Jinchao Guo

The Development and Standardization of Testing Methods for Genetically Modified Organisms and their Derived Products

As the worldwide commercialization of genetically modified organisms (GMOs) increases and consumers concern the safety of GMOs, many countries and regions are issuing labeling regulations on GMOs and their products. Analytical methods and their standardization for GM ingredients in foods and feed are essential for the implementation of labeling regulations. To date, the GMO testing methods are mainly based on the inserted DNA sequences and newly produced proteins in GMOs. This paper presents an overview of GMO testing methods as well as their standardization.

Development of the visual loop-mediated isothermal amplification assays for seven genetically modified maize events and their application in practical samples analysis.

As more and more genetically modified (GM) crops are approved for commercialization and planting, the development of quick and on-spot methods for GM crops and their derivates is required. Herein, we established the polymerase chain reaction and agarose gel electrophoresis-free system for the identification of seven GM maize events (DAS-59122-7, T25, BT176, TC1507, MON810, BT11, and MON863) employing a loop-mediated isothermal amplification (LAMP) technique. The LAMP assay was performed using a set of four specific primers at 60-65 °C in less than 40 min, and the results were observed by direct visual observation. In these developed assays, the specificity targeted at each GM maize event based on the event-specific sequence was well confirmed, and the limits of detection were as low as four copies of maize haploid genomic DNA with an exception of 40 copies for MON810 assay. Furthermore, these developed assays were successfully used to test six practical samples with different GM maize events and contents (ranged from 0.0 to 2.0%). All of the results indicated that the established event-specific visual LAMP assays are more convenient, rapid, and low-cost for GM maize routine analysis.

Development of one novel multiple-target plasmid for duplex quantitative PCR analysis of roundup ready soybean.

To enforce the labeling regulations of genetically modified organisms (GMOs), the application of reference molecules as calibrators is becoming essential for practical quantification of GMOs. However, the reported reference molecules with tandem marker multiple targets have been proved not suitable for duplex PCR analysis. In this study, we developed one unique plasmid molecule based on one pMD-18T vector with three exogenous target DNA fragments of Roundup Ready soybean GTS 40-3-2 (RRS), that is, CaMV35S, NOS, and RRS event fragments, plus one fragment of soybean endogenous Lectin gene. This Lectin gene fragment was separated from the three exogenous target DNA fragments of RRS by inserting one 2.6 kb DNA fragment with no relatedness to RRS detection targets in this resultant plasmid. Then, we proved that this design allows the quantification of RRS using the three duplex real-time PCR assays targeting CaMV35S, NOS, and RRS events employing this reference molecule as the calibrator. In these duplex PCR assays, the limits of detection (LOD) and quantification (LOQ) were 10 and 50 copies, respectively. For the quantitative analysis of practical RRS samples, the results of accuracy and precision were similar to those of simplex PCR assays, for instance, the quantitative results were at the 1% level, the mean bias of the simplex and duplex PCR were 4.0% and 4.6%, respectively, and the statistic analysis ( t-test) showed that the quantitative data from duplex and simplex PCR had no significant discrepancy for each soybean sample. Obviously, duplex PCR analysis has the advantages of saving the costs of PCR reaction and reducing the experimental errors in simplex PCR testing. The strategy reported in the present study will be helpful for the development of new reference molecules suitable for duplex PCR quantitative assays of GMOs.

International Collaborative Study of the Endogenous Reference Gene, Sucrose Phosphate Synthase (SPS), Used for Qualitative and Quantitative Analysis of Genetically Modified Rice

One rice ( Oryza sativa ) gene, sucrose phosphate synthase (SPS), has been proven to be a suitable endogenous reference gene for genetically modified (GM) rice detection in a previous study. Herein are the reported results of an international collaborative ring trial for validation of the SPS gene as an endogenous reference gene and its optimized qualitative and quantitative polymerase chain reaction (PCR) systems. A total of 12 genetically modified organism (GMO) detection laboratories from seven countries participated in the ring trial and returned their results. The validated results confirmed the species specificity of the method through testing 10 plant genomic DNAs, low heterogeneity, and a stable single-copy number of the rice SPS gene among 7 indica varieties and 5 japonica varieties. The SPS qualitative PCR assay was validated with a limit of detection (LOD) of 0.1%, which corresponded to about 230 copies of haploid rice genomic DNA, while the limit of quantification (LOQ) for the quantitative PCR system was about 23 copies of haploid rice genomic DNA, with acceptable PCR efficiency and linearity. Furthermore, the bias between the test and true values of eight blind samples ranged from 5.22 to 26.53%. Thus, we believe that the SPS gene is suitable for use as an endogenous reference gene for the identification and quantification of GM rice and its derivates.

Applicability of the chymopapain gene used as endogenous reference gene for transgenic huanong no. 1 papaya detection.

The virus-resistant papaya (Carica papaya L.), Huanong no. 1, was the genetically modified (GM) fruit approved for growing in China in 2006. To implement the labeling regulation of GM papaya and its derivates, the development of papaya endogenous reference gene is very necessary for GM papaya detection. Herein, we reported one papaya specific gene, Chymopapain (CHY), as one suitable endogenous reference gene, used for GM papaya identification. Thereafter, we established the conventional and real-time quantitative PCR assays of the CHY gene. In the CHY conventional PCR assay, the limit of detection (LOD) was 25 copies of haploid papaya genome. In the CHY real-time quantitative PCR assay, both the LOD and the limit of quantification (LOQ) were as low as 12.5 copies of haploid papaya genome. Furthermore, we revealed the construct-specific sequence of Chinese GM papaya Huanong no. 1 and developed its conventional and quantitative PCR systems employing the CHY gene as endogenous reference gene. This work is useful for papaya specific identification and GM papaya detection.

A multiplex degenerate PCR analytical approach targeting to eight genes for screening GMOs

Currently, the detection methods with lower cost and higher throughput are the major trend in screening genetically modified (GM) food or feed before specific identification. In this study, we developed a quadruplex degenerate PCR screening approach for more than 90 approved GMO events. This assay is consisted of four PCR systems targeting on nine DNA sequences from eight trait genes widely introduced into GMOs, such as CP4-EPSPS derived from Acetobacterium tumefaciens sp. strain CP4, phosphinothricin acetyltransferase gene derived from Streptomyceshygroscopicus (bar) and Streptomyces viridochromogenes (pat), and Cry1Ab, Cry1Ac, Cry1A(b/c), mCry3A, and Cry3Bb1 derived from Bacillus thuringiensis. The quadruplex degenerate PCR assay offers high specificity and sensitivity with the absolute limit of detection (LOD) of approximate 80targetcopies. Furthermore, the applicability of the quadruplex PCR assay was confirmed by screening either several artificially prepared samples or samples of Grain Inspection, Packers and Stockyards Administration (GIPSA) proficiency program.

International Collaborative Study of the Endogenous Reference Gene LAT52 Used for Qualitative and Quantitative Analyses of Genetically Modified Tomato

One tomato ( Lycopersicon esculentum) gene, LAT52, has been proved to be a suitable endogenous reference gene for genetically modified (GM) tomato detection in a previous study. Herein are reported the results of a collaborative ring trial for international validation of the LAT52 gene as endogenous reference gene and its analytical systems; 14 GMO detection laboratories from 8 countries were invited, and results were finally received from 13. These data confirmed the species specificity by testing 10 plant genomic DNAs, less allelic variation and stable single copy number of the LAT52 gene, among 12 different tomato cultivars. Furthermore, the limit of detection of LAT52 qualitative PCR was proved to be 0.1%, which corresponded to 11 copies of haploid tomato genomic DNA, and the limit of quantification for the quantitative PCR system was about 10 copies of haploid tomato genomic DNA with acceptable PCR efficiency and linearity. Additionally, the bias between the test and true values of 8 blind samples ranged from 1.94 to 10.64%. All of these validated results indicated that the LAT52 gene is suitable for use as an endogenous reference gene for the identification and quantification of GM tomato and its derivates.

Collaborative Ring Trial of the Papaya Endogenous Reference Gene and Its Polymerase Chain Reaction Assays for Genetically Modified Organism Analysis

The papaya (Carica papaya L.) Chymopapain (CHY) gene has been reported as a suitable endogenous reference gene for genetically modified (GM) papaya detection in previous studies. Herein, we further validated the use of the CHY gene and its qualitative and quantitative polymerase chain reaction (PCR) assays through an interlaboratory collaborative ring trial. A total of 12 laboratories working on detection of genetically modified organisms participated in the ring trial and returned test results. Statistical analysis of the returned results confirmed the species specificity, low heterogeneity, and single-copy number of the CHY gene among different papaya varieties. The limit of detection of the CHY qualitative PCR assay was 0.1%, while the limit of quantification of the quantitative PCR assay was ∼25 copies of haploid papaya genome with acceptable PCR efficiency and linearity. The differences between the tested and true values of papaya content in 10 blind samples ranged from 0.84 to 6.58%. These results indicated that the CHY gene was suitable as an endogenous reference gene for the identification and quantification of GM papaya.

Metabolic changes in transgenic maize mature seeds over-expressing the Aspergillus niger phyA2.

Key messageNon-targeted metabolomics analysis revealed only intended metabolic changes in transgenic maize over-expressing theAspergillus niger phyA2.AbstractGenetically modified (GM) crops account for a large proportion of modern agriculture worldwide, raising increasingly the public concerns of safety. Generally, according to substantial equivalence principle, if a GM crop is demonstrated to be equivalently safe to its conventional species, it is supposed to be safe. In this study, taking the advantage of an established non-target metabolomic profiling platform based on the combination of UPLC-MS/MS with GC–MS, we compared the mature seed metabolic changes in transgenic maize over-expressing the Aspergillus niger phyA2 with its non-transgenic counterpart and other 14 conventional maize lines. In total, levels of nine out of identified 210 metabolites were significantly changed in transgenic maize as compared with its non-transgenic counterpart, and the number of significantly altered metabolites was reduced to only four when the natural variations were taken into consideration. Notably, those four metabolites were all associated with targeted engineering pathway. Our results indicated that although both intended and non-intended metabolic changes occurred in the mature seeds of this GM maize event, only intended metabolic pathway was found to be out of the range of the natural metabolic variation in the metabolome of the transgenic maize. Therefore, only when natural metabolic variation was taken into account, could non-targeted metabolomics provide reliable objective compositional substantial equivalence analysis on GM crops.

Collaborative trial for the validation of event-specific PCR detection methods of genetically modified papaya Huanong No.1.

For transferring the event-specific PCR methods of genetically modified papaya Huanong No.1 to other laboratories, we validated the previous developed PCR assays of Huanong No.1 according to the international standard organization (ISO) guidelines. A total of 11 laboratories participated and returned their test results in this trial. In qualitative PCR assay, the high specificity and limit of detection as low as 0.1% was confirmed. For the quantitative PCR assay, the limit of quantification was as low as 25 copies. The quantitative biases among ten blind samples were within the range between 0.21% and 10.04%. Furthermore, the measurement uncertainty of the quantitative PCR results was calculated within the range between 0.28% and 2.92% for these ten samples. All results demonstrated that the Huanong No.1 qualitative and quantitative PCR assays were creditable and applicable for identification and quantification of GM papaya Huanong No.1 in further routine lab analysis.