New SYBR®Green methods targeting promoter sequences used for screening of several GM events pending for authorisation in Europe

Broeders, Sylvia; Barbau-Piednoir, Elodie; Vandermassen, Els; Debode, Frédéric; Mazzara, Marco; Roosens, Nancy

doi:10.1007/s00217-013-1910-4

Cited by 12 publications

(10 citation statements)

References 12 publications

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“…Specificity, sensitivity and repeatability [35] were tested in-house. The specificity was tested using 25 ng of the CRM gDNA in the qPCR reaction.…”

Section: Methods Acceptance Parametersmentioning

confidence: 99%

“…Using these data would lead to an incorrect calculation of the amplification efficiency and linearity. Therefore, these parameters were calculated using the points until the LOD 6 of each run (431-4 HGE except for runs 1 and 2 were the lowest HGE was, respectively, 2 and 1 HGE) as described previously [35]. The results, as presented in Table 4, show that the efficiency (ε) of the method is 97 % and the R 2 coefficient 0.999, which still fulfil the set criteria.…”

Section: Amplification Efficiency and Linearitymentioning

confidence: 98%

“…Sequencing of the amplicon and plasmid registration was performed as outlined in [35]. The Sybricon names of the plasmids can be found in Table 1B.…”

Section: Cloning Sequencing and Plasmid Registrationmentioning

confidence: 99%

“…the Combinatory SYBR ® Green qPCR Screening (CoSYPS) system) [7] to form a whole with the previously validated and published qualitative SYBR ® Green methods targeting reference genes [34], generic elements [13,35,36] and trait sequences [16]. On the one hand, the gat-tpinII method will allow covering and discriminating GM events that do not possess any of the targets that are currently included in the CoSYPS, i.e.…”

mentioning

confidence: 99%

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New qualitative trait-specific SYBR®Green qPCR methods to expand the panel of GMO screening methods used in the CoSYPS

Broeders

Fraiture

Vandermassen

et al. 2015

Eur Food Res Technol

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specificity and repeatability were assessed as well as the robustness of the methods. Additionally, the reproducibility was evaluated by transferring the methods to a second laboratory. Both methods allow a specific, sensitive and repeatable detection of the respective targets in food and feed samples and can be easily applied in a routine laboratory. Moreover, they can be used together with previously validated SYBR ® Green qPCR methods to expand the panel of screening methods. This allows an extended coverage of the GM events authorised in the EU and adds discriminative power to the screening phase.

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“…Specificity, sensitivity and repeatability [35] were tested in-house. The specificity was tested using 25 ng of the CRM gDNA in the qPCR reaction.…”

Section: Methods Acceptance Parametersmentioning

confidence: 99%

Section: Amplification Efficiency and Linearitymentioning

confidence: 98%

“…Sequencing of the amplicon and plasmid registration was performed as outlined in [35]. The Sybricon names of the plasmids can be found in Table 1B.…”

Section: Cloning Sequencing and Plasmid Registrationmentioning

confidence: 99%

mentioning

confidence: 99%

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New qualitative trait-specific SYBR®Green qPCR methods to expand the panel of GMO screening methods used in the CoSYPS

Broeders

Fraiture

Vandermassen

et al. 2015

Eur Food Res Technol

Self Cite

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show abstract

“…Some recent examples of additional qPCR screening methods are the element bar and construct ctp2/cp4epsps [21], the elements cry1A.105 and cry2Ab2 [15], promoters P-FMV, P-nos, P-SSuAra, P-TA29, P-ubi, P-rice actin and terminators T-35S, T-E9, T-ocs, T-g7 [14], element vip3A [29], and element cry1Ab/Ac and construct P-ubi/cry [22]. Recent examples of SYBR ® Green detection methods are P-nos and P-FMV [9], cry3Bb and gat/T-pinII [10], and the application of Combinatory SYBR ® Green PCR Screening (CoSYPS) [4,30,42]. Detection and identification of GMOs is not static.…”

mentioning

confidence: 99%

Novel TaqMan PCR screening methods for element cry3A and construct gat/T-pinII to support detection of both known and unknown GMOs

Prins

Hoof

Scholtens

et al. 2016

Eur Food Res Technol

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Inter-laboratory Testing of GMO Detection by Combinatory SYBR®Green PCR Screening (CoSYPS)

et al. 2014

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Combinatory SYBR®Green real-time PCR Screening (CoSYPS) is an efficient, sensitive approach for detecting complex targets such as genetically modified organisms (GMOs) in food and feed products. GMO analysis for legal purposes has become increasingly complex and costly due to the diversity in recombinant targets present in the different GMOs. For this reason, screening for the presence of GMOs is in general the first step in the detection of GM material in a product. CoSYPS allows detecting the large majority of globally commercial GMOs using SYBR®Green real-time PCR methods for six GM targets (P35S, Tnos, CryIAb, CP4-EPSPS, PAT and BAR) combined with species-specific PCR methods (e.g., maize, soy, rapeseed). Here, the results of an inter-laboratory trial on seven samples with different GMO mixtures at different levels are presented. In total, 13 laboratories participated in the trial and the currently most frequently used PCR analysis platforms are represented. The inter-laboratory study clearly demonstrates that PCR methods used in CoSYPS form a very robust GMO screening system. Sensitivity, specificity, positive and negative predictive values are for all PCR methods higher than 95 % for all samples. Together, these results show that the SYBR®Green real-time PCR methods used in CoSYPS are effectively applicable to different PCR platforms and amendable to configuration into a sensitive high-throughput GMO screening and decision support tool.

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New SYBR®Green methods targeting promoter sequences used for screening of several GM events pending for authorisation in Europe

Cited by 12 publications

References 12 publications

New qualitative trait-specific SYBR®Green qPCR methods to expand the panel of GMO screening methods used in the CoSYPS

New qualitative trait-specific SYBR®Green qPCR methods to expand the panel of GMO screening methods used in the CoSYPS

Novel TaqMan PCR screening methods for element cry3A and construct gat/T-pinII to support detection of both known and unknown GMOs

Inter-laboratory Testing of GMO Detection by Combinatory SYBR®Green PCR Screening (CoSYPS)

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