Correction of the lack of commutability between plasmid DNA and genomic DNA for quantification of genetically modified organisms using pBSTopas as a model

Zhang, Li; Wu, Yuhua; Wu, Gang; Cao, Yanwei; Lu, Changming

doi:10.1007/s00216-014-8056-5

Cited by 10 publications

(6 citation statements)

References 23 publications

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“…To assess the discrepancy between experimental and theoretical Cf values, we constructed PCR standard curves (as shown in Figure and Table S2) using genomic DNA templates of each of the eight GM canola events at different concentrations (50.0, 25.0, 5.0, 2.5, 0.5 ng/reaction). We found that the deviations of slopes and intercepts of plasmid and genomic DNA standard curves (Table S2) might cause the difference between the experimental and theoretical Cf values, especially for the deviation of intercepts . For instance, in RF1 event and endogenous PEP gene assays, standard curves between reactions using genomic DNA and pCAN plasmid DNA templates had similar slopes (−3.4471 and −3.4466) but different intercepts of 39.583 and 37.052, the intercept of 2.531 was the mainly origin caused the high Cf value.…”

Section: Resultsmentioning

confidence: 94%

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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Section: Resultsmentioning

confidence: 94%

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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“…Compared with the genetic RMs that are commonly used, the genetic RM system established in this study can to some extent address the disadvantages of easy contamination, short preservation time, and inaccurate quantification of plasmid or synthetic DNA. 14,34 Compared with commercial cell lines, 8 this system eliminated the complicated cell line construction process and cell immortalization operation. In addition, the system obtained a genetic reference cell line with the same clear mutation frequency and copy number and convenient DNA extraction, amplification, and detection.…”

Section: Discussionmentioning

confidence: 99%

Development of a new genetic reference material system based on Saccharomyces cerevisiae cells

Ding

et al. 2021

Molecular Therapy - Methods & Clinical Development

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As an important quality control link of molecular diagnosis, genetic reference materials (RMs) are widely used in various gene detection platforms such as mutation detection, gene quantification, and second generation sequencing. However, contamination, construction, and storage of existing genetic RMs still remain challenges. Here, we established a new genetic RM system based on Saccharomyces cerevisiae. We chose the non-small cell lung cancer (NSCLC) mutation hotspots in Kirsten rat sarcoma viral oncogene (KRAS) and epidermal growth factor receptor (EGFR), using clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR-Cas9) system-mediated gene editing technology, combined with the high homologous recombination efficiency of Saccharomyces cerevisiae. A single copy of the target gene was inserted into the yeast genome, and the inserted target gene was stably inherited with the passage of yeast cells. The copy number calculation for the target gene can replays by cell counting. The RM system was evaluated by sequence, copy number, stability, and homogeneity. In summary, the recombinant yeast cell line has ease of construction and screening, stable genetic characteristics, accurate copy number calculation, and convenient culture and preservation. Our findings may provide new ideas and directions for the research and industrialization of genetic RMs.

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“…The disadvantages of matrix-based CRMs in GMO quantitative analysis include the narrow dynamic range of quantification, multiple and complex preparation procedures, high cost, and difficulty in obtaining the homogeneous candidate [ 15 , 16 ]. Compared with matrix-based CRMs, plasmid-DNA-based CRMs are good substitutes in GMO analysis since they have a well-characterized transgene sequence and copy number, and they can be easily maintained in and produced from bacterium stocks [ 17 , 18 , 19 , 20 , 21 ]. In the detection of foodborne bacteria and viruses, plasmid-DNA-based CRMs are also widely used instead of inactivated bacteria and viruses [ 22 , 23 ].…”

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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Correction of the lack of commutability between plasmid DNA and genomic DNA for quantification of genetically modified organisms using pBSTopas as a model

Cited by 10 publications

References 23 publications

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

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

Development of a new genetic reference material system based on Saccharomyces cerevisiae cells

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

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