A calibrator plasmid for quantitative analysis of insect resistant maize (Yieldgard MON 810)

Ballari, Rajashekhar V.; Martin, Asha; Gowda, Lalitha R.

doi:10.1016/j.foodchem.2013.02.067

Cited by 13 publications

(5 citation statements)

References 30 publications

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“…(1) large differences between plasmid DNA and plant genomic DNA in quantitative PCR efficiencies, and some studies involving crops validate this as Cf values ranging from 0.53 to 0.83 have been reported [14,21,23]; (2) these samples' GM content was defined as 100% positive by the FAPAS, but the fact was there was less than 100% GM content; (3) flue-cured tobacco DNA partially degrades after curing [24]. e SD and RSD values of the Cf were 0.03 and 6.77%, respectively, which were all in the acceptable range.…”

Section: Discussionmentioning

confidence: 93%

“…Samples' Test. In order to minimize the differences between plasmid DNA and plant genomic DNA in PCR efficiencies, calculating the Cf value is a prerequisite [14]. For the unknown samples' test, 100% transgenic flue-cured tobacco DNA was diluted into a gradient of 100%, 50%, 25%, 12.5%, and 6.25% (V/V) with the content of 0% transgenic fluecured tobacco DNA, reactions were run in ViiA TM 7 Real-Time PCR System, and then target gene and endogenous gene copy number were calculated according to the pGMT27 standard curve.…”

Section: Conversion Factor (Cf ) Calculation and Unknownmentioning

confidence: 99%

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A New Reference Plasmid “pGMT27” Provides an Efficient Transgenic Detection Method for Flue-Cured Tobacco

Zhang

Adil

et al. 2021

Journal of Food Quality

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Owing to the economic value of its foliage, tobacco (Nicotiana tabacum) is cultivated all across the world. For the detection of genetically modified (GM) tobacco, there is a lack of universal standard material which ultimately limits the detection methods because the accuracy and comparability of the results cannot be ensured. Here, we prepared a reference plasmid “pGMT27” for the detection of GM tobacco, which was 18,296 bp in length harboring two of the tobacco endogenous and seven exogenous genes. By using qualitative PCR test for the nine genes, 10 copies were used for plasmid sensitivity. In the quantitative real-time PCR (qPCR) assays with pGMT27 as a calibrator, the reaction efficiencies for P-35S and NR were 101.427% and 98.036%, respectively, whereas the limit of detection (LOD) and limit of quantification (LOQ) were 5 copies and 10 copies per reaction. For standard deviation (SD) and relative standard deviation (RSD) of the Ct values, the repeatability values were from 0.04 to 0.42 and from 0.18% to 1.29%, respectively; and the reproducibility values were from 0.04 to 0.39 and from 0.18% to 1.14%, respectively. For the unknown sample test, the average conversion factor (Cf) was 0.39, and the accuracy bias was from −15.55% to 1.93%; for precision, the SD values ranged from 0.02 to 0.62, while RSD values were from 1.34% to 10.6%. We concluded that using the pGMT27 plasmid as a calibrator provided a highly efficient transgenic detection method for flue-cured tobacco.

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Section: Discussionmentioning

confidence: 93%

Section: Conversion Factor (Cf ) Calculation and Unknownmentioning

confidence: 99%

A New Reference Plasmid “pGMT27” Provides an Efficient Transgenic Detection Method for Flue-Cured Tobacco

Zhang

Adil

et al. 2021

Journal of Food Quality

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“…Often results in scientific publications on GMO quantification are given without numerical information about the software settings (e.g. [ 21 - 24 ]). Sometimes it is mentioned that baseline and threshold were set according to the manufacturer’s or other published instructions (e.g.…”

Section: Discussionmentioning

confidence: 99%

A statistical approach to quantification of genetically modified organisms (GMO) using frequency distributions

Gerdes¹,

Busch²,

Pecoraro³

2014

BMC Bioinformatics

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BackgroundAccording to Regulation (EU) No 619/2011, trace amounts of non-authorised genetically modified organisms (GMO) in feed are tolerated within the EU if certain prerequisites are met. Tolerable traces must not exceed the so-called ‘minimum required performance limit’ (MRPL), which was defined according to the mentioned regulation to correspond to 0.1% mass fraction per ingredient. Therefore, not yet authorised GMO (and some GMO whose approvals have expired) have to be quantified at very low level following the qualitative detection in genomic DNA extracted from feed samples. As the results of quantitative analysis can imply severe legal and financial consequences for producers or distributors of feed, the quantification results need to be utterly reliable.ResultsWe developed a statistical approach to investigate the experimental measurement variability within one 96-well PCR plate. This approach visualises the frequency distribution as zygosity-corrected relative content of genetically modified material resulting from different combinations of transgene and reference gene Cq values. One application of it is the simulation of the consequences of varying parameters on measurement results. Parameters could be for example replicate numbers or baseline and threshold settings, measurement results could be for example median (class) and relative standard deviation (RSD). All calculations can be done using the built-in functions of Excel without any need for programming. The developed Excel spreadsheets are available (see section ‘Availability of supporting data’ for details). In most cases, the combination of four PCR replicates for each of the two DNA isolations already resulted in a relative standard deviation of 15% or less.ConclusionsThe aims of the study are scientifically based suggestions for minimisation of uncertainty of measurement especially in —but not limited to— the field of GMO quantification at low concentration levels. Four PCR replicates for each of the two DNA isolations seem to be a reasonable minimum number to narrow down the possible spread of results.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-014-0407-x) contains supplementary material, which is available to authorized users.

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“…The amount of DNA reliable detected using DNA extraction protocol B was sufficient to detect 0.1% GM content, which makes it suitable for quantitative analysis of GM samples even with low GMO content. Other publications on plasmid calibrator development reported LOD around 10 copy number (Ballari et al, 2013;Chaouachi et al, 2014;Meng et al, 2012;Wang et al, 2011).…”

Section: Limit Of Detection and Limit Of Quantitationmentioning

confidence: 99%

“…CRM commonly used for GMO analysis are dried powders, either genomic DNA (gDNA) or plasmid DNA (pDNA) (Caprioara-Buda et al, 2012). Dried powder CRM are mixtures of GM and non-GM seed powders gravimetrically determined, gDNA calibrators are extracted from leaves of a single GM plant and pDNA calibrators are recombinant plasmids containing an event-specific sequence and an endogenous reference gene sequence (Ballari, Martin, & Gowda, 2013;Meng et al, 2012). These pDNA calibrators were single-target or multiple-target, some pDNA are commercially available as CRM (Table S1).…”

Section: Introductionmentioning

confidence: 99%

New plasmid calibrators for geminivirus-resistant (EMB-PV051-1 event) common bean (Phaseolus vulgaris L.) quantitation using simplex and duplex qPCR

et al. 2018

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The geminivirus-resistant common bean, Embrapa 5.1, was the first commercial genetically modified (GM) event developed in Latin America by the Brazilian Agricultural Research Corp.. Therein novel standard reference plasmids were constructed for species-specific (pLEC) and event-specific (pFGM) qualitative and quantitative detection of Embrapa 5.1 GM common bean. To establish these plasmids as certified reference materials (CRM) for Embrapa 5.1 GM common bean, two DNA extraction protocols, simplex and duplex qPCR using two different operators (experimenters) were tested. The efficiency values ranged from 92% to 110% for the simplex and duplex reactions considering both operators. The limit of detection was enough to detect at least 0.1% GM content. These plasmids are suitable to be used as CRM for Embrapa 5.1 GM common bean. They will be useful for survey of food labels for compliance with legislation about GMO content in Brazil and in other countries where GM common bean is not yet approved for commercialization.

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A calibrator plasmid for quantitative analysis of insect resistant maize (Yieldgard MON 810)

Cited by 13 publications

References 30 publications

A New Reference Plasmid “pGMT27” Provides an Efficient Transgenic Detection Method for Flue-Cured Tobacco

A New Reference Plasmid “pGMT27” Provides an Efficient Transgenic Detection Method for Flue-Cured Tobacco

A statistical approach to quantification of genetically modified organisms (GMO) using frequency distributions

New plasmid calibrators for geminivirus-resistant (EMB-PV051-1 event) common bean (Phaseolus vulgaris L.) quantitation using simplex and duplex qPCR

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