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This study was designed to evaluate the effects of γ-irradiated Astragalus polysaccharides (IAPS) on growth performance, cecal microbiota populations, and concentrations of cecal short-chain fatty acids of immunosuppressed broilers. A total of 144 one-day-old broiler chicks were randomly assigned into 3 groups: nontreated group (control), cyclophosphamide (CPM)-treated groups fed either a basal diet or the diets containing 900 mg/kg IAPS, respectively. On day 16, 18, and 20, broilers in the control group were intramuscularly injected with 0.5 mL sterilized saline (0.75%, wt/vol), and those in the CPM and IAPS groups were intramuscularly injected with 0.5 mL CPM (40 mg/kg of BW). The trial lasted 21 d. Compared with the control group, CPM treatment decreased the broiler average daily gain (ADG) and feed intake (P 0.05). Dietary IAPS supplementation increased broiler ADG, Shannon index, and decreased Simpson index (P

With the large-scale planting of genetically modified (GM) crops, the development of a rapid and convenient method for on-site monitoring GM crops is needed. In this study, a duplex recombinase polymerase amplification (DRPA)-based, quick and simple detection system is presented for on-site detection of GM crops. In this system, a rapid DNA extraction method, a DRPA, and a lateral flow biosensor (LFB) are integrated to allow for rapid DNA extraction and amplification, and fast visualization of the detection results. Using the rapid DNA extraction method, high-quality DNA suitable for RPA and polymerase chain reaction (PCR) was quickly isolated from various crops within 5 min. Utilizing the optimal DRPA assay, the universal screening sequences (Cauliflower mosaic virus 35S promoter [35S] and Agrobacterium tumefaciens NOS terminator [NOS]) were rapidly and simultaneously amplified with high selectivity and sensitivity. The sensitivity threshold of the DRPA assay was ∼10 copies for GM soybean genomic DNA and 100 ng DNA of 0.1% GM soybean. In combination with the LFB in an enclosed cassette, the entire detection process was performed in approximately 20–30 min and eliminated the carryover contamination. No special or expensive equipment was needed for the detection process. The system was successfully applied and validated for on-site detection of GM rice, demonstrating its suitability for on-site testing of GM crops and high potential for application to other fields, especially in low-resource regions that require rapid detection of DNA targets.

To assess the effects of food processing on the detection and quantification of transgenic rice TT51-1 in processed food by polymerase chain reaction (PCR) technology, we monitored the presence of TT51-1 components in rice crackers at different processing stages using conventional PCR, quantitative real-time PCR (qPCR), and droplet digital PCR (ddPCR) with standard or validated primers and probes. In conventional PCR, relatively longer amplification targets, such as the Bacillus thuringiensis (Bt) gene (301 bp) and the event-specific target (274 bp), were barely detected in baked, fried or microwaved samples. In qPCR, the amplification fluorescence signal was detected in boiled, dried, baked, and microwaved samples, but barely observed in fried samples. Conventional PCR with the same primers used in qPCR detected the corresponding shorter targets in all samples. The conventional PCR results were mainly consistent with the results of qPCR. The results indicate that food processing directly affects the detection of transgenic components, and suggest that relatively shorter fragments should be selected as the amplification targets for this type of analysis. We established qPCR and duplex ddPCR methods for quantifying TT51-1. The results of an orthogonal experiment indicated that the optimal conditions for TT51-1/PLD duplex ddPCR were 500/250 nM of primers/probe combined with 58 °C annealing temperature. Both methods were feasible for quantitative detection of TT51-1 in processed samples, with duplex ddPCR being a more attractive method for detecting transgenic components in processed food due to its stability, accuracy, PCR inhibitor resistance, and the lack of a need for reference materials.

Genetically modified organisms (GMOs) need to be evaluated for safety before their release. Metabolome techniques provide effective methods to evaluate unintended effects. In this study, the grain metabolome of six transgenic rice lines containing secticidal cry and glyphosate tolerance epsps genes were tested by using an non-targeted metabolite profiling, and 161 and 138 metabolites were identified in grains at grain-filling stage and mature stage, respectively. The metabolic profiles of genetically modified (GM) lines and non-GM lines were all significantly different from each other at grain-filling stage. Although the levels of many metabolites were significantly changed in each transgenic line, only seven of them were simultaneously changed in all the six lines compared with those in non-GM lines at grain-filling stage. The number of significantly changed metabolites was much less at mature stage than that at grain-filling stage. Besides, none of these metabolites was simultaneously changed in all the six lines, suggesting that the metabolites of GMOs are different at different stages of GMO development. This study provides useful information about the metabolic variation between GMO and non-GMO in two development stages of rice grains. These findings suggest that non-targeted metabolite profiling can strengthen the assessment of risk based on metabolites.

This study was conducted to investigate the individual and combined effects of xylo-oligosaccharides (XOS) and gamma-irradiated Astragalus polysaccharides (IAPS) on the growth performance and intestinal mucosal barrier function of broiler chickens. A total of 240 1-day-old Ross-308 chicks were allocated into 5 groups for 21 d: control group (basal diet), antibiotic growth promoter (AGP) group (basal diet supplemented with 50 mg/kg chlortetracycline), XOS group (basal diet supplemented with 100 mg/kg XOS), IAPS group (basal diet supplemented with 600 mg/kg IAPS), and XOS + IAPS group (basal diet supplemented with 100 mg/kg XOS and 600 mg/kg IAPS). The results showed that birds in the XOS + IAPS group showed higher ADG and lower feed-to-gain ratio than those in the control group (P

Gene-editing techniques are becoming powerful tools for modifying target genes in organisms. Although several methods have been reported that detect mutations at targeted loci induced by the CRISPR/Cas system in different organisms, they are semiquantitative and have difficulty in the detection of mutants in processed food samples containing low initial concentrations of DNA and may not accurately quantify editing frequency, especially at very low frequencies in a complex polyploid plant genome. In this study, we developed a duplexed dPCR-based method for the detection and evaluation of gene-editing frequencies in plants. We described the design, performance, accurate quantification, and comparison with other detection systems. The results show that the dPCR-based method is sensitive to different kinds of gene-editing mutations induced by gene-editing. Moreover, the method is applicable to polyploid plants and processed food samples containing low initial concentrations of DNA. Compared with qPCR and NGS-based methods, the dPCR method has a lower limit of detection (LOD) of the editing frequency and a better relationship with the expected editing frequency in detecting the edited region of gene-edited rice samples. Taken together, the duplexed dPCR assay is accurate and precise, and it will be a powerful tool for the detection and evaluation of gene-editing frequencies in plants in gene-editing technology.

In this study, droplet digital polymerase chain reaction (ddPCR) was used to detect adulteration of acacia honey with canola honey. DNA extraction from pollen in acacia honey and canola honey was performed using four different pollen treatment methods, including, enzymatic hydrolysis, freeze-thaw, mechanical, and ultrasonic. The mechanical extraction method was the most preferred method of DNA extraction from honey. A duplex ddPCR method developed based on the specific target gene in acacia and canola was performed. The detection results showed that the established duplex ddPCR had a high detection sensitivity, detecting up to 1% adulteration. The accuracy of duplex ddPCR was consistent with the traditional melissopalynologic method. All results indicated that ddPCR was a powerful method for assessing the authenticity and quality of honey.

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.

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.

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.

The rice cultivar Zhejing 22 has been transformed to carry the Bacillus thuringiensis Cry1A(b) gene in 2007. Although the cultivation of this line (referred to as Bt-ZJ22) is yet to be authorized, it is likely to be approved in China in the near future. The transgene-host 3’ integration junction was isolated using thermal asymmetric interlaced PCR, and its sequence was used to design PCR primers and an appropriate TaqMan probe for a real-time PCR quantitative assay of the transgene. The limit of detection for a standard PCR assay was estimated at ten gene copies, while that for the real-time PCR assay was 5–10 copies. The latter assay was tested on samples of rice DNA with either 5, 3, 1, or 0.5% Bt-ZJ22; this produced estimates for the relative content of Bt-ZJ22 of, respectively, 5.3, 3.2, 0.97, and 0.48%. The precision of these estimates demonstrated that this real-time PCR assay should be suitable for the detection and monitoring of this transgenic event.

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.