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The presence of genetically modified organisms (GMOs) in food and feed products is regulated in many countries. Various countries have issued labeling threshold levels to supervise GMO commercialization, and efficient detection methods to quantify GMO contents are critical to meet labeling regulations. The aim of this study was to develop event-specific methods to measure the event-specific/lectin gene (Le1) copy number ratio in soybean using droplet digital PCR (ddPCR), which is a recently developed technology that offers high accuracy. Duplex ddPCR methods were developed and optimized using a central composite design for the five most widely authorized GM soybean events, in which the target gene was labeled with one fluorescent reporter and the endogenous gene with another. The methods produced specific results, and the performance complied with international GMO labeling regulations. Further, the duplex ddPCR methods are compatible with chip-based digital PCR platforms and original real-time PCR, but allow higher throughput and are more sensitive in identification and quantification of these five widely authorized genetically modified soybean events.

Salt stress is an important abiotic factor that causes severe losses in soybean yield and quality. Therefore, breeding salt-tolerant soybean germplasm resources via genetic engineering has gained importance. Aspergillus glaucus, a halophilic fungus that exhibits significant tolerance to salt, carries the gene AgGlpF. In this study, we used the soybean cotyledonary node transformation method to transfer the AgGlpF gene into the genome of the soybean variety Williams 82 to generate salt-tolerant transgenic soybean varieties. The results of PCR, Southern blot, ddPCR, and RT-PCR indicated that AgGlpF was successfully integrated into the soybean genome and stably expressed. When subjected to salt stress conditions via treatment with 250 mM NaCl for 3 d, the transgenic soybean plants showed significant tolerance compared with wild-type plants, which exhibited withering symptoms and leaf abscission after 9 d. The results of this study indicated that the transfer of AgGlpF into the genome of soybean plants produced transgenic soybean with significantly improved salt stress tolerance.

Background: The gut microbiota plays a critical role in metabolism and growth of piglets, and was modifiable by dietary tryptophan (Trp) in previous study. However, no studies focused on the investigation of whether additional dietary Trp supplementation would modulate the composition of ileum and cecum microbiota of piglets challenged by lipopolysaccharide (LPS). We conducted to investigate whether dietary tryptophan could alleviate dysbacteriosis of piglets after challenged with LPS.Methods: A total of 40 28 days old male weaning piglets were randomly allotted to five groups, include Con group (basal diet), LPS group (basal diet), 0.2% Trp group (0.2% Trp diet), LPS+0.2% Trp group (0.2% Trp diet) and LPS+0.4% group (0.4% Trp diet). On day 10, 20 and 29, Con and 0.2% Trp groups were injected with saline, LPS, LPS+0.2% Trp and LPS+0.4% Trp groups were injected with LPS respectively. The experiment lasted for 30 days.Results: These results showed that the major three phyla in ileum were Firmicutes, Proteobacteria and Actinobacteria, in cecum were Firmicutes, Bacteroidetes and Proteobacteria. Dietary with 0.2% Trp could attenuate the effect of LPS on the alpha diversity (diversity and richness) in ileum (P＜0.05). The alpha diversity of cecum microbiota was not affected by LPS or Try. The relative abundance of Turicibacte (P＜0.05) and unclassified_f__Peptostreptococccaceae (P＜0.05) were decreased but Lactobacillu (P＜0.05) was increased in ileum by LPS. Compared with LPS group, the relative abundance of Actinobacillus (P=0.07) was decreased in LPS+0.2% Trp group, and the level of Blautia (P=0.08) was increased in LPS+0.4% Trp group. The complexity of ileum microbiota was decreased by LPS. But the complexity of ileum microbiota was increased in LPS+0.2% and LPS+0.4% groups. The relative abundance of Lactobacillu was negative correlation with the majority of genus in ileum and positive correlation with the antioxidant ability of liver (P＜0.05). Conclusions: These finding indicated that Trp could alleviate dysbacteriosis induced by LPS, and ileum microbiota might have a self-protective mechanism to resist the injury induced by LPS through enriching Lactobacillu.

Among all of the high-throughput detection methods, PCR-based methodologies are regarded as the most cost-efficient and feasible methodologies compared with the next-generation sequencing or ChIP-based methods. However, the PCR-based methods can only achieve multiplex detection up to 15-plex due to limitations imposed by the multiplex primer interactions. The detection throughput cannot meet the demands of high-throughput detection, such as SNP or gene expression analysis. Therefore, in our study, we have developed a new high-throughput PCR-based detection method, multiplex enrichment quantitative PCR (ME-qPCR), which is a combination of qPCR and nested PCR. The GMO content detection results in our study showed that ME-qPCR could achieve high-throughput detection up to 26-plex. Compared to the original qPCR, the Ct values of ME-qPCR were lower for the same group, which showed that ME-qPCR sensitivity is higher than the original qPCR. The absolute limit of detection for ME-qPCR could achieve levels as low as a single copy of the plant genome. Moreover, the specificity results showed that no cross-amplification occurred for irrelevant GMO events. After evaluation of all of the parameters, a practical evaluation was performed with different foods. The more stable amplification results, compared to qPCR, showed that ME-qPCR was suitable for GMO detection in foods. In conclusion, ME-qPCR achieved sensitive, high-throughput GMO detection in complex substrates, such as crops or food samples. In the future, ME-qPCR-based GMO content identification may positively impact SNP analysis or multiplex gene expression of food or agricultural samples. Graphical abstract For the first-step amplification, four primers (A, B, C, and D) have been added into the reaction volume. In this manner, four kinds of amplicons have been generated. All of these four amplicons could be regarded as the target of second-step PCR. For the second-step amplification, three parallels have been taken for the final evaluation. After the second evaluation, the final amplification curves and melting curves have been achieved.

The insect-resistant transgenic maize event IE034 has been proved to be one of the most commercially developed transgenic maize events in China. This study was aimed to develop a stable and reliable quantitative detection method to monitor this new transgenic maize event. Here, we developed a novel event-specific real-time PCR method for this genetically modified maize event IE034. The resulting 134 base pair (bp) amplicon was designed according to the 5′ junction of inserted sequence and flanking maize genome sequence. Standard curve of the IE034 5′ event-specific sequence showed good linear regression and high PCR efficiency when using the IE034 pure line samples as calibrator. The limit of detection (LOD) for the IE034 detection method was estimated at approximately 8 initial template copies, and the limit of quantification (LOQ) was estimated at about 40 copies. The accuracy of this quantitative real-time PCR method was verified by screening four mixed DNA samples with known levels of the IE034 event (5, 1, 0.5, and 0.23 %, respectively). The quantified biases deviated from 8.7 to −12.2 %, and the relative standard deviation (RSD) ranged from 2.7 to 12.7 %. These data indicated that this new-developed IE034 event-specific real-time PCR method is suitable and reliable for the quantification of IE034 maize and its derivates.

Loop-mediated isothermal amplification (LAMP) is a widely used isothermal nucleic acid amplification method. Here we developed a new closed-tube colorimetric method for judging LAMP with a novel metal indicator. First, the metal indicator, acid chrome blue K (ACBK), was evaluated in the LAMP reaction with various combinations of reaction reagents, such as reaction buffer, dNTP mixtures, primer mixtures, or Mg2+. We found that the solution color of the LAMP reaction with ACBK changed from red to blue based on a decrease in the Mg2+ concentration in the reaction solution. We then optimized the LAMP with ACBK method for detecting the Cauliflower Mosaic Virus 35S promoter. Further, the specificity of the new colorimetric assay using ACBK in the LAMP reaction for detecting the 35S promoter was tested with diverse transgenic events in different crops, and the sensitivity threshold of the assay was ∼50 copies for transgenic rice genomic DNA and 100 ng of 0.1 % DNA from rice, soybean, rapeseed, and maize. Finally, the applicability of the LAMP assay was successfully validated using practical maize samples. All the detection results could be easily discerned either by UV–vis spectroscopy or the naked eye.

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.

MicroRNAs (miRNAs) have been widely demonstrated to play fundamental roles in gene regulation in most eukaryotes. To date, there has been no study describing the miRNA composition in genetically modified organisms (GMOs). In this study, small RNAs from dry seeds of two GM soybean lines and their parental cultivars were investigated using deep sequencing technology and bioinformatic approaches. As a result, several differentially expressed gma-miRNAs were found between the GM and non-GM soybeans. Meanwhile, more differentially expressed gma-miRNAs were identified between distantly relatednon-GM soybeans, indicating that the miRNA components of soybean seeds varied among different soybean lines, including the GM and non-GM soybeans, and the extent of difference might be related to their genetic relationship. Additionally, fourteen novel gma-miRNA candidates were predicted in soybean seeds including a potential bidirectionally transcribed miRNA family with two genomic loci (gma-miR-N1). Our findings firstly provided useful data for miRNA composition in edible GM crops and also provided valuable information for soybean miRNA research.

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.

Insertion sequence IS1112 is a repetitive element with a relatively high number of copies in Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight of rice (Oryza sativa L.). Three new loci of IS1112 were identified in seven Chinese strains of Xoo using a single oligonucleotide primer J3; 5'-GCTCAGGTCAGGTGGCCTGG-3' by insertion-sequence-based polymerase chain reaction (IS-PCR). Among the three new loci of IS1112, two were located in the open-reading frame region of genes fhuA and cirA, which encode TonB-dependent receptors, and the third in ISXo2, another type of insertion sequence in Xoo genome. Three variants of IS1112 were identified in those three loci based on their sequence similarities: two were identical to IS1112a and IS1112b, reported in strain PXO86 from the Philippines, while the third was a new member of IS1112, defined as IS1112d. Inserting IS1112 in gene fhuA caused three bases, GGT, to be duplicated at the target site, but inserting it in gene cirA did not cause any duplication in the target site. The diversity of IS1112 sequence and insertion loci in Xoo genome and their potential effects are discussed.