scholarly journals A real-time PCR method for detection of CpTI (Cowpea Trypsin Inhibitor) gene in the genetically modified rice originating from China

2015 ◽  
Vol 18 (2) ◽  
pp. 74-86
Author(s):  
Linh Thi My Nguyen ◽  
Thanh Nguyen Chu ◽  
Le Van Bui
Keyword(s):  
Real Time ◽  
Transgenic Rice ◽  
Trypsin Inhibitor ◽  
Real Time Pcr ◽  
Genetically Modified Organisms ◽  
Limit Of Detection ◽  
Genetically Modified ◽  
Positive Control ◽  
Amplification Efficiency ◽  
Cowpea Trypsin Inhibitor

Labelling and traceability of genetically modified organisms (GMOs) are necessary for trade and regulation in the world and Vietnam. Cowpea Trypsin Inhibitor (CpTI) gene encodes a trypsin inhibitor which is considered as a suitable candidate for developing insect-resistant transgenic plants, especially transgenic rice lines originating from China. In this study, we established a real-time PCR protocol to detect the CpTI gene in transgenic rice. The protocol with CpTI-F and CpTI-R primers, 300 nM primers, 0.5 X SYBR Green I, annealing temperature at 62 0C showed the best results. Amplification efficiency is 94.64 % and the limit of detection is 50 copies. Moreover, PCR product of CpTI gene was cloned into pBluescript plasmid using as a positive control

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.

Two FAST multiplex real-time PCR reactions to assess the presence of genetically modified organisms in food

2019 ◽  
Vol 274 ◽  
pp. 760-765 ◽  
Author(s):  
Geoffrey Cottenet ◽  
Carine Blancpain ◽  
Véronique Sonnard ◽  
Poh Fong Chuah
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Genetically Modified Organisms ◽  
Genetically Modified

scholarly journals Verification of Real-Time PCR Methods for Qualitative and Quantitative Testing of Genetically Modified Organisms

2012 ◽  
Vol 35 (6) ◽  
pp. 442-447 ◽  
Author(s):  
S. Del Gaudio ◽  
A. Cirillo ◽  
G. Di Bernardo ◽  
U. Galderisi ◽  
M. Cipollaro
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Genetically Modified Organisms ◽  
Genetically Modified ◽  
Qualitative And Quantitative

scholarly journals Evaluation of a Real Time PCR Assay Method for the Detection of Genetically Modified Organisms in Food Products

2020 ◽  
Vol 9 (2) ◽  
pp. 1
Author(s):  
Eleni Spanea ◽  
Theofania Tsironi ◽  
Efstathia Tsakali ◽  
Anthimia Batrinou ◽  
Valentina Stefanou ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Genetically Modified Organisms ◽  
Genetically Modified ◽  
Food Products ◽  
Assay Method ◽  
Genetically Modified Maize ◽  
Nos Terminator ◽  
Repeatability And Reproducibility ◽  
Pcr Method

The objective of the study was to determine qualitatively by validated Real Time PCR method the occurrence of genetically modified maize and soybean in commercial food products from the Greek market. 70 independent samples were collected, including products from different categories (i.e. cereal based, biscuits and snacks) which declared either corn or soybean on the labelling. The result of the study indicated that 37.1% of maize and soy products (n=70) displayed in the Greek market have detectable levels of genetically modified maize or soy. These products were identified by specific primers and included common GMΟ detection primers for 35S and NOS terminator. Adequate repeatability and reproducibility was demonstrated for the applied Real Time PCR method, as evaluated by intra- and inter-laboratory tests.

Evaluation of Systems for Nopaline Synthase Terminator in Fast and Standard Real-Time PCR to Screen Genetically Modified Organisms

2015 ◽  
Vol 9 (4) ◽  
pp. 1009-1019 ◽  
Author(s):  
Elisa Pierboni ◽  
Ludovica Curcio ◽  
Gloria Raquel Tovo ◽  
Martina Torricelli ◽  
Cristina Rondini
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Genetically Modified Organisms ◽  
Genetically Modified

scholarly journals New Triplex Real-Time PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Bovine Feces

2008 ◽  
Vol 74 (9) ◽  
pp. 2751-2758 ◽  
Author(s):  
H. Schönenbrücher ◽  
A. Abdulmawjood ◽  
K. Failing ◽  
M. Bülte
Keyword(s):  
Real Time ◽  
Mycobacterium Avium ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Detection System ◽  
Locked Nucleic Acid ◽  
Amplification Efficiency ◽  
Bacterial Strains ◽  
Fecal Samples ◽  
Bovine Feces

ABSTRACT In the present study, a robust TaqMan real-time PCR amplifying the F57 and the ISMav2 sequences of Mycobacterium avium subsp. paratuberculosis from bovine fecal samples was developed and validated. The validation was based on the recommendations of International Organization for Standardization protocols for PCR and real-time PCR methods. For specificity testing, 205 bacterial strains were selected, including 105 M. avium subsp. paratuberculosis strains of bovine, ovine, and human origin and 100 non-M. avium subsp. paratuberculosis strains. Diagnostic quality assurance was obtained by use of an internal amplification control. By investigating six TaqMan reagents from different suppliers, the 100% detection probability was assessed to be 0.1 picogram M. avium subsp. paratuberculosis DNA per PCR. The amplification efficiency was 98.2% for the single-copy gene F57 and 97.8% for the three-copy insertion sequence ISMav2. The analytical method was not limited due to instrument specificity. The triplex real-time PCR allowed the reliable detection of M. avium subsp. paratuberculosis DNA using the ABI Prism 7000 sequence detection system, and the LightCycler 1.0. TaqManmgb and locked nucleic acid fluorogenic probes were suitable for fluorescent signal detection. To improve the detection of M. avium subsp. paratuberculosis from bovine fecal samples, a more efficient DNA extraction method was developed, which offers the potential for automated sample processing. The 70% limit of detection was assessed to be 102 CFU per gram of spiked bovine feces. Comparative analysis of 108 naturally contaminated samples of unknown M. avium subsp. paratuberculosis status resulted in a relative accuracy of 98.9% and a sensitivity of 94.4% for fecal samples containing <10 CFU/g feces compared to the traditional culture method.

scholarly journals Development and validation of a highly sensitive real-time PCR assay for rapid detection of parapoxviruses

2016 ◽  
Vol 29 (4) ◽  
pp. 499-507 ◽  
Author(s):  
Amaresh Das ◽  
Gordon Ward ◽  
Andre Lowe ◽  
Lizhe Xu ◽  
Karen Moran ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Positive Control ◽  
Nucleotide Sequencing ◽  
Amplification Efficiency ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Highly Sensitive ◽  
Development And Validation

Parapoxviruses (PaPVs) cause widespread infections in ruminants worldwide. All PaPVs are zoonotic and may infect humans after direct or indirect contact with infected animals. Herein we report the development and validation of a highly sensitive real-time PCR assay for rapid detection of PaPVs. The new assay (referred to as the RVSS assay) was specific for PaPVs only and had no cross-reactivity against other pox viruses. Using a recombinant plasmid as positive control, the analytical sensitivity of the assay was determined to be 16 genome copies of PaPV per assay. The amplification efficiency estimate (91–99%), the intra- and interassay variability estimate (standard deviation [SD]: 0.28–1.06 and 0.01–0.14, respectively), and the operator variability estimate (SD: 0.78 between laboratories and 0.28 between operators within a laboratory) were within the acceptable range. The diagnostic specificity was assessed on 100 specimens from healthy normal animals and all but 1 tested negative (99%). The diagnostic sensitivity (DSe) was assessed on 77 clinical specimens (skin/scab) from infected sheep, goats, and cattle, and all tested positive (100%). The assay was multiplexed with beta-actin as an internal positive control, and the multiplex assay exhibited the same DSe as the singleplex assay. Further characterization of the PaPV specimens by species-specific real-time PCR and nucleotide sequencing of the PCR products following conventional PCR showed the presence of Orf virus not only in sheep and goats but also in 1 bovid. The validated RVSS assay demonstrated high specificity, sensitivity, reproducibility, and ruggedness, which are critical for laboratory detection of PaPVs.

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