scholarly journals Novel Reference Molecules for Quantitation of Genetically Modified Maize and Soybean

2002 ◽  
Vol 85 (5) ◽  
pp. 1077-1089 ◽  
Author(s):  
Hideo Kuribara ◽  
Yoichiro Shindo ◽  
Takeshi Matsuoka ◽  
Ken Takubo ◽  
Satoshi Futo ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genetically Modified ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Specific Primers ◽  
Gm Maize ◽  
True Value ◽  
Qualitative And Quantitative Pcr ◽  
Relative Standard ◽  
Polymerase Chain

Abstract New quantitation methods based on a real-time polymerase chain reaction (PCR) technique were developed for 5 lines of genetically modified (GM) maize, including MON810, Event176, Bt11, T25, and GA21, and a GM soy, Roundup Ready. Oligonucleotide DNA, including specific primers and fluorescent dye, labeled probes, were designed for PCRs. Two plasmids were constructed as reference molecules (RMs) for the detection of GM maize and GM soy. The molecules contain the DNA sequences of a specific region found in each GM line, universal sequences used in various GM lines, such as cauliflower mosaic virus 35S promoter and nopaline synthase terminator, and the endogenous DNA sequences of maize or soy. By using these plasmids, no GM maize and GM soy were required as reference materials for the qualitative and quantitative PCR technique. Test samples containing 0, 0.10, 0.50, 1.0, 5.0, and 10% GM maize or GM soy were quantitated. At the 5.0%level, the bias (mean–true value) ranged from 2.8 to 19.4% and the relative standard deviation was <5.2%. These results show that our method involving the use of these plasmids as RMs is reliable and practical for quantitation of GM maize and GM soy.

Interlaboratory Study of Qualitative PCR Methods for Genetically Modified Maize Events MON810, Bt11, GA21, and CaMV P35S

2013 ◽  
Vol 96 (2) ◽  
pp. 346-352 ◽  
Author(s):  
Reona Takabatake ◽  
Kaori Takashima ◽  
Takeyo Kurashima ◽  
Junichi Mano ◽  
Satoshi Furui ◽  
...  
Keyword(s):  
Quantitative Methods ◽  
Genetically Modified ◽  
False Negative ◽  
Cauliflower Mosaic Virus ◽  
Interlaboratory Study ◽  
35S Promoter ◽  
Gm Maize ◽  
Detection And Identification ◽  
Pcr Products ◽  
Qualitative Pcr

Abstract Qualitative PCR methods for the genetically modified (GM) maize events MON810, Bt11, and GA21, and the 35S promoter (P35S) region of the cauliflower mosaic virus (CaMV) were evaluated in an interlaboratory study. Real-time PCR-based quantitative methods for these GM events using the same primer pairs had already been validated in previous studies. Fifteen laboratories in Japan participated in this interlaboratory study. Each participant extracted DNA from blind samples, performed qualitative PCR assays, and then detected the PCR products with agarose gel electrophoresis. The specificity, sensitivity, and false-negative and false-positive rates of these methods were determined with different concentrations of GM mixing samples. LODs of these methods for MON810, Bt11, GA21, and the P35S segment calculated as the amount of MON810 were 0.2, 0.2, 0.1, and 0.2% or less, respectively, indicating that the LODs of MON810, Bt11, and P35S were lower than 10 copies, and the LOD of GA21 was lower than 25 copies of maize haploid genome. The current study demonstrated that the qualitative methods would be fit for the detection and identification of these GM maize events and the P35S segment.

scholarly journals Detection of Genetic Modification in Cured Tobacco Leaf: Proficiency Testing Using Polymerase Chain Reaction-Based Methods

2006 ◽  
Vol 89 (3) ◽  
pp. 693-707
Author(s):  
Ferruccio Gadani ◽  
Martin Ward ◽  
Sue Black ◽  
Neil Harris ◽  
David McDowell ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Proficiency Testing ◽  
Quantitative Methods ◽  
Genetically Modified Organisms ◽  
Genetically Modified ◽  
Cauliflower Mosaic Virus ◽  
Chain Reaction ◽  
Tobacco Leaf ◽  
Qualitative And Quantitative ◽  
Polymerase Chain

Abstract The Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA; Paris, France) Task Force Genetically Modified TobaccoDetection Methods investigated the performance of qualitative and quantitative methods based on the polymerase chain reaction (PCR) for the detection and quantitation of genetically modified (GM) tobacco. In the 4 successful rounds of proficiency testing, the cauliflower mosaic virus 35S RNA promoter (CaMV 35S) and the Agrobacterium tumefaciens nopaline synthase terminator (NOS) were selected as target sequences. Blind-coded reference materials containing from 0.1 to 5.0% and from 0.15 to 4% GM tobacco were used in 2 rounds of qualitative and quantitative PCR, respectively. Eighteen laboratories from 10 countries participated in this study. Considering all methods and 2 rounds, the different laboratories were able to detect GM tobacco at the 0.1% level in 46 out of 58 tests in qualitative assays. The results of the proficiency test indicate that both end point screening and real-time quantitative methods are suitable for the detection of genetically modified organisms in tobacco leaf samples having a GM content of 0.1% or higher. The CORESTA proficiency study represents a first step towards the interlaboratory evaluation of accuracy and precision of PCR-based GM tobacco detection, which may lead to the harmonization of analytical procedures and to the enhancement of comparability of testing results produced by different laboratories.

scholarly journals Quantification of the 35S Promoter in DNA Extracts from Genetically Modified Organisms Using Real-Time Polymerase Chain Reaction and Specificity Assessment on Various Genetically Modified Organisms, Part I: Operating Procedure

2005 ◽  
Vol 88 (2) ◽  
pp. 547-557 ◽  
Author(s):  
Sophie Fernandez ◽  
Chrystèle Charles-Delobel ◽  
Angèle Geldreich ◽  
Georges Berthier ◽  
Francine Boyer ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Real Time ◽  
Genetically Modified Organisms ◽  
Limit Of Detection ◽  
Genetically Modified ◽  
Collaborative Study ◽  
Cauliflower Mosaic Virus ◽  
Performance Criteria ◽  
35S Promoter ◽  
Conserved Sequence

Abstract A highly sensitive quantitative real-time assay targeted on the 35S promoter of a commercial genetically modified organism (GMO) was characterized (sF/sR primers) and developed for an ABI Prism® 7700 Sequence Detection System and TaqMan® chemistry. The specificity assessment and performance criteria of sF/sR assay were compared to other P35S-targeted published assays. sF/sR primers amplified a 79 base pair DNA sequence located in a part of P35S that is highly conserved among many caulimovirus strains, i.e., this consensus part of CaMV P35S is likely to be present in many GM events. According to the experimental conditions, the absolute limit of detection for Bt176 corn was estimated between 0.2 and 2 copies of equivalent genome (CEG). The limit of quantification was reached below 0.1% Bt176 content. A Cauliflower Mosaic Virus control (CaMV) qualitative assay targeted on the ORF III of the viral genome was also used as a control (primers 3F/3R) to assess the presence of CaMV in plant-derived products. The specificity of this test was assessed on various CaMV strains, including the Figwort Mosaic Virus (FMV) and solanaceous CaMV strains. Considering the performance of sF/sR quantification test, the highly conserved sequence, and the small size of the amplicon, this assay was tested in a collaborative study in order to be proposed as an international standard.

scholarly journals A simple and accurate PCR method for detection of genetically modified rice

2019 ◽  
Vol 17 (2) ◽  
pp. 847-851 ◽  
Author(s):  
Payam Safaei ◽  
Ebrahim Molaee Aghaee ◽  
Gholamreza Jahed Khaniki ◽  
Setareh Agha Kuchak Afshari ◽  
Sassan Rezaie
Keyword(s):  
Genetically Modified ◽  
Sucrose Phosphate Synthase ◽  
Cauliflower Mosaic Virus ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Rice Varieties ◽  
Camv 35S ◽  
Nos Terminator ◽  
Gm Rice ◽  
Pcr Method

Abstract Background Legislation regulating for labeling and use of genetically modified (GM) crops are increased considerably worldwide in order to health and safety assurance of consumers. For this purpose, a polymerase chain reaction (PCR) method has been developed for detection of GM rice in people’s food diet. Methods In this study, eighty-one non-labeled rice samples were collected randomly from different market sites of Tehran, Iran. In order to analysis, rice genomic DNA was extracted using MBST DNA extraction kit and subsequently, sucrose phosphate synthase (SPS) gene was used to confirm the quality of extracted DNA. Then, cauliflower mosaic virus (CaMV) 35S promoter and Agrobacterium nopaline synthase (NOS) terminator were selected as screening targets for detection of GM rice sequences by PCR. Results According to our results, 2 out of 81 (2.4%) samples tested were positive for CaMV 35S promoter while no positive result was detected for NOS terminator. Conclusion The obtained data indicated that this method is capable to identify the GM rice varieties. Furthermore, it can demonstrate the possibility of the presence of GM rice in Tehran’s market, thus putting emphasis on the requirement for developing a precise approach to evaluate this product.

scholarly journals Real-Time Polymerase Chain Reaction Detection of Cauliflower mosaic virus to Complement the 35S Screening Assay for Genetically Modified Organisms

2005 ◽  
Vol 88 (3) ◽  
pp. 814-822 ◽  
Author(s):  
Katarina Cankar ◽  
Maja Ravnikar ◽  
Jana Žel ◽  
Kristina Gruden ◽  
Nataša Toplak
Keyword(s):  
Polymerase Chain Reaction ◽  
Mosaic Virus ◽  
Real Time ◽  
Genetically Modified Organisms ◽  
Genetically Modified ◽  
Pcr Amplification ◽  
Cauliflower Mosaic Virus ◽  
Chain Reaction ◽  
Screening Assay ◽  
Polymerase Chain

Abstract Labeling of genetically modified organisms (GMOs) is now in place in many countries, including the European Union, in order to guarantee the consumer's choice between GM and non-GM products. Screening of samples is performed by polymerase chain reaction (PCR) amplification of regulatory sequences frequently introduced into genetically modified plants. Primers for the 35S promoter from Cauliflower mosaic virus (CaMV) are those most frequently used. In virus-infected plants or in samples contaminated with plant material carrying the virus, false-positive results can consequently occur. A system for real-time PCR using a TaqMan minor groove binder probe was designed that allows recognition of virus coat protein in the sample, thus allowing differentiation between transgenic and virus-infected samples. We measured the efficiency of PCR amplification, limits of detection and quantification, range of linearity, and repeatability of the assay in order to assess the applicability of the assay for routine analysis. The specificity of the detection system was tested on various virus isolates and plant species. All 8 CaMV isolates were successfully amplified using the designed system. No cross-reactivity was detected with DNA from 3 isolates of the closely related Carnation etched ring virus. Primers do not amplify plant DNA from available genetically modified maize and soybean lines or from different species of Brassicaceae or Solanaceae that are natural hosts for CaMV. We evaluated the assay for different food matrixes by spiking CaMV DNA into DNA from food samples and have successfully amplified CaMV from all samples. The assay was tested on rapeseed samples from routine GMO testing that were positive in the 35S screening assay, and the presence of the virus was confirmed.

Event-specific quantitative polymerase chain reaction methods for detection of double-herbicide-resistant genetically modified corn MON 87419 based on the 3′-junction of the insertion site

2021 ◽  
Author(s):  
Likun Long ◽  
Wei Yan ◽  
Congcong Li ◽  
Liming Dong ◽  
Na Liu ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Genetically Modified ◽  
Quantitative Polymerase Chain Reaction ◽  
Insertion Site ◽  
Chain Reaction ◽  
Herbicide Resistant ◽  
Qualitative Polymerase Chain Reaction ◽  
Relative Standard ◽  
Junction Site ◽  
Polymerase Chain

ABSTRACT MON 87419 was one of the new transgenic corn events developed in US with the trait of herbicide resistance to both dicamba and glyphosate. To monitor unintended release of genetically modified organism in the future, as well as to meet GM-labeling requirements, it is requisite to develop a reliable method for the detection and quantification of MON 87419, an event-specific primer pair was designed to amplify the 3′-junction site between the endogenous genome sequence and the transferred DNA of GM event MON 87419, amplicons of desired size were produced by qualitative polymerase chain reaction (PCR) assay. For the validation of this quantitative method, the mixed samples containing 10%, 1%, and 0.1% MON 87419 ingredient were quantified. The precisions were expressed as relative standard deviations, deviated by 7.87%, 12.94%, and 19.98%, respectively. These results clearly demonstrate that the PCR methods we developed herein can be used for event-specific quantitative testing of the double-herbicide-resistant corn MON 87419.

Detection of specific DNA sequences in Maize (Zea mays L.) based on phosphorescent quantum-dot exciton energy transfer

2019 ◽  
Vol 43 (14) ◽  
pp. 5308-5314 ◽  
Author(s):  
Jinzhi Lv ◽  
Yanming Miao ◽  
Guiqin Yan
Keyword(s):  
Quantum Dots ◽  
Zea Mays ◽  
Dna Sequences ◽  
Room Temperature ◽  
Zea Mays L ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Exciton Energy ◽  
Complementary Sequence ◽  
Cauliflower Mosaic Virus 35S

The complementary sequence of genetically-modified marker sequence cauliflower mosaic virus 35S promoter (Ca MV 35S) DNA was trimmed and designed into sequences S1 and S2, which were separately modified onto the surfaces of room-temperature phosphorescent (RTP) quantum dots (QDs), forming QDs-S1 (P1) and QDs-S2 (P2), respectively.

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