Xiaofu Wang

Endpoint Visual Detection of Three Genetically Modified Rice Events by Loop-Mediated Isothermal Amplification

Genetically modified (GM) rice KMD1, TT51-1, and KF6 are three of the most well known transgenic Bt rice lines in China. A rapid and sensitive molecular assay for risk assessment of GM rice is needed. Polymerase chain reaction (PCR), currently the most common method for detecting genetically modified organisms, requires temperature cycling and relatively complex procedures. Here we developed a visual and rapid loop-mediated isothermal amplification (LAMP) method to amplify three GM rice event-specific junction sequences. Target DNA was amplified and visualized by two indicators (SYBR green or hydroxy naphthol blue [HNB]) within 60 min at an isothermal temperature of 63 °C. Different kinds of plants were selected to ensure the specificity of detection and the results of the non-target samples were negative, indicating that the primer sets for the three GM rice varieties had good levels of specificity. The sensitivity of LAMP, with detection limits at low concentration levels (0.01%–0.005% GM), was 10- to 100-fold greater than that of conventional PCR. Additionally, the LAMP assay coupled with an indicator (SYBR green or HNB) facilitated analysis. These findings revealed that the rapid detection method was suitable as a simple field-based test to determine the status of GM crops.

Citrinin Determination in Red Fermented Rice Products by Optimized Extraction Method Coupled to Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS).

A rapid and sensitive method was developed and validated for citrinin determination in red fermented rice products by liquid chromatography tandem mass spectrometry (LC-MS/MS) under the selected reaction monitoring mode. Sample preparation was especially focused, and the quantitative methods of LC-MS/MS and high-performance liquid chromatography with fluorescence detection (HPLC-FLD) were compared. In red fermented rice samples, the limit of detection was 1.0 μg/kg for LC-MS/MS compared to 250 μg/kg for HPLC-FLD, the limit of quantification was 3.0 μg/kg for LC-MS/MS compared to 825 μg/kg for HPLC-FLD. High correlation coefficient was obtained (R(2) = 0.999) within the linear range (0.1 to 100 μg/L) in the MS method. The recoveries ranging from 80.9% to 106.5% were obtained in different spiking concentrations. The average intra- and inter-day accuracy ranged from 75.4% to 103.1%, and the intra- and inter-day precisions were from 3.3% to 7.9%. The developed method was applied to 12 commercial red fermented rice products, and citrinin was found in 10 samples ranging from 0.14 to 44.24 mg/kg. Compared to traditional qualitative and quantitative methods, the newly developed LC-MS/MS method for citrinin determination includes the merits of using a small amount of extraction solvent, simple preparation steps, and high sensitivity.

Multiplex event-specific qualitative polymerase chain reaction for detecting three transgenic rice lines and application of a standard plasmid as a quantitative reference molecule

The three most well-known genetically modified (GM) rice lines in China are TT51-1, KMD1, and KF6. The purposes of this study were to establish a multiplex event-specific qualitative polymerase chain reaction (meqPCR) system for simultaneous detection of the three transgenic rice events and to construct a plasmid as the reference molecule for quantitative analysis. Event-specific primers for each event were selected or designed by focusing on the transgene borders between the inserted DNA and the flanking rice DNA. The developed meqPCR was anticipated to detect distinct amplicons as 454, 398, 301, and 250bp from KF6, KMD1, TT51-1, and the rice endogenous reference gene, respectively. The robustness of the meqPCR was tested with different levels of the three transgenic rice genomic DNAs, and the sensitivity threshold of the meqPCR was at least 50ng of 0.1% rice DNA for each event when the three transgenic rice events present and with other GM materials together. The constructed plasmid was evaluated using mixed samples with known GM contents in real-time quantitative PCR. The results indicated that the constructed plasmid was acceptable and suitable for GM rice quantitative analysis.

Comparison of droplet digital PCR with quantitative real-time PCR for determination of zygosity in transgenic maize

This study evaluated the applicability of droplet digital PCR (ddPCR) as a tool for maize zygosity determination using quantitative real-time PCR (qPCR) as a reference technology. Quantitative real-time PCR is commonly used to determine transgene copy number or GMO zygosity characterization. However, its effectiveness is based on identical reaction efficiencies for the transgene and the endogenous reference gene. Additionally, a calibrator sample should be utilized for accuracy. Droplet digital PCR is a DNA molecule counting technique that directly counts the absolute number of target and reference DNA molecules in a sample, independent of assay efficiency or external calibrators. The zygosity of the transgene can be easily determined using the ratio of the quantity of the target gene to the reference single copy endogenous gene. In this study, both the qPCR and ddPCR methods were used to determine insect-resistant transgenic maize IE034 zygosity. Both methods performed well, but the ddPCR method was more convenient because of its absolute quantification property.

Degradation and detection of transgenic Bacillus thuringiensis DNA and proteins in flour of three genetically modified rice events submitted to a set of thermal processes.

This study aimed to investigate the degradation of three transgenic Bacillus thuringiensis (Bt) genes (Cry1Ab, Cry1Ac, and Cry1Ab/Ac) and the corresponding encoded Bt proteins in KMD1, KF6, and TT51-1 rice powder, respectively, following autoclaving, cooking, baking, or microwaving. Exogenous Bt genes were more stable than the endogenous sucrose phosphate synthase (SPS) gene, and short DNA fragments were detected more frequently than long DNA fragments in both the Bt and SPS genes. Autoclaving, cooking (boiling in water, 30 min), and baking (200 °C, 30 min) induced the most severe Bt protein degradation effects, and Cry1Ab protein was more stable than Cry1Ac and Cry1Ab/Ac protein, which was further confirmed by baking samples at 180 °C for different periods of time. Microwaving induced mild degradation of the Bt and SPS genes, and Bt proteins, whereas baking (180 °C, 15 min), cooking and autoclaving led to further degradation, and baking (200 °C, 30 min) induced the most severe degradation. The findings of the study indicated that degradation of the Bt genes and proteins somewhat correlated with the treatment intensity. Polymerase chain reaction, enzyme-linked immunosorbent assay, and lateral flow tests were used to detect the corresponding transgenic components. Strategies for detecting transgenic ingredients in highly processed foods are discussed.

Comparative analysis of miRNA expression profiles in transgenic and non-transgenic rice using miRNA-Seq

Safety assessment for genetically modified organisms (GMOs) is required before their release. To date, miRNAs that play important roles in eukaryotic gene regulation have not been considered in the current assessment system. In this study, we identified 6 independent Bt and EPSPS GM rice lines using PCR and immune strip. We analyzed the expression levels of Cry1Ac and EPSPS using quantitative real-time PCR and western blot. Further, miRNAs from the developing seeds of the 6 GM rice lines and the wild-type line were investigated using deep sequencing and bioinformatic approaches. Although these GM lines have different types of integration sites, copy numbers, and levels of gene expression, 21 differentially expressed miRNAs have been found compared to wild type. There is no correlation between transgenic protein expression level and the quantity of differentially expressed miRNAs. This study provides useful data about the miRNA composition of GM plants, and it might be helpful for future risk assessments of miRNA-based GM plants.

High-throughput detection and screening of plants modified by gene editing using quantitative real-time polymerase chain reaction

Gene editing techniques are becoming powerful tools for modifying target genes in organisms. Although several methods have been developed to detect gene-edited organisms, these techniques are time and labour intensive. Meanwhile, few studies have investigated high-throughput detection and screening strategies for plants modified by gene editing. In this study, we developed a simple, sensitive and high-throughput quantitative real-time (qPCR)-based method. The qPCR-based method exploits two differently labelled probes that are placed within one amplicon at the gene editing target site to simultaneously detect the wild-type and a gene-edited mutant. We showed that the qPCR-based method can accurately distinguish CRISPR/Cas9-induced mutants from the wild-type in several different plant species, such as Oryza sativa, Arabidopsis thaliana, Sorghum bicolor, and Zea mays. Moreover, the method can subsequently determine the mutation type by direct sequencing of the qPCR products of mutations due to gene editing. The qPCR-based method is also sufficiently sensitive to distinguish between heterozygous and homozygous mutations in T0 transgenic plants. In a 384-well plate format, the method enabled the simultaneous analysis of up to 128 samples in three replicates without handling the post-polymerase chain reaction (PCR) products. Thus, we propose that our method is an ideal choice for screening plants modified by gene editing from many candidates in T0 transgenic plants, which will be widely used in the area of plant gene editing.

Event-Specific Qualitative and Quantitative Detection of Genetically Modified Rice G6H1

The genetically modified rice G6H1 expressing a fused protein of Cry1Ab/Vip3H and a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) G6 is a genetically modified event that has been approved for preproduction field testing in China. The purpose of this study was to establish an event-specific qualitative and quantitative detection method that could provide a stable, reliable system for monitoring this new transgenic event. In this study, event-specific qualitative and quantitative detection methods based on the 3′ integration junction sequence between host plant DNA and the integrated gene were developed. The limit of detection (LOD) of qualitative PCR was assessed to be 0.1%. The LOD of quantitative PCR was estimated to be ten haploid genome copies. The quantitative PCR detection method was verified with three mixed rice samples with known G6H1 contents, and the results agreed with the expected values. Evaluation of specificity and sensitivity indicated that the developed qualitative and quantitative PCR methods are reliable and can be used for the detection and quantification of G6H1.

Reclassification of Solimonas soli (Kim et al., 2007) and Singularimonas variicoloris (Friedrich et al., 2008) as Sinobacter soli comb. nov. and Sinobacter variicoloris comb. nov. and emended description of the genus Sinobacter (Zhou et al., 2008)

Three types of strains; CW-KD 4 T , MN28 T and DCY12 T which were published by Zhou et al. (2008) as Sinobacter flavus gen. nov., sp. nov., Friedrich et al. (2008) as Singularimonas variicoloris gen. nov., sp. nov., and Kim et al. (2007) as Solimonas soli gen. nov., sp. nov., respectively, were selected and subjected to reclassification analyses using the polyphasic taxonomy. Phylogenetic analysis displayed that the three strains represented a deep-separated lineage within the family, Sinobacteraceae. The 16S rRNA gene sequence similarities among the three strains were 99.7% (CW-KD 4 T and MN28 T ), 98.0% (CW-KD 4 T and DCY12 T ) and 98.1 (MN28 T and DCY12 T ). The isoprenoid quinone of the three strains was Q-8. The major fatty acids of strains CW-KD 4 T and MN28 T were C16:0 and Sum in Feature 5, but the major fatty acids for strain DCY12 T were anteiso-C15:0 and anteiso-C17:0. The G+C contents of the DNA were 68.1 mol% (MN28 T ), 65.8 mol% (CW-KD 4 T ) and 63.5 mol% (DCY12 T ). The co-predominant polar lipids of the three type strains were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. DNA–DNA hybridization results showed the levels of relatedness of 59.0% (MN28 T -DCY12 T ), 21.4% (DCY12 T -CW-KD 4 T ) and 37.5% (MN28 T -CW-KD 4 T ) among each other. On basis of phenotypic and genotypic characteristics of the present study, the three type strains were reclassified as different species of the same genus Sinobacter, and Sinobacter variicoloris comb. nov and Sinobacter soli comb. nov. were proposed for the strains MN28 T and DCY12 T , respectively. The type strains are MN28 T (=DSM 15731 T =LMG 22844 T ) and DCY12 T (=DSM 21787 T =LMG 24014 T =KCTC 12834 T ).