To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. There are currently 24 reported detection methods, targeting different regions of the P35S promoter. Initial assessment revealed that due to the absence of primer binding sites in the P35S sequence, 19 of the 24 reported methods failed to detect P35S in MON cotton and the other two methods could only be applied to certain GMOs.
In this study, we discovered a conserved region in the P35S sequence through sequencing of P35S promoters from multiple transgenic events and developed new qualitative and quantitative detection systems targeting this conserved region. The quantitative method was suitable for measurement of P35S promoter, exhibiting good agreement between the amount of template and Ct values for each testing event.
This study provides a general P35S screening method, with greater coverage than existing methods. Ever since the first genetically modified GM crop was commercially planted, genetically modified organisms GMOs have come under suspicion from governments and citizens because of potential safety risks 1 , 2. The core of GMO regulation involves detecting GMOs, analyzing legality of their components in a particular region and determining the need for labeling.
This is largely because of its ability to amplify specific DNA fragments from highly processed materials 2. PCR-based GMO detection strategies include element screening, construct-specific and transgenic event-specific methods 3. The construct-specific detection method involves targeting the junction between two elements and it is not able to distinguish two different events transformed with the same plasmid 4.
Event-specific detection can precisely distinguish legitimate transgenic events from related illegal varieties transformed with similar or identical transgenic constructs, thus it is often used to evaluate the legality of a GMO sample 3. The screening method targets the most frequently used elements in transgenic constructs, has the lowest specificity and is mainly used for rapid evaluation of high numbers of GMOs.
In , GM crop varieties from 27 different species were commercialized worldwide 5. This number is rising as ever more GM crops in the research stage enter field trials, in which, only partial varieties were developed event-specific detection methods. This revealed that In commercially important transgenic crops, the presence percentage of these two components is higher.
Because of the importance of the P35S promoter in screening detection of GMOs, a large variety of GMO screening tests have been established and published 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , Some of these methods have been adopted by ISO, EU, China and other countries and regions as standard methods for GMO detection 10 , 11 , 13 , 14 , 15 , 16 , 28 , 31 , 32 , 33 , 34 , In the GMDD, some primer pairs are simultaneously used by both qualitative and quantitative assays and some methods are actually repeats of the same method using different primer names.
The P35S promoter sequences in different GMOs and vectors may be different from each other owing to origination from different strains or from modification in vector construction or mutation during the breeding process Morisett et al. Although many P35S-based methods are available for the testing laboratories, only partial methods have gone through necessary validation processes and inter-laboratory studies against a small number of transgenic events 13 , 14 , 19 , 24 , 31 , 34 , 43 , Indeed, no one method has been systematically verified for accuracy and sensitivity across all commercially available transgenic events.
Some laboratories encountered methodological flaws of P35S in their testing, such as low sensitivity, low reproducibility and false positives or negatives 9.
Holden et al. The human immunodeficiency virus HIV often generates drug resistance mutations owing to highly variable gene and drug selection pressure. During detection of HIV-1 drug resistance mutations, the genotyping assay, combining reverse transcription PCR with sequencing technology or high resolution melting HRM analysis, is commonly used to detect all the possible mutations in HIV genome 47 , 48 , the design of RT-PCR primers should target conserved regions flanking mutational hot spots and the used primers must be specific to the region of interest.
Similarly, the primers for GMO detection also should lie within conserved regions and be specific to the target sequence and the detection of P35S requires amplification of a conserved region across different transgene events. Otherwise, the P35S-based methods would exhibit the above flaws, even result in false testing results during GMO screening.
P35S-based methodologies play an important role during the GMO screening phase. Currently, GMO detection laboratories select methods from different sources, including published literature, ISO standards, databases, or in-house developed methods. This heterogeneity in methodology may result in divergent test results if the P35S sequence carried by the testing sample was altered during construction of the transforming vector or in the breeding process.
The ILSI survey revealed that most participating laboratories were interested in adopting a standardized method, which could generate consistent testing results and lead to better inter-laboratory reproducibility 9. Currently, no optimal method is available that is based on the comprehensive comparison of the existing methods.
The isolated sequences were submitted to the GenBank database and accession numbers and sequences are summarized in Table 1. Three transgenic events Bt11, T25 and MON had two copies of P35S in their transforming constructs, with the sequence of these two copies different from each other for each event Table 1.
In transgenic events selected for this study, all of the isolated P35S promoters normally drive the target genes to express functional proteins. We therefore concluded that all P35S sequences were complete. In accordance with the results of the sequence alignment, we obtained the conserved region of P35S across different transgenic events, corresponding to the genomic region of CaMV between positions and Twenty-four different detection methods targeting the P35S promoter were identified from published papers and detection standards and labeled as M1 to M24 Table 2.
Primer and probe sequences are given in Table 2. S1a-f online. Sequence alignment revealed that 21 of the 24 published P35S-based methods had defects, resulting in missed detection of partial transgenic events; the exceptions were M2, M7 and M12, where both primers and probes were located within the conserved region of P35S.
For the M7 method, the binding site of the probe was a high variability region containing four SNP mutations, resulting in a mismatch with most of the transgenic events and vectors. We speculated that the nucleotide mutation in the primer binding sites would cause inefficient amplification of methods M2, M7 and M12 and that the M7 probe located in a highly variable region could give rise to an abnormal fluorescent signal when detecting mutated P35S targets.
Sequence comparison indicated that the above 19 methods would also fail to detect P35S in maize, whereas, this was unable to be confirmed due to unavailable to maize Supplementary Fig.
S1a—f online. S1h online. The detection results for P35S in GM crops were in agreement with the above sequence alignment results. The qualitative detection of P35S demonstrated that most existing P35S-based methods had flaws resulting in missed detection of partial GM crops harboring P35S.
No gradient change and poor repeatability among the three parallel reactions were visualized using Bt11 with the M7 method, resulting in standard curves with a poor correlation coefficient 0.
Partial dilutions of pMCG were assayed using the three methods, the characteristic parameters of the standard curves were in the acceptable range for methods M2 and M12, whereas, the M7 method generated poor fluorescent signals and relatively large Ct values.
Standard curves were constructed based on the amplification plot. Specificity testing revealed that no amplification occurred in samples lacking the promoter data not shown. Lanes 1—4 correspond to , 50, 20 and 10 haploid genome copies, respectively; each template was run with two parallel PCR reactions. These results indicated that the detection sensitivity of our new qualitative method was as low as 50 copies or fewer in the tested species.
The serial dilutions were used as calibrators to establish standard curves for P35S detection and each dilution was assayed in triplicate. Standard curves were created by plotting Ct values against the logarithm of transgene copy numbers, good agreement was observed between the quantity of template and the Ct values for each event Fig. The R 2 values ranged from 0. Based on the slope of the standard curve, the efficiency of this P35S PCR method was estimated to be from The real-time PCR assays verified that SNPs in conserved regions did not influence the amplification efficiency of the newly developed quantitative method.
We therefore conclude that our real-time assay is suitable for quantifying the P35S promoter copy number in various GM crops. Host cell processes to accomplish mechanical and non-circulative virus transmission. Protoplasma , — Early interactions during the encounter of plants, aphids and arboviruses. Plant Signal. Ethylene signaling mediates Potyvirus spread by aphid vector in potato. Oecologia , — Balique, F. Tobacco mosaic virus in cigarettes and saliva of smokers.
Can plant viruses cross the kingdom border and be pathogenic to humans? Viruses 7, — Bawa, A. Genetically modified foods: safety, risks and public concerns—a review. Food Sci. Becker, R. Improved detection and quantification of Cauliflower mosaic virus in food crops: assessing false positives in GMO screening based on the 35S promoter. Food Res. Interactions between drought and plant genotype change epidemiological traits of Cauliflower mosaic virus. Front Plant Sci. Bertsch, C.
Retention of the virus-derived sequences in the nuclear genome of grapevine as a potential pathway to virus resistance. Organic food in the diet: exposure and health implications.
Public Health 38, — Broadbent, L. Cambridge: Cambridge University Press. Broeders, S. Hernandez-Rodriguez Rijeka: IntechOpen — Chaouachi, M. Monitoring of genetically modified food and feed in the Tunisian market using qualitative and quantitative real-time PCR. Chiter, A. DNA stability in plant tissues: implications for the possible transfer of genes from genetically modified food.
FEBS Lett. Colson, P. Pepper mild mottle virus , a plant virus associated with specific immune responses, fever, abdominal pains, and pruritus in humans. Cottenet, G. Food Chem. Covey, S. Host regulation of the Cauliflower mosaic virus multiplication cycle. Dancer, S. Walker Lanarkshire: Woodhead Publishing , — Datukishvili, N. New multiplex PCR methods for rapid screening of genetically modified organisms in foods. Davison, J. GM plants: science, politics and EC regulations.
Plant Sci. Demeke, T. Critical assessment of digital PCR for the detection and quantification of genetically modified organisms. Difonzo, C. Crop borders reduce potato virus Y incidence in seed potato. Dixon, L. Oligonucleotide directed mutagenesis of Cauliflower mosaic virus DNA using a repair-resistant nucleoside analogue: identification of an agnogene initiation codon. Gene 41, — Dong, W. BMC Bioinformatics Doumayrou, J. An experimental test of the transmission-virulence trade-off hypothesis in a plant virus.
Evolution 67, — Emerson, J. Microbiome Escobar, M. Agrobacterium tumefaciens as an agent of disease. Trends Plant Sci. Farzadfar, S. A phylogeographical study of the Cauliflower mosaic virus population in mid-Eurasia Iran using complete genome analysis.
Occurrence of Cauliflower mosaic virus in different cruciferous plants in Iran. Plant Pathol. Fereres, A. Barrier crops as a cultural control measure of non-persistently transmitted aphid-borne viruses. Virus Res. Fraiture, M. Current and new approaches in GMO detection: challenges and solutions. Biomed Res. Fu, W. A highly sensitive and specific method for the screening detection of genetically modified organisms based on digital PCR without pretreatment. Gachet, E. Trends Food Sci.
Geering, A. Endogenous florendoviruses are major components of plant genomes and hallmarks of virus evolution. Gong, Z. Hidden diversity and macroevolutionary mode of Caulimoviridae uncovered by euphyllophyte paleoviruses.
Grohmann, L. Detection and identification of genome editing in plants: challenges and opportunities. Haas, M. Nucleic Acids Res. Binary Agrobacterium vectors for plant transformation. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene.
J Mol Appl Genet. Nopaline synthase: transcript mapping and DNA sequence. Expression of bacterial genes in plant cells. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. Transcription of Cauliflower mosaic virus DNA: detection of promoter sequences, and characterization of transcripts. Chimeric genes as dominant selectable markers in plant cells. A rapid boiling method for the preparation of bacterial plasmids. Inheritance of functional foreign genes in plants.
These results indicate that the lacZ gene linked to a strong promoter such as P35S is useful as a biochemical and histochemical marker gene in plant cells. Abstract Several lacZ chimeric genes were constructed by fusing the truncated lacZ sequence of Escherichia coli to N-terminal sequences of few other genes.
0コメント