Taqman-MGB实时荧光PCR定量检测转基因玉米MON863

运用实时荧光聚合酶链式反应技术(polymerase chain reaction,PCR)对转基因玉米MON863进行品种特异性检测和定量分析。通过设计玉米内源基因和外源基因边界序列特异性引物和Taqman-MGB探针,验证了内源基因的物种特异性和外源基因边界序列的品种特异性。利用已知转基因百分含量的MON863玉米作为标准品,进行荧光定量反应,建立定量标准曲线,通过标准曲线对玉米样品MON863玉米成份进行含量分析。结果表明,该方法重复性好,检测特异性强,最低检测限浓度达到0.001 ng/μL,即14个拷贝。由于使用实时荧光PCR技术,检测周期短,操作简便,可广泛运用于转基因玉米MON863的进出口检测和转基因产品的含量分析。     

长期低剂量饲喂稀土对大白鼠体重及基因组DNA的影响

利用Operon公司C ,D ,E 3组共 60个引物对饲喂不同浓度稀土的大白鼠基因组DNA进行了RAPD分析 ,共获得了 1 71个DNA片段 ,其中有 4个片段呈多态性。实验表明这些片段均与饲喂稀土无关 ,提示稀土对大白鼠基因组DNA并不造成影响 ;但是大白鼠体重测定显示 ,高浓度时大白鼠增长明显低于对照组 ,说明稀土具有一定的毒性。     

非线性化学指纹图谱技术用于菖蒲的鉴别及二元混合物中不同菖蒲含量的测定

采用非线性化学指纹图谱技术获得了石菖蒲、水菖蒲和九节菖蒲的特征指纹图谱,直观差异明显;将石菖蒲分别与水菖蒲和九节菖蒲进行二元掺杂,利用指纹图谱定量信息和各菖蒲含量拟合得到线性回归方程,相关系数0.9990.用该方程预测了二元混合物中不同菖蒲的含量,相对误差≤4.17%,RSD≤2.73%.非线性化学指纹图谱技术具有良好的重现性和特征性,同种菖蒲指纹图谱间系统相似度≥0.9989,不同种菖蒲指纹图谱间相似度≤0.9123.此外,该技术摒弃传统方法复杂的前处理过程,可高效、快速进行鉴别,从整体性角度对样本进行定性鉴别与定量分析.     

基于定量指纹图谱技术的中药质量控制

定量指纹图谱技术是中药指纹图谱技术与多指标成分定量分析相结合的中药质量控制模式。定量指纹图谱技术的发展包括定量组分的制备、过程控制的指纹图谱技术和产品含量测定3个主要部分。本文以丹参为例,通过水提、醇沉、过膜、大孔树脂分离和工业色谱分离5个工艺流程制备了丹参定量组分,对各个工艺步骤以指纹图谱技术考察其稳定性和重复性。对丹参定量组分中的原儿茶醛、迷迭香酸、丹酚酸B 3个成分进行含量测定。3个成分的含量总和大于50%。定量组分的制备以现有的活性成分为目标,经过去粗存精的工艺过程,其质量标准得到了有效的提高。     

朝鲜淫羊藿的色谱指纹图谱及液相色谱/电喷雾串联质谱分析

建立了长白山区朝鲜淫羊藿药材的高效液相色谱指纹图谱的分析方法. 确定了18批朝鲜淫羊藿药材的13个共有峰, 该指纹图谱的精密度、稳定性和重现性的相对标准偏差均低于3.0%. 结合液相色谱/电喷雾串联质谱对特征峰进行了结构确认, 并根据电喷雾串联质谱数据推测了13个特征化合物的结构. 结果表明采用高效液相色谱与质谱联用技术对朝鲜淫羊藿色谱指纹图谱中的特征峰进行结构确认, 使其色谱指纹图谱的特征性更强, 更适合于药材质量的鉴别与评价.     

复方板蓝根颗粒抗病毒成分HPLC-UV指纹图谱及LC-ESI-MSn研究

建立了复方板蓝根颗粒提取物高效液相色谱(HPLC-UV)指纹图谱分析方法. 确定了10批不同来源复方板蓝根颗粒提取物的19个共有峰. 各提取物的HPLC-UV指纹图谱与对照指纹图谱比较, 相似度均在93%以上. 利用液相色谱与质谱联用(LC-ESI-MSⁿ)技术对主要共有峰的结构进行了鉴定. 药效学研究表明, 复方板蓝根颗粒提取物有抗病毒活性. 该指纹图谱精密度、稳定性和重现性良好, 可作为复方板蓝根颗粒的质量评价方法.     

不同产地三七的~1H NMR指纹图谱对比分析

通过提取方法和检测方法的探讨,选用体积分数90%的甲醇水溶液,对三七主根样品进行超声提取,并用noesypr1d脉冲序列预饱和压制水峰实验,获取了8种不同产地三七的1H NM R指纹图谱,对三七主要成分特征信号进行了初步指认。通过图谱分析看出,不同产地三七的三萜皂苷含量和各种三萜皂苷组成比例不同,黄酮、糖类含量比重也不一样。由于1H NM R具有提供信息丰富、简便、快速和重现性好的特点,可以作为中药指纹图谱研究的方法。     

外源野生大豆DNA导入栽培大豆及RAPD分子验证

报道了利用大豆自花受粉后形成的花粉管通道,将外源高蛋白野生大豆总DNA直接导入栽培大豆的实验结果及对导入转化后代的分子验证。 结果显示:在D_1代所获7个单株中有1株发生明显变异,其结荚习性、株型、叶型、花色等与受体基本相同:百粒重、种皮色及茎杆强度介于双亲之间:单株荚数明显增多,连续3年测定蛋白质含量比受体提高12.5％,从D_1代起已稳定遗传3个世代。对该组合的受体、供体及转化后代进行RAPD分析,从150个引物中找到24个引物检测出DNA的多态性,证明外源DNA导入受体后引起后代基因组的显著变异,并推测大片段外源DNA同源重组可能是变异的主要原因。     

单细胞分析技术在人类细胞图谱计划中的应用

2016年10月人类细胞图谱计划正式启动,该计划旨在区分不同类型的细胞及其在人体内的位置,研究激活、分化等细胞行为的分子机制,定义病理表征和标记分子.人类细胞图谱的建立将推动对人类健康和疾病诊断、监测、治疗的发展,从而促进对生命最基本单元的理解.建立系统的单细胞分析方法是完成人类细胞图谱的重要前提和保证.微流控细胞分离、单细胞DNA/RNA测序、细胞原位分析、质谱流式细胞分析等技术已经逐渐建立并用于单细胞分析.然而人类细胞图谱仍然面临诸多挑战,需要具有更高灵敏度和准确度的分析方法以及高效的数据处理技术.本文简要介绍了人类细胞图谱计划的发展现状及现有单细胞分析技术,并对挑战所需的新技术提出了展望.     

正常组织和胰腺癌组织中差异表达蛋白的鉴定

采用双向凝胶电泳和生物质谱技术, 对12对胰腺癌组织和癌旁组织样品、3个胰腺良性疾病样品、3个正常胰腺组织样品的蛋白质进行了分离和鉴定, 获得了重复性较好的双向凝胶电泳图谱; 鉴定了胰腺癌和癌旁组织的差异表达蛋白质, 发现了30个差异表达蛋白质; 应用MALDI-TOF-MS/MS技术对差异表达蛋白质进行鉴定, 共有24个蛋白质得到鉴定, 其中15个蛋白质在胰腺癌组织中表达上调, 9个蛋白质表达下调. 这些蛋白质与胰腺癌的发生相关, 可能成为胰腺癌的分子标志物和药物治疗的靶蛋白.     

Genetic and Phytochemical Analysis of Armillaria mellea by RAPD,ISSR, and HPLC

Abstract

Molecular genetic and phytochemical methods were combined to investigate 17 Chinese strains of Armillaria mellea. Ten random amplified polymorphic DNA (RAPD) primers yielded 106 bands, of which 94 were polymorphic; 80 out of the 84 bands produced by nine inter-simple sequence repeats (ISSR) markers were polymorphic. Contents of water-soluble constituents of mycelia were characterized by high-performance liquid-chromatography–diode array detection (HPLC-DAD) analyses. Cluster analyses of the genetic and phytochemical variation revealed that Armillaria mellea exhibited four chemotypes and four clusters from phytochemical contents. Phylogenetic groupings displayed in tree plots calculated from the RAPD and ISSR data matrix correlated with the geographical origin of the fungi materials. Genetic profiles partially correlated with the chemotypes. Phytochemical contents mainly correlated with the strains. A method based on RAPD and HPLC is described to establish genetic and chemical quality control of Armillaria mellea simultaneously. Ten RAPD primers and a coefficient of >0.95 can be used in authentication of Armillaria mellea. The fingerprints obtained with HPLC consist of 22 common peaks within 45 min. This method could be readily utilized as a quality-control method for pharmaceutical-grade Armillaria mellea.     

Screening of Tea (Camellia sinensis) for Trait-Associated Molecular Markers

This study was done to identify random amplified polymorphic DNA (RAPD) markers that may associate with seven important traits in tea. Sixty RAPD primers were first screened using 18 cultivars under each of the 7 traits, followed by confirmatory screening of 20 promising primers with 32 tea cultivars. Six RAPD primers generated a total of nine specific bands that associated with six desired traits: black tea quality and tolerance to drought, high temperature, low temperature, Phomopsis theae, and high yield. These markers would allow early identification of plant material with the desired traits that can be advanced to the next stage of selection and enhance targeted choice of breeding stocks with the desirable traits. The nine RAPD markers identified in this study could improve precision and efficiency in tea breeding and selection and are an important contribution towards the establishment of marker-assisted selection in tea breeding programmes.     

RAPD and ISSR Marker-Based Comparative Evaluation of Genetic Diversity Among Indian Germplasms of <Emphasis Type="Italic">Euryale ferox</Emphasis>: an Aquatic Food Plant

Euryale ferox Salisbury is an important aquatic food plant cultivated largely in eastern India. E. ferox is a monotypic genus, and breeding programmes have mostly relied on the variability present in the primary gene pool. Knowledge of the genetic structure of the population is limited, and there are very few reports available on the genetic diversity of E. ferox. In this study, comprehensive research on the genetic diversity of 16 germplasms of E. ferox was carried out using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Out of 320 RAPD and 95 ISSR primers screened initially, 61 primers (40 RAPD and 21 ISSR) gave reproducible bands and were selected for further work. Amplification of the 40 RAPD primers gave 533 polymorphic bands with an average of 13.32 polymorphic bands per primer. The percentage of polymorphism ranged from 37.5 to 100, with an average of 88.3 %. The 21 ISSR primers produced 259 bands, of which 214 were polymorphic, with an average of 10.19 polymorphic bands per primer. The percentage of polymorphism using ISSR primers ranged from 50 to 100, with a mean of 82.6 %. Jaccard’s coefficient ranged from 0.45 to 0.69 (RAPD), 0.50 to 0.77 (ISSR) and 0.48 to 0.71 (RAPD and ISSR). Molecular characterization of different germplasms of E. ferox not only is essential for its conservation but also can be used in further breeding programmes.     

Novel reference molecules for quantitation of genetically modified maize and soybean

New quantitation methods based on a real-time polymerase chain reaction (PCR) technique were developed for 5 lines of genetically modified (GM) maize, including MON810, Event176, Bt11, T25, and GA21, and a GM soy, Roundup Ready. Oligonucleotide DNA, including specific primers and fluorescent dye-labeled probes, were designed for PCRs. Two plasmids were constructed as reference molecules (RMs) for the detection of GM maize and GM soy. The molecules contain the DNA sequences of a specific region found in each GM line, universal sequences used in various GM lines, such as cauliflower mosaic virus 35S promoter and nopaline synthase terminator, and the endogenous DNA sequences of maize or soy. By using these plasmids, no GM maize and GM soy were required as reference materials for the qualitative and quantitative PCR technique. Test samples containing 0, 0.10, 0.50, 1.0, 5.0, and 10% GM maize or GM soy were quantitated. At the 5.0% level, the bias (mean-true value) ranged from 2.8 to 19.4% and the relative standard deviation was <5.2%. These results show that our method involving the use of these plasmids as RMs is reliable and practical for quantitation of GM maize and GM soy.     

Studies on the genetic variation of the green unicellular alga Haematococcus pluvialis (Chlorophyceae) obtained from different geographical locations using ISSR and RAPD molecular marker

Haematococcus pluvialis (Flotow) is a unicellular green alga, which is considered to be the best astaxanthin-producing organism. Molecular markers are suitable tools for the purpose of finding out genetic variations in organisms; however there have been no studies conducted on ISSR or RAPD molecular markers for this organism. The DNA of 10 different strains of H. pluvialis (four strains from Iran, two strains from Finland, one strain from Switzerland and three strains from the USA) was extracted. A genetic similarity study was carried out using 14 ISSR and 12 RAPD primers. Moreover, the molecular weights of the bands produced ranged from 0.14 to 3.4 Kb. The PCA and dendrogram clustered the H. pluvialis strains into various groups according to their geographical origin. The lowest genetic similarity was between the Iran2 and USA2 strains (0.08) and the highest genetic similarity was between Finland1 and Finland2 (0.64). The maximum numbers of bands produced by the ISSR and RAPD primers were 35 and 6 bands, respectively. The results showed that ISSR and RAPD markers are useful for genetic diversity studies of Haematococcus as they showed geographical discrimination.     

芝麻致敏原的实时荧光PCR检测

针对芝麻Sesamum indicum 2S albumin mRNA基因设计引物、探针,在实时荧光PCR仪上进行扩增、检测和分析.结果显示:该组探针和引物对芝麻有很强的特异性,除黑芝麻和白芝麻外,其余6种对照材料均未检测到荧光信号,黑白芝麻成分检测灵敏度达到0.1%.该方法具有灵敏度高、快速、简便的特点,可用于芝麻致敏原成分的定量检测.     

A two primers random amplified polymorphic DNA procedure to obtain polymerase chain reaction fingerprints of bacterial species

Polymerase chain reation (PCR) fingerprints are used to characterize and recognize bacteria and are generally obtained using universal primers that generate an array of DNA amplicons, which can be separated by electrophoresis. Universal primers 8F and 1491 R have been used to amplify specifically 16S rDNA. We have used these primers at an annealing temperature of 50 degrees C. Agarose gel electrophoresis of PCR products revealed several bands. The band pattern of each bacterial species was different and the strains belonging to the same species shared an identical pattern. The patterns obtained did not show variations with plasmid DNA content or the growth stage of the bacteria. The peculiarity of the randomly amplified polymorphic DNA (RAPD) described in this work lies in the use of two large primers (proximately 20 nt) to obtain the pattern, since normally a only smaller primer is used, and in the new application for the primers used to amplify 16S rDNA. This new procedure, called two primers (TP)-RAPD fingerprinting, is thus rapid, sensitive, reliable, highly reproducible and suitable for experiments with a large number of microorganisms, and can be applied to bacterial taxonomy, ecological studies and for the detection of new bacterial species.     

巴西果仁致敏原的实时荧光PCR检测

针对巴西果仁2Salbumin mRNA基因设计引物、探针,在实时荧光PCR仪上进行扩增、检测和结果分析.结果显示:该组探针和引物对巴西果仁有很强的特异性,除巴西果仁外,其余8种对照树果材料均未检测到荧光信号,巴西果仁成分检测灵敏度达到0.1%.该方法具有灵敏度高、快速、简便的特点,可用于巴西果仁致敏原成分的定量检测.     

Genetic Diversity Caused by Environmental Stress in Natural Populations of Niupidujuan as Revealed by RAPD Technique

Multiplex environmental factors are generally expected to have significant effects on genetic diversity of plant populations.In this study,randomly amplified polymorphic DNA(RAPD) technique was used to reveal the genetic diversity in the same species of four populations collected from Niupidujuan(Rhododendron chrysanthum) at different altitudes,an endangered species,endemic to Northeast China.Initially,twenty informative and reproducible primers were chosen for final RAPD analysis.A total of 152 clear bands...