Dataset Information

Event-specific qualitative polymerase chain reaction analysis for two T-DNA copies in genetically modified orange Petunia.

ABSTRACT: In 2017, various orange coloured petunia on the market turned out to be genetically modified (GM) without an official authorization for commercialization. Sequence analysis suggested these undeclared plants most probably originated from a plant transformation experiment performed in the 1980s. For a deeper understanding how GM petunia entered classical breeding programmes worldwide, and whether they originated from a single source or not, we undertook a molecular genetic characterization of the T-DNA integration sites in different GM petunia cultivars and breeding lines. By means of genome walking, we isolated different T-DNA sequences, which are located at the junctions between the T-DNA(s) and the petunia DNA. Based on the results obtained we conclude that there are at least two T-DNA copies of different lengths. This is supported by Southern blot analysis. For T-DNA1, the 3'-junction sequence was isolated, whereas the 5'-junction remained unclear. In contrast, for T-DNA2, the 5'-junction sequence was isolated, whereas the sequence isolated from the 3'-region consists only of T-DNA, but did not include the junction from the T-DNA to the petunia DNA. We developed primers for event-specific PCRs and screened a set of three orange GM petunia cultivars and 126 GM offspring from a commercial breeding program. We show that both T-DNA copies are present in all our tested GM petunia samples, which underpins the assumption of a single transgenic origin of the undeclared GM petunia. Most likely, the two T-DNAs are integrated in close proximity into the petunia genome.

SUBMITTER: Haselmair-Gosch C

PROVIDER: S-EPMC7359168 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Event-specific qualitative polymerase chain reaction analysis for two T-DNA copies in genetically modified orange <i>Petunia</i>.

Haselmair-Gosch Christian C Nitarska Daria D Walliser Benjamin B Flachowsky Henryk H Marinovic Silvija S Halbwirth Heidi H

Plant cell, tissue and organ culture 20200619 2

In 2017, various orange coloured petunia on the market turned out to be genetically modified (GM) without an official authorization for commercialization. Sequence analysis suggested these undeclared plants most probably originated from a plant transformation experiment performed in the 1980s. For a deeper understanding how GM petunia entered classical breeding programmes worldwide, and whether they originated from a single source or not, we undertook a molecular genetic characterization of the ...[more]

PMID: 32684656

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Project description:Digital PCR in droplets (ddPCR) is an emerging method for more and more applications in DNA (and RNA) analysis. Special requirements when establishing ddPCR for analysis of genetically modified organisms (GMO) in a laboratory include the choice between validated official qPCR methods and the optimization of these assays for a ddPCR format. Differentiation between droplets with positive reaction and negative droplets, that is setting of an appropriate threshold, can be crucial for a correct measurement. This holds true in particular when independent transgene and plant-specific reference gene copy numbers have to be combined to determine the content of GM material in a sample. Droplets which show fluorescent units ranging between those of explicit positive and negative droplets are called 'rain'. Signals of such droplets can hinder analysis and the correct setting of a threshold. In this manuscript, a computer-based algorithm has been carefully designed to evaluate assay performance and facilitate objective criteria for assay optimization. Optimized assays in return minimize the impact of rain on ddPCR analysis. We developed an Excel based 'experience matrix' that reflects the assay parameters of GMO ddPCR tests performed in our laboratory. Parameters considered include singleplex/duplex ddPCR, assay volume, thermal cycler, probe manufacturer, oligonucleotide concentration, annealing/elongation temperature, and a droplet separation evaluation. We additionally propose an objective droplet separation value which is based on both absolute fluorescence signal distance of positive and negative droplet populations and the variation within these droplet populations. The proposed performance classification in the experience matrix can be used for a rating of different assays for the same GMO target, thus enabling employment of the best suited assay parameters. Main optimization parameters include annealing/extension temperature and oligonucleotide concentrations. The droplet separation value allows for easy and reproducible assay performance evaluation. The combination of separation value with the experience matrix simplifies the choice of adequate assay parameters for a given GMO event.

Project description:Background and aimDifferent polymerase chain reaction (PCR) techniques have and are still being used for the direct detection of Brucella DNA in serum samples of different animal species and humans without being validated or properly validated, resulting in discrepancies. Thus, this study aimed to evaluate the diagnostic performance of the TaqMan Real-Time-PCR (RT-PCR) targeting the bcsp31 gene versus conventional PCR for the accurate diagnosis of brucellosis at the genus level in cattle sera.Materials and methodsOne hundred and eighty-four serum samples were collected from bacteriologically positive and negative cows with ages ranging from 1 to 5 years old at some infected private farms in the Nile Delta under quarantine measures as well as brucellosis free farms. These samples were classified into four groups after serological diagnosis and investigated by TaqMan RT-PCR and conventional PCR targeting the IS711 gene for Brucella DNA detection. The diagnostic performance characteristics of both PCR techniques were estimated considering the bacteriological results as a gold standard.ResultsTaqMan RT-PCR revealed superiority over conventional PCR; it was able to detect Brucella DNA in 95% (67/70) and 89% (25/28) of the cattle sera samples belonging to Group 1 (serologically and bacteriologically positive) and Group 2 (serologically negative but bacteriologically positive), respectively. On evaluating the diagnostic performance, TaqMan RT-PCR showed superior diagnostic sensitivity (93.9%), diagnostic specificity (88.4%), performance index (182.3), almost perfect kappa agreement (0.825±0.042), strong positive correlation (r=0.826), high accuracy based on the receiver operating characteristic (ROC) curve, and area under the ROC curve (0.911) at p<0.05 and CI of 95%.ConclusionA cattle serum sample is not the metric of choice for targeting Brucella genomic DNA by conventional PCR. The time-saving and rapid TaqMan RT-PCR method revealed a better diagnostic performance in the detection of Brucella DNA in cattle sera. Such performance offered by TaqMan RT-PCR may be considered a step toward the possibility of using such technology in the direct differentiation between Brucella-infected and -vaccinated cattle immunized by smooth vaccines from cattle sera using primers specific for such vaccines.

Dataset Information

Event-specific qualitative polymerase chain reaction analysis for two T-DNA copies in genetically modified orange Petunia.

Publications

Event-specific qualitative polymerase chain reaction analysis for two T-DNA copies in genetically modified orange <i>Petunia</i>.

Similar Datasets

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