LAMP实时浊度法快速检测转基因玉米MON810

LAMP实时浊度法是采用环介导等温扩增（Loop Mediated Isothermal Amplification,LAMP）技术通过实时浊度仪实时检测反应过程中所产生的白色沉淀,从而实现对扩增全过程的监控,弥补了显色法只能观看反应终点的缺陷,使引物筛选和反应体系的优化有数据可依。本研究以转基因玉米MON810为研究对象,针对外源基因苏云金芽孢杆菌杀虫毒蛋白CryIA(b)与内源基因边界序列设计6条特异性引物,通过实时浊度法在63 ℃的恒温条件下完成检测,对检测的灵敏度、特异性、稳定性进行了评价。建立了转基因玉米MON810的LAMP实时浊度检测方法,该方法最低检出限为0.5%,与LAMP显色法和实时荧光PCR法进行结果比对,符合率为100%,经评价具有特异性高、稳定性强、准确简便等优点,非常适合转基因玉米MON810的快速检测,有较好的应用价值。     

实时荧光PCR定量检测加工产品中转基因玉米Mon810成分

采用实时荧光PCR技术，建立了定量(性)鉴定检测加工产品中转基因玉米Mon810成分的方法。实验设计的可以扩增玉米自身基因和外源基因边界序列的引物和探针具有品种和品系特异性，特异性地检测出食品、饲料等加工产品中转基因玉米Mon810成分。某些检测样品不仅检出转基因玉米Mon810成分，还同时检出其它转基因玉米品系或其它转基因品种。本研究实验建立的转基因玉米Mon810品系鉴定检测方法，即可以用于加工产品中转基因成分的定量检测(检测低限为0．1％)，也可以用于定性检测，或作为常规PCR定性检测后的确证实验方法。     

Statistical Evaluation of Real-Time PCR Protocols Applied to Quantify Genetically Modified Maize

Real-time PCR (RTi-PCR) is the technique of choice for event-specific quantification of genetically modified organisms (GMOs) by determining the amount of event with respect to a species-specific reference gene. Reference genes can be amplified from the genome extracted from Certified Reference Materials (CRMs) or from ad hoc designed plasmids. In the present study, we statistically evaluate the performance of RTi-PCR protocols for GM maize MON810 event by using both genomic DNA from conventional CRMs and a plasmid containing sequences representative of four maize species-specific reference genes. The significance of simple and interaction effects of several variables included in the experimental design on DNA quantification methods and RTi-PCR were evaluated and discussed. Statistically significant differences on Ct values may have an impact on the GMOs quantification and consequently on the compliance of GM quantification-established legal thresholds. Our results confirm the reliability of the plasmid as alternative calibrant for the calculation of GMOs copy number.     

Molecular Identification of Four Genetically Modified Maize (Bt11, Bt176, Mon810 and T25) by Duplex Quantitative Real-Time PCR

In response to the increasing number of genetically modified (GM) events released on the market, control laboratories explore various strategies to simplify and reduce the number of tests needed to characterise the content in genetically modified organism (GMO) of a given sample. Lastly, multiplexing is considered as one of the possible ways to decrease the time and cost of analysis. Here, we report the development of four duplex polymerase chain reaction (PCR) tests for the identification and the quantification of four maize transformation events from which commercial lines have been authorised in Europe namely, Bt11 and Bt176 (Syngenta, DE, USA), Mon810 MaisGard? (Monsanto, MO, USA) and T25 Liberty Link? (Bayer CropScience, Monheim, Germany). The duplex PCR tests combine a maize-specific PCR test hybridising in the Adh1 locus with an event-specific detection system designed on a junction fragment for each of these four GM maize. Real-time PCR tests, suitable to comply with the European regulation, were designed by using Taqman® chemistry.     

实时荧光定量PCR检测转基因玉米59122的方法建立及测量不确定度评估

为了准确定量检测非故意扩散的转基因玉米59122,建立转基因玉米59122及产品的实时荧光定量聚合酶链式反应（polymerase chain reaction,PCR）检测方法,并用特异性、准确度、灵敏度以及测量不确定度等指标评价该方法。结果显示,利用该方法只能检出转基因玉米59122;16 次重复测量含量为1%的转基因玉米59122样品,平均测量值（0.9%）接近真实值（1%）,相对误差为10%,测试回收率为90%,测量不确定度为0.002;能检测到最低含量为2 拷贝的59122分子片段;除去3 次偏离平均值较大的测量值外,13 次重复测量值的变异系数为0.05。因此,实验建立的转基因玉米59122实时荧光定量PCR方法具有较高的特异性、准确度、灵敏度、精密度,较低的测量不确定度。     

Comparison of High-throughput and Manual DNA Extraction Methods for the Qualitative and Quantitative Analysis of MON810 Maize

PCR-based methods are widely used in the European Union and in other countries for the detection, identification, and quantification of genetically modified organisms (GMOs). The preparation of good-quality DNA from plant samples for GMO detection can be a challenging task, particularly if the DNA will be used for quantitative analysis. Two DNA extraction methods, namely manual (NucleoSpin Food kit from Machery-Nagel) and high-throughput partially automated (NucleoMag Plant kit from Machery-Nagel) methods, which utilize different DNA separation principles, were used for the isolation of DNA from maize flour samples. Despite the higher DNA recovery obtained using the high-throughput isolation method, a lower PCR efficiency was achieved, most likely due to the presence of PCR inhibitors in the extracts. We found both DNA extraction methods suitable for GMO analysis.     

转sck基因大米对小型猪肠道菌群和3种消化酶的影响

用表达修饰后的CpTI(豇豆胰蛋白酶抑制剂)基因大米(sck)喂养小型猪,研究其与亲本大米相比对小型猪消化功能的影响,从而评价转sck基因大米与亲本大米的实质等同性。选择断乳期雄性中国实验用小型猪15只,初始体重(6.22±0.42)kg。按窝别体重分成3组,分别为正常对照组(NG),仅喂饲市售猪饲料;亲本大米对照组(CG),喂饲70%亲本大米+基础饲料;转基因大米组(GG),喂饲70%转sck基因大米+基础饲料。基础饲料包括12%小麦麸,8%豆粕,6%酪蛋白和4%的矿物质和维生素混合物。喂养62天,测定粪便菌群及胰腺和粪便中胰蛋白酶、糜蛋白酶和淀粉酶的活性。对胃、小肠、结肠、胰腺称重并做常规组织学和病理学检查。结果表明:与亲本大米组动物相比,转基因大米组动物肠道菌群、胰腺和粪便中胰蛋白酶、糜蛋白酶和淀粉酶活性,胃肠道组织和胰腺组织均未见明显差异(P>0.05),也未见到明显非期望效应。含有CpTI的转sck基因大米未对大米在哺乳动物体内的消化过程产生明显不良影响,为进一步证明转sck基因大米与亲本大米的实质等同性提供了新的证据。     

Method Validation and Quality Management in the Flexible Scope of Accreditation: An Example of Laboratories Testing for Genetically Modified Organisms

Quality assurance is a prerequisite for accurate and reliable results in food and feed testing, ISO/IEC 17025 being recognized worldwide as the base standard. A flexible scope of accreditation enables testing laboratories to react quickly to customer demand and to cope with the large number of new methods, which have to be introduced in the laboratory. Precisely defined procedures for the validation of methods, together with performance and acceptance criteria, are the key points for flexible scope of accreditation. Testing for genetically modified organisms (GMO) is a challenging exercise, especially with more GMOs entering the world market. We describe here the organization and performance of validating quantitative detection methods for GMO testing in the context of the European Union legislation. Operational procedures for method validation organized by the Community Reference Laboratory for Genetically Modified Food and Feed, assisted by the European Network of GMO Laboratories, are described. A protocol for validating methods within an individual laboratory is proposed and discussed in terms of the requirements of flexible scope of accreditation. The system setup can be an example for other similar fields of analytical work.     

Medical and biological assessment of the safety of genetically modified corn lines MON 810 and GA 21: a toxicological-biochemical study

The rats were fed with the flour of corn from genetically modified corn MON 810 and from genetically modified corn GA 21 (Monsanto Co, USA) 3 g/rat/day for 6 months. Their blood, urea and liver were investigated to measure total protein and glucose levels, aminotransferase and alkaline phosphatase activities, pH, creatinine level as well as hepatic enzyme activity of the I and II phases of xenobiotic metabolism and whole and non-sedimentated lysosomal enzyme activities, the level of processes of lipids peroxidation and activity of antioxidant system.     

Medical and biological assessment of genetically modified corn line MON 810 resistant to European corn borer and line GA 21 resistant to glyphosate: a chemical study

Chemical analysis of genetically modified corns MON 810 resistance to European corn borer and GA 21 tolerance to glyphosphate was performed. Results of these studies showed that there is no difference between genetically modified and conventional corn products.     

转Bt玉米暴露对亲代及子代大鼠免疫细胞分型及细胞因子的影响

为研究转苏云金芽胞杆菌(Bacillus thuringiensis,Bt)基因抗虫玉米长期食用对大鼠免疫的影响,以其亲本非转基因玉米饲料及商业饲料为对照,分别采用流式细胞术及液相芯片法检测喂养90 d后亲代及子代刚断乳大鼠外周血、脾脏免疫细胞分型及相关血清细胞因子的表达。结果发现,转Bt基因抗虫玉米饲料组亲代大鼠外周血和脾脏及子代脾脏各淋巴细胞亚群(CD4~+、CD8~+、TCRαβ~+、TCRγδ~+)的细胞数量比例及亲代血清中10种细胞因子表达水平均与非转基因玉米饲料组无显著差异(P0.05);子代外周血B淋巴细胞数量比例显著增高,巨噬细胞炎症蛋白-1α、单核细胞趋化因子-1β、白细胞介素-5表达水平明显降低(P0.05)。结果表明:转Bt基因抗虫玉米暴露90 d对亲代大鼠免疫系统影响不大;对子代刚断乳大鼠的细胞免疫及体液免疫有一定的影响。     

Next-Generation Sequencing as a Tool for Detailed Molecular Characterisation of Genomic Insertions and Flanking Regions in Genetically Modified Plants: a Pilot Study Using a Rice Event Unauthorised in the EU

Precise molecular characterisation of genetic modifications integrated into the genomes of genetically modified organisms (GMOs) and of their flanking genomic regions forms a key component for the development of event-specific detection methods. In the EU, this information is of particular importance for risk management in cases where genetic modifications of unauthorised GM food, feed or seeds are detected. PCR-based chromosome walking approaches are commonly used for DNA sequence determination of the genetic modifications and of the flanking genomic regions in yet undescribed GM plants. If the plant contains complex and re-arranged modifications, sequencing and molecular characterisation are often difficult and laborious. Next-generation sequencing (NGS) of DNA is a powerful alternative tool to rapidly generate primary sequence data on the genome of so far uncharacterised sample material if pure GMO material is available. Recently, robust NGS platforms and affordable sequencing services are accessible for food and feed control laboratories. We here present a NGS-based study for whole-genome sequencing of the GM rice event LLRice62 as a proof-of-principle experiment to develop bioinformatics easy-to-use data analysis tools for rapid molecular characterisation. A total of 171,657,155 read mate pairs of approximately 75 bp each were obtained. Sequence reads belonging to the genetic modifications and their flanking genomic regions in LLRice62 were identified by bioinformatic comparison to the corresponding Oryza sativa ssp. japonica reference genome sequence using the Illumina InDel caller software and subsequent iterative mapping of retrieved NGS reads. An entire genetic modification of 1,493 bp in the genome of the LLRice62 sample material was determined and correctly mapped on chromosome 6. The determined nucleotide sequence coincides to the genetic modification described by the developer of this rice event. This study demonstrates for the first time the applicability of NGS for molecular characterisation of uncharacterised GMOs.     

Adaptation of the ToxRTool to Assess the Reliability of Toxicology Studies Conducted with Genetically Modified Crops and Implications for Future Safety Testing

To determine the reliability of food safety studies carried out in rodents with genetically modified (GM) crops, a Food Safety Study Reliability Tool (FSSRTool) was adapted from the European Centre for the Validation of Alternative Methods’ (ECVAM) ToxRTool. Reliability was defined as the inherent quality of the study with regard to use of standardized testing methodology, full documentation of experimental procedures and results, and the plausibility of the findings. Codex guidelines for GM crop safety evaluations indicate toxicology studies are not needed when comparability of the GM crop to its conventional counterpart has been demonstrated. This guidance notwithstanding, animal feeding studies have routinely been conducted with GM crops, but their conclusions on safety are not always consistent. To accurately evaluate potential risks from GM crops, risk assessors need clearly interpretable results from reliable studies. The development of the FSSRTool, which provides the user with a means of assessing the reliability of a toxicology study to inform risk assessment, is discussed. Its application to the body of literature on GM crop food safety studies demonstrates that reliable studies report no toxicologically relevant differences between rodents fed GM crops or their non-GM comparators.     

Decision Support for the Comparative Evaluation and Selection of Analytical Methods: Detection of Genetically Modified Organisms as an Example

The selection of the best-fit-for-purpose analytical method to be implemented in the laboratory is difficult due to availability of multiple methods, targets, aims of detection, and different kinds and sources of more or less reliable information. Several factors, such as method performance, practicability, cost of setup, and running costs need to be considered together with personnel training when selecting the most appropriate method. The aim of our work was to prepare a flexible multicriteria decision analysis model suitable for evaluation and comparison of analytical methods used for the purpose of detecting and/or quantifying genetically modified organisms, and to use this model to evaluate a variety of changing analytical methods. Our study included selection of PCR-, isothermal-, protein-, microarray-, and next-generation sequencing-based methods in simplex and/or multiplex formats. We show that the overall result of their fitness for purpose is relatively similar; however, individual criteria or a group of related criteria exposed more substantial differences between the methods. The proposed model of this decision support system enables easy modifications and is thus suitable for any other application of complex analytical methods.     

酸碱两步法改性硅灰石的表征及对纸张性能的影响

研究了经酸碱两步法改性处理后的硅灰石形态结构变化及作为填料对纸张性能的影响。结果表明,先用H2SO4使硅灰石部分溶解,然后调节p H值至碱性,以使溶出物质再沉积到颗粒表面,形成疏松多孔包覆层,在保持硅灰石原有针状形态的基础上,实现了硅灰石颗粒表面结构的重建。与未改性硅灰石相比,改性后硅灰石的粒径有所增加,白度提高;其加填纸的白度高、松厚度好、强度降低少。虽然改性硅灰石在纸张中的留着率低于未改性硅灰石,但高于GCC填料。     

Analytical Methods Applied to the Chemical Characterization and Antioxidant Properties of Three Wild Edible Mushroom Species from Northeastern Portugal

The chemical composition and the antioxidant potential of three species of wild mushrooms from Northeastern Portugal, namely Agaricus albertii, Agaricus urinascens var. excellens, and Pleurotus eryngii, were compared. Standard procedures were followed in the nutritional value evaluation, while chromatographic procedures were used to analyze free sugars, fatty acids, tocopherols, phenolic compounds, and organic acids. To assess the antioxidant potential, reducing power, radical-scavenging activity, and lipid peroxidation inhibition were evaluated. P. eryngii revealed the highest levels of macronutrients, except proteins, as also the highest sugars, tocopherols, and monounsaturated fatty acids contents. A. albertii and A. urinascens var. excellens showed similar macronutrients composition. However, A. albertii revealed the highest content in PUFA and phenolic compounds. P. eryngii revealed the highest reducing power and radical-scavenging activity and A. albertii the highest lipid peroxidation inhibition. This study provides a detailed chemical characterization and antioxidant potential evaluation of three species of wild mushrooms from Portugal not yet previously reported. Thus, this work intended to contribute to the increase of information concerning species of edible mushrooms (directed to the scientific community and general population) as well as contribute to the conservation of these resources as sources of compounds of interest.     

Technical Approach to Simplify the Purification Method and Characterization of Microbial Transglutaminase Produced from Streptoverticillium ladakanum

In order to fast and economically purify MTGase from Streptoverticillium ladakanum , a stepwise elution method was developed and compared with linear gradient elution method. MTGase was purified to electrophoretical homogeneity by using CM Sepharose CL-6B and Blue Sepharose Fast Flow chromatographies by linear gradient or stepwise methods. The recovery of MTGase by linear gradient and stepwise methods were 68.4% and 81.0%, respectively. The optimal temperature and pH were 40 °C and 5.5, respectively. It was stable at pH 5.0 to 7.0 and had a rate constant (KD) of 6.21 °o 10^-5 min^-1 for thermal inactivation at 45 °C. The purified MTGase was activated by K⁺ Na⁺, Ca²⁺, Mn²⁺, and Mg²⁺, not affected by Fe³⁺, EDTA, but inhibited by Cu²⁺, Zn²⁺, Hg²⁺, Ni²⁺, Co²⁺, Cd²⁺, PCMB, NEM, IAA, and PMSF. A simple stepwise method was developed for the purification of MTGase from S. ladakanum.     

Synthesis and Characterization of Amino-Terminated Chloration Modified Peanut Shell and Its Application to Preconcentrate and Detect the Concentration of Sunset Yellow in Drink and Jelly Samples

Sunset yellow, as a common pigment, is harmful to people’s health. In this work, a new sensitive adsorbent based on the chlorination peanut shell and HBP-NH₂ was fabricated via one-step synthesis. Optimization of the modified peanut shell was carried out at different temperatures, different molar ratios of reagents, and different time intervals in HBP-NH₂ using Response Surface Methodology (RSM) for the first attempt. The structure of modified peanut shell was characterized by FT-IR, SEM, and TGA analysis. The adsorption properties of the modified peanut shell for Sunset yellow were investigated by batch and column experiments. Batch adsorption’s results suggested that modified peanut shell had a high adsorption capability for Sunset yellow and its maximum saturated adsorption capacity was 468.0 mg/g at 308 K, estimated from the Langmuir model. Furthermore, the modified peanut shell can be eluted easily using 3 mol/L NaOH. The proposed method was successfully applied to quantify the amount of SY in jelly and soft drinks with good recovery valuing from 98 to 104%. Hence, the proposed sensor shows a rapid and sensitive method, low cost, and simple, which is appropriate for alternative technique use for the analysis of food colorants in commercial products.     

Modified curcumin with hyaluronic acid: Combination of pro-drug and nano-micelle strategy to address the curcumin challenge

Curcumin is well known for its pleiotropic activities such as anti-oxidant, anti-inflammatory and anti-tumor properties. But curcumin has extremely low solubility in aqueous medium which limits its potential applications in food and pharmaceutical industries. In this study, the combined pro-drug and nano-micelle strategy was attempted to address the limitation. We modified curcumin with hyaluronic acid (HA) based on the understanding that the hydrophilic polymer facilitates dissolution of curcumin in water. The amphiphilic curcumin derivative self-assembled into nano-micelle in water with the size around 100 nm and low PDI (< 0.3) which indicated that it had narrow size distribution. FT-IR spectrum verified that curcumin was successfully conjugated onto the backbone of HA. On the other hand, curcumin was also loaded into the hydrophobic core of the nano-micelle which worked as carrier for it. Using this combined strategy, the water solubility of curcumin was increased more than 1100 times compared to the pure curcumin. The morphology characteristic of curcumin loaded nano-micelle was examined by TEM which confirmed that curcumin loaded nano-micelle has spherical shape and unified size distribution. Meanwhile, the antioxidant activities of the nano-micelle and the curcumin loaded nano-micelle were examined by DPPH radical scavenging assay and reducing power assay the results of which indicated that the antioxidant potentials were significantly enhanced. The pro-drug and nano-micelle combined method reported here may be promising to address the curcumin challenge such as low water solubility and low availability.