玉米变性淀粉两种不同DNA提取方法的比较研究

采用两种不同方法对玉米变性淀粉基因组DNA提取比较,并经特异性引物进行实时荧光定量-聚合酶联反应(qRT-PCR)检测,从中选择出更适合玉米变性淀粉后续转基因成分检测的DNA提取方法。通过采用磁珠法和试剂盒法提取玉米变性淀粉基因组DNA,比较不同方法的提取效果。结果表明,试剂盒法的提取效果优于磁珠法。实时荧光定量PCR结果显示,试剂盒法提取的基因组DNA均可扩增出标准的S形曲线,而磁珠法提取的基因组DNA则无任何扩增信号。该研究为检测玉米变性淀粉中的转基因成分奠定了基础。     

植物源食品DNA制备方法的比较研究

目的：探索可用作PCR方法检测和鉴定植物源转基因食品模板的DNA抽提方法。方法：分别用改良CTAB法、SDS法制备黄豆、玉米、土豆和番茄及其加工产品的DNA，PCR扩增叶绿体基因片段及黄豆、玉米的内参照基因lectin和zein10。结果：改良CTAB法所得DNA作为PCR扩增模板，叶绿体基因片段及lectin和zein10均呈阳性，SDS法所得DNA作为模板时，部分样品内参照基因lectin或zein10扩增阴性。结论：PCR方法检测转基因食品时，应用改良CTAB法制备DNA模板。     

Schwefeldioxidaufnahme bei der Maisquellung. 2. Technologische Aspekte

Absorption of SO₂ During Swelling of Maize. Part 2. Technological Aspects . Investigations had been made by means of a modified method for practical starch extraction from maize kernels. Already after steeping in 0.8% sulfur dioxide in steeping water a final sulfur dioxide content in the starch lower than 8 mg/kg was observed. Studies in three german starch plants showed that the sulfur dioxide content of products from different process steps before the hydrocyclon station varied. After refining of raw milk this differences could not be observed any longer. Due to a further addition of sulfur dioxide during the refining steps the industrially produced starches contained much more sulfur dioxide than those extracted by the laboratory method. These results have been confirmed by processing industrially steeped maize in the laboratory without further addition of sulfur dioxide during the refining steps.     

肉制品中志贺氏菌DNA的快速提取方法

肉制品中致病菌DNA的提取对PCR检测具有重要意义。以污染志贺氏菌的猪肉为材料,比较了沸水浴法、裂解液煮沸法、CTAB/SDS法、改良的碱性异硫氰酸胍法4种提取方法对志贺氏菌DNA的提取效果,并以提取的DNA为模板进行PCR检测。结果表明:改良的碱性异硫氰酸胍法提取志贺氏菌DNA的效果较好,猪肉中志贺氏菌的PCR最低检出限为5×100CFU/g,整个检测时间<8h。经改良的碱性异硫氰酸胍法操作简便、经济省时,可用于肉类的PCR检测。     

GRANULE SIZE DISTRIBUTION AND RHEOLOGICAL BEHAVIOR OF HEATED MODIFIED WAXY AND UNMODIFIED MAIZE STARCH DISPERSIONS

The effect of granule size distribution, supported by microscopic analysis, on the steady shear and dynamic shear rheological behavior of 5% gelatinized modified waxy maize starch (MWMS) dispersions heated at 80C for time periods from 0.5 to 30 min was examined and data obtained with 5% unmodified maize starch (UMS) dispersions was used to provide additional insight. The MWMS dispersions showed a faster and more extensive swelling than the UMS dispersions at 80C. They also exhibited rheopectic (anti-thixotropic) time-dependent and shear thinning with yield stress behavior. UMS dispersions showed shear thickening characteristics after short heating times that gradually turned to shear thinning as the heating time was increased. Dynamic tests on MWMS dispersions showed G' was about ten times higher than G". G' showed a small dependence on frequency over 0.6–63 rad/s while G", after an increase at low frequencies, became less sensitive. The gelatinized MWMS dispersions can therefore be classified as 'weak gels.' The flow behavior index of UMS dispersions was strongly     

高直链玉米III型抗性淀粉制备及其结构和特性

以高直链玉米淀粉G50和G70为原料,经酸解、糊化、脱支和重结晶步骤获得III型抗性淀粉,通过退火与压热处理以进一步提升淀粉的抗性比例。采用扫描电子显微镜、X射线衍射、差示扫描量热、快速黏度分析等方法,研究淀粉颗粒形貌、结晶结构、热特性及糊化特性,利用Englyst法测试淀粉消化特性。结果表明：高直链玉米淀粉G50和G70酸解后的得率分别为77.9%和84.5%,重结晶后的得率降为54.4%和70.2%。原G50和G70改性后,淀粉颗粒形貌被破坏,形成大小不等、颗粒形貌不规则的团聚体;淀粉结晶型由B＋V型转变为A＋V型,且结晶度升高;淀粉糊化温度升高,且加热过程中黏度几乎消失。溶解与膨胀特性结果表明,经酸解、糊化、脱支和老化处理后原G50和G70的溶解性显著升高,退火和压热处理后降低了III型抗性淀粉的溶解性和膨胀度。体外消化特性分析表明,改性后的G50和G70具备更强的抗消化性能,抗性淀粉含量最高可达80.5%（G70-RS3-压热20%）。本研究的改性处理能有效提高高直链玉米淀粉G50和G70中抗性淀粉含量,同时抗性淀粉含量与结晶度和糊化温度呈显著正相关。     

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.     

Development of a CTAB buffer-based automated gDNA extraction method for the surveillance of GMO in seed

Seed imported into the EU from countries growing genetically modified (gm) plants may contain traces of these gm crops. As a result of the zero tolerance policy of the EU, these products must be removed from the market. Along with the amount of biotech crops produced worldwide, the work load for seed surveillance authorities increases. Since the commonly used CTAB buffer-based extraction methods are manual and laborious, a large part of the work load is caused by DNA extraction. In order to reduce labour input and accelerate the DNA analysis workflow, we developed an automated CTAB buffer-based DNA isolation method for seed. Several isolation and chemistry parameters were altered to combine a thorough cell lysis, removal of inhibitors and a highly efficient binding of gDNA to paramagnetic beads. This optimised procedure was compared with manual CTAB buffer-based and Wizard-based DNA extraction methods for maize, soya bean and rapeseed. Automated DNA extraction was faster, less laborious and resulted, on average, in higher DNA yield and purity. The applicability of our method was successfully proven with in-house routine samples.     

Effect of Modified Starches on Rheological Properties of Ketchup

The aim of this work was to study the effect of commercial modified starches of different origin on rheological properties of ketchup. The following starches were used to produce the ketchup samples: chemically modified potato (acetylated distarch adipate from potato starch), waxy maize (acetylated distarch adipate from waxy maize starch and hydroxypropyl distarch phosphate from waxy maize starch), and cassava (acetylated distarch adipate from cassava starch) starches and physically modified cassava and waxy maize starches (physically modified cassava starch and physically modified waxy maize starch). The SEM microphotographs revealed that swollen or disrupted starch granules were present in the ketchup samples. As was evaluated by particle size distribution analysis, two peaks characteristic for different starch granule sizes were observed, the first peak at about 100 μm for ketchup thickened with potato starch and the second one at about 50 μm for the rest of the samples. Ketchups showed non-Newtonian, shear-thinning flow with tendency to yield stress. Values of the rheological parameters describing the flow curves significantly correlated with Bostwick consistency. Ketchup samples exhibited different susceptibility for temperature changes, while values of flow activation energy were from 4.18 to 9.00 kJ/mol. On the basis of mechanical spectra, it is noted that values of G′ were higher than these of G″ showing that elastic properties dominated over the viscous ones. Ketchup samples exhibited properties of weak gels which were estimated from the values of G′ and G″ moduli and their relation and from values of tangent of phase angle (tan δ = 0.14???0.37). Principal component analysis revealed both similarities and differences in rheological behavior of the examined ketchup samples thickened with different modified starches.     

超声法联合微波辅助提取玉米须甾醇的工艺优化

应用响应面法对超声与微波联合提取玉米须甾醇的工艺进行优化,建立了相应的回归模型。在单因素试验的基础上,考察超声时间、超声波功率、液料比和微波时间对玉米须甾醇得率的影响。通过中心组合试验优化获得最佳工艺条件为:超声时间55 min,超声波功率200W,料液比1:40(g/mL),微波时间16 min,通过五次验证试验,玉米须甾醇的得率为0.76%,与理论预测值基本符合,为玉米须甾醇的开发提供理论依据。     

Rapid small‐scale starch isolation using a combination of ultrasonic sonication and sucrose density separation

The proposed rapid small‐scale starch isolation technique in the laboratory was a combination of dry grinding of grain, suspension of the resulting flour in extraction buffer, application of ultrasonic sonication, then separation by sucrose density centrifugation. Light microscopy of separated fractions showed intact starch granules in the pellet and proteins and damaged starch in the top layer. The extraction method yielded 61% starch from sorghum and 63% from maize. The isolated starch showed lower starch damage and proteins content than by the conventional method. The gelatinization enthalpy of isolated starch was slightly higher than by wet grinding conventional method. In addition to low amount of starting flour (100 mg) the new starch isolation method was performed in less than 2 h from dry grinded seed to dried starch. Thus, it could be a useful method for cereal chemists and plant genetists.     

变性淀粉对玉米饮料稳定性的影响

将变性淀粉作为稳定剂添加至玉米饮料中以提高玉米饮料的稳定性。采用单因素和正交试验研究3种变性淀粉对玉米饮料稳定性的影响。结果表明:向玉米饮料中添加0.8%羟丙基复合变性淀粉、0.8%乙酰化变性淀粉和0.6%羟丙基辛烯基琥珀酸酯化淀粉,可以提高玉米饮料的稳定性,降低其离心沉淀率。对玉米饮料混浊稳定性和离心沉淀率效果影响主次顺序为:羟丙基复合变性淀粉,羟丙基辛烯基琥珀酸酯化淀粉,乙酰化变性淀粉。     

DNA extraction method using a silica-base resin type kit for the detection of genetically modified papaya

Genetically modified (GM) papaya has not yet been approved for importation into, or cultivation in the European Union (EU) and Japan. A DNA extraction method using the Qiagen DNeasy Plant Mini Kit (PM method) and a method using a buffer containing cetyltrimethyl ammonium bromide (CTAB method) have been adopted as the official Japanese methods for detecting GM foods. However, the amounts of DNA extracted from papaya by these methods are very low. Therefore, we investigated an extraction method to obtain a high yield of DNA from raw or freeze-dried fresh papaya using the Promega Wizard DNA Clean-Up Resin System (WCR). The incubation for the extraction was carried out at 58 degrees C without proteinase K for 15 min. The extract was applied to a mini-column, then the column was washed with 80% isopropyl alcohol, and genomic DNA adsorbed on the column was eluted with TE buffer. The WCR method gave a higher yield of genomic DNA, and was simpler and faster than the PM method or CTAB method. In addition, it could be used to extract genomic DNA from fresh papaya at various stages of ripeness. Based on these results, we propose that the present method using WCR is the most practical and useful way to extract genomic DNA for the purpose of detecting GM papaya.     

Laboratory-performance study of quantitative PCR methods to analyze an approved genetically modified maize (Mon810 Line)

A laboratory-performance study was carried out to investigate factors affecting the reliability of the quantitative PCR method to analyze an approved genetically modified (GM) maize (Mon810 line). Test maize powdered samples were prepared as blind samples containing a high (assigned value; 5.45%) or low (assigned value; 0.35%) concentration of the Mon810 line. After confirmation of their homogeneity, they were provided to 27 laboratories participating in the collaborative study. The data were collected from all laboratories and statistically analyzed. Two laboratories, which used a Roche LightCycler (LC), reported significantly high test values. A further examination showed that the LC method is greatly affected by the equipment itself or PCR reagents, resulting in poor repeatability. On the other hand, some laboratories, which used ABI quantitative PCR equipment, reported erroneous test values. In these laboratories, the errors appeared to have been due to inadequate quality and/or yield of DNA. To identify factors affecting the test values, analysis of the measured values for the taxon-specific gene will be useful. Furthermore, the modified silica-gel membrane DNA extraction method made it possible to extract the required amounts of DNA more easily and in a shorter time than before.     

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。     

紫薯淀粉结构与理化性质研究

目的 了解紫薯淀粉的结构和理化性质。方法 利用碱提取法从紫薯中提取淀粉, 与普通玉米淀粉进行对比, 分别对淀粉结构(分子链结构、结晶结构等)和理化性质(透明度、凝沉性、冻融稳定性、热稳定性)进行研究。结果 紫薯淀粉直链含量(24.5%)比玉米淀粉(26.7%)低, 两者均为A型结晶结构, 但紫薯淀粉的结晶度和分子有序程度比玉米淀粉高; 紫薯淀粉糊的透明度高于玉米淀粉糊, 且随时间延长其透明度下降程度比玉米淀粉糊低; 紫薯淀粉糊不易发生凝沉现象, 但其析水率(21.4%)比玉米淀粉糊高, 即冻融稳定性弱于玉米淀粉糊; 此外, 紫薯淀粉部分结构的热稳定性大于玉米淀粉。结论 紫薯淀粉在分子链结构和结晶结构上与玉米淀粉有较小差异, 但在理化性质上与玉米淀粉差别较大, 可为其工业应用提供指导基础。     

Systematic comparisons of genetically modified organism DNA separation and purification by various functional magnetic nanoparticles

DNA extraction is always an important and key step in bioanalytical fields for target nucleic acid detection. Traditional organic solvent‐based extraction methods are toxic and time‐consuming. In this work, we report a systematic study of magnetic nanoparticles (MNPs) as probes for genomic DNA extraction from genetically modified organism (GMO) plants. Different surface‐functionalised MNPs with controllable diameters have been used to extract the genomic DNA directly from GMO plants for detections. Systematic comparison of the results obtained under different extraction conditions has indicated that carboxyl‐modified MNPs with smaller diameters are more suitable for genomic DNA extractions. The successful qualitative detection of GMO and non‐GMO products based on the MNP extracted DNA is also achieved and discussed in this article. In view of the advantages of magnetic extraction, such as nontoxicity, ease of operation, and rapid and high throughput, this systematic research has demonstrated the great potential of the method and provides theoretical guidance for practical MNP applications.     

A simple DNA extraction method suitable for PCR detection of genetically modified maize

BACKGROUND: The polymerase chain reaction (PCR) is a powerful tool that is being increasingly used for detection of transgenic DNA. PCR requires only a minute quantity of template, but sensitive and accurate testing requires DNA of sufficient purity and free from inhibitors such as plant polysaccharides. Several standard protocols are available for this purpose, but they usually involve several steps, imply destruction of the maize kernel, or are time‐consuming. Our aim was to develop a fast and simple extraction method to isolate a raw DNA‐containing solution from maize tissues suitable for use as a template in a PCR‐based detection assay with specific oligonucleotides directed to the identification of event MON810. RESULTS: The NaOH‐based DNA extraction method we report here is time‐saving (5 min) and can be used to isolate DNA‐containing solutions from a small maize leaf portion (down to 1 mg) or from a single overnight‐germinated kernel. PCR performed with selected primers yielded reproducible detection of transgenic DNA. CONCLUSION: The main advantages of the procedure are the quick extraction step, the possibility of non‐destructive testing of maize kernels, and the robustness of the PCR‐based detection, a consequence of the selection of MON810‐matching oligonucleotides yielding intense and highly specific amplicons. Copyright © 2007 Society of Chemical Industry     

Use of commercial protease preparations to reduce protein and lipid content of maize starch

A method was developed to produce pure maize starch from maize flour using a protease processing step, and additional salt washing and sulphite steeping steps. A range of commercial protease enzymes were evaluated for this purpose. The laboratory scale procedure that was developed, using one protease in particular (Promod P25P, thermolysin), produced relatively pure starch (<0.45% protein). Using the same procedure, but applying to starches which had been produced in advance using traditional wet milling, starch protein contents could be reduced further by 25–50% with the lipid content reduced by up to 25%. The amount of starch damage was minimal using this approach (<1%). This procedure could be developed industrially for a ‘greener approach’ to starch extraction – although it may still be necessary to incorporate sulphite steeping stages to facilitate protein solubilisation and extraction.