substitute for the matrix-based CRM of MON810.

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Section: Introductionmentioning

confidence: 99%

Collaborative Ring Trial of the Applicability of a Reference Plasmid DNA Calibrant in the Quantitative Analysis of GM Maize Event MON810

Meng

Wang

Guo

et al. 2022

Foods

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“…DNA-based PCR techniques were mainly used in GMO identification and quantification, such as PCR, real-time PCR, − and digital PCR, etc. − Real-time PCR employing the TaqMan probe technique is the gold standard for GMO analysis because of high sensitivity, accuracy, and practicability. A lot of real-time PCR assays were developed and validated to detect and quantify GM contents in raw materials and processed food/feed samples. − Also, the collaborative ring trial validation is one prerequisite step for proposing a method to be used in routine lab analysis and included as one national or ISO standard. , …”

Section: Introductionmentioning

confidence: 99%

Development and Interlaboratories Validation of Event-Specific Quantitative Real-Time PCR Method for Genetically Modified Rice G6H1 Event

Liu

Yang

Jin

et al. 2018

J. Agric. Food Chem.

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The transgenic rice G6H1 was a new event with the traits of herbicide-tolerance and insect-resistant. Herein, we developed one event-specific real-time PCR method with high specificity and sensitivity for G6H1 event quantitative analysis, and validated its performance on practical samples quantification through a collaborative ring trial. A total of eight laboratories participated in this validation and quantified three blind G6H1 powder samples including DNA extraction and real-time PCR analysis. The statistically analyzed results from returned data confirmed its high PCR efficiency and good linearity, trueness, and precision, indicating that the developed G6H1 real-time PCR assay was accurate, reliable, and comparable for G6H1 identification and quantification.

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“…To implement the labeling regulation, prevalidated event-specific detection methods and reference materials for each GM line need to be well developed. Currently, several techniques have been developed for GMO analysis in the past decade, such as PCR, , real-time PCR, digital PCR, , microarray, loop-mediated isothermal amplification (LAMP), , and next generation sequencing . Regardless of which detection method to choose, reference materials (RMs) which are used as calibrator are essential for quality insurance to deliver accurate GM contents.…”

Section: Introductionmentioning

confidence: 99%

One Novel Multiple-Target Plasmid Reference Molecule Targeting Eight Genetically Modified Canola Events for Genetically Modified Canola Detection

Wang

et al. 2017

J. Agric. Food Chem.

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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 efficiencies of all event-specific and PEP assays are between 91% and 97%, and the squared regression coefficients (R) are all higher than 0.99. The quantification bias values varied from 0.47% to 20.68% with relative standard deviation (RSD) from 1.06% to 24.61% in the quantification of simulated samples. Furthermore, 10 practical canola samples sampled from imported shipments in the port of Shanghai, China, were analyzed employing pCAN as the calibrator, and the results were comparable with those assays using commercial certified materials as the calibrator. Concluding from these results, we believe that this newly developed pCAN plasmid is one good candidate for being a plasmid DNA reference material in the detection and quantification of the eight GM canola events in routine analysis.

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Collaborative trial for the validation of event-specific PCR detection methods of genetically modified papaya Huanong No.1

Cited by 12 publications

References 21 publications

Collaborative Ring Trial of the Applicability of a Reference Plasmid DNA Calibrant in the Quantitative Analysis of GM Maize Event MON810

Collaborative Ring Trial of the Applicability of a Reference Plasmid DNA Calibrant in the Quantitative Analysis of GM Maize Event MON810

Development and Interlaboratories Validation of Event-Specific Quantitative Real-Time PCR Method for Genetically Modified Rice G6H1 Event

One Novel Multiple-Target Plasmid Reference Molecule Targeting Eight Genetically Modified Canola Events for Genetically Modified Canola Detection

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