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1.
一种改良的从LongSAGE标签中鉴定猪新基因的GLGI方法 总被引：3，自引：0，他引：3
将长的基因表达序列标签(LongSAGE)和结合标签的基因序列延伸(GLGI)技术相结合,对GLGI的方法进行改良。(1)将原始GLGI方法中正向引物中长为10bp的SAGE标签特异序列改为17bp的LongSAGE标签;(2)针对不同LongSAGE标签,在PCR反应中给定相应不同的退火温度;(3)在PCR反应中用普通的DNA聚合酶代替高保真酶(Pfu);(4)在PCR反应的克隆中,使用普通的T载体和感受态细胞代替原始方法中特异的载体和感受态细胞。利用改良的GLGI方法分离的EST位于cDNA的3′末端并带有加尾信号和ploy(dA)尾巴。该方法在鉴定低丰度表达的基因时比普通的EST测序方法具有更高的灵敏性。 相似文献
2.
基于超高频RFID双天线双标签对照的果园单轨运输机定位 总被引：1，自引：1，他引：0
针对果园单轨运输机在轨位置精准感知的应用需求,该文基于超高频射频识别技术(radio frequency identification,RFID)接收信号强度数据进行了运输机定位试验研究。该研究通过分析RFID通信特征,设计了RFID双天线双标签对照的运输机定位方法,构建了能量传输定位模型和路径损耗定位模型;通过阅读器单天线试验,得到当前RFID设备的安装参数,确定了阅读器天线与轨道标签之间的最优垂直距离为20 cm,双标签之间的最优水平在轨距离为45 cm;通过双天线试验及数据分析,得到适用于试验环境的最优定位模型系数。试验结果表明,该研究提出的定位模型能够有效降低噪声干扰,减少定位误差。RFID设备在最优定位参数条件下,使用路径损耗定位模型得到的最小定位误差均值为1.0070 cm。该研究验证了基于超高频RFID对果园单轨运输机定位的可行性,提升了运输机运行安全性和可靠性。 相似文献
3.
激活标签技术（activation tagging）在植物功能基因组学的研究中具有重要的作用。本文以拟南芥bzr1-D为试材，用农杆菌介导的转化方法，构建了一个含有30000个独立抗Basta株系的激活标签突变体库。其中有47个株系有明显的表型改变，包括花期的改变、株型矮小、叶片形状改变、叶柄变长、叶表皮毛消失、育性降低以及恢复了bzr1-D茎叶处的打折等。并通过TAIL-PCR和Nested PCR的方法得到了若干T-DNA侧翼序列，为克隆引起突变表型的基因以及进行后代遗传分析打下基础。 相似文献
4.
苹果酸-乳酸酶是进行MLF的关键酶.该研究以酒类酒球菌31DH(Oenococcus oeni 31DH)的基因组DNA为模板进行其苹果酸-乳酸酶基因mleA的PCR扩增.PCR引物为5'-CGGAATTCATGACAGATC-CAGTAAGTAT-3'和5'-TAGGTACCACACTCTCAACACTCGTAAT-3',引物的5端分别引入EcoRI和KpnI酶切位点.得到的PCR产物约1.6kb.PCR产物回收后用EcoRI-KpnI双酶切,与经同样双酶切的质粒YEp352(大肠杆菌-酵母穿梭载体)进行连接并转化大肠杆菌感受态细胞,筛选重组质粒并用酶切及PCR验证.获得的酒类酒球菌苹果酸-乳酸酶基因的重组质粒命名为pLmleA. 相似文献
5.
Ac/Ds双元激活标签体系用于建立水稻突变体材料 总被引：1，自引：0，他引：1
将含有两个35S启动子和4个串联重复的35S增强子的Ac／Ds双元转座子标签载体转入水稻(O／yza sativa L．)基因组。100个转化植株的分析结果表明，Ds在T0植株的体细胞中切离频率高达60％。20个T1株系的600个T1植株的PCR分析结果表明，14个株系(70％)表现出较高的切离频率(约43％～100％)。Southem杂交发现在同一T1株系的不同植株中Ds转座既有相同的转座位点(约90％)，也有不同的转座位点(约10％)。 相似文献
6.
本研究中用融合标签技术通过单质粒转化和双质粒转化分别促进Aβ1-42淀粉样蛋白的可溶性表达。构建重组载体pET（yd-b42）、pET（Msb-b42）、pET（Od-b42）、pAY-sls[ydb42]、pAY-sls[tydb42],并在大肠杆菌中表达,通过SDS-PAGE验证分析。标签yd、Msb、Od、tyd都能很好的促进Aβ1-42淀粉样蛋白可溶性表达,其中yd、tyd的促溶效果最好。Aβ1-42淀粉样蛋白能在大肠杆菌中可溶性表达。为阿尔茨海默病的治疗提供进一步理论基础。 相似文献
7.
改进AOA模式的大田农机无人驾驶导航参数检测系统设计 总被引：1，自引：1，他引：0
卫星导航、视觉导航和雷达导航的成本昂贵、系统构成复杂和适用作业场景有限,在生产特征呈现区域化、适度小规模和分布零散的国内南方水田难以实现便捷跨区域作业和无法适用多农业场景。针对上述问题,该研究以大田环境下无人驾驶农机的牛耕式往复作业路径模式为背景,提出了改进AOA(信号到达角度,Angle-of-Arrival)模式的农业机械无人驾驶导航参数检测系统。该系统采用UWB(超宽带通信,Ultra Wide Band)基站-标签作为检测传感器,设计了TBZ(田边双基站-车身纵向双标签)和TBH(田边双基站-车身横向双标签)2种传感器布置方式,实现农业机械无人驾驶过程中导航参数的快速精准检测。静态试验结果表明:对于2种传感器布置方式,在固定的基站间距和标签间距下,随着标签间距或基站间距的增大导航参数检测精度均有所提高,横向偏差检测误差≤8 cm,航向偏差趋近于0,但不大于1°,并通过正交组合试验方差分析明确了2种传感器布置方式的关键参数对横向偏差和航向偏差检测精度影响的显著性,确定了主次因素和较优参数组合。动态试验结果表明:随着车速增大,横向偏差和航向偏差的检测精度有所降低,横向偏差误差均不超过10 cm,航向偏差的检测误差均小于3°,变异系数均小于10%,说明动态环境下自主导航参数检测系统仍具有较高的检测精度,可满足农机大田自主导航作业需求。研究结果可为研制低成本、高精度和便捷的无人驾驶系统提供参考。 相似文献
8.
黄泽颖 《农产品质量与安全》2020,(3)
北欧国家实施的Keyhole标签系统的建立与发展,为世界各国发展食品包装正面(Front of Package, FOP)标签系统提供了丰富的经验。本文介绍了北欧国家食品Keyhole标签系统的主要做法,分析了Keyhole标签系统发展面临的挑战,并由此建议我国实施FOP标签系统需要具备充分的科学依据、制定指导性规定、实施政府监管以及不断的革新等。 相似文献
9.
10.
本文重组和构建了新城疫病毒 ( NDV)融合蛋白基因 ,并对该基因进行了鉴定 :将新城疫病毒 F基因片段经 RT— PCR扩增 ,插入经 Eco R I/ Sal 酶切的克隆载体 p UC18及表达载体 p GEMEX,转化大肠杆菌 JM10 9株。用氨苄青霉素平板法初步筛选克隆 ,再用双酶切法、核酸探针、 PCR及核苷酸序列分析法鉴定 ,表明插入成功并且阅读框架正确 相似文献
11.
Qualitative and quantitative event-specific PCR detection methods for oxy-235 canola based on the 3' integration flanking sequence 总被引：2，自引：0，他引：2
As more genetically modified plant events are approved for commercialization worldwide, the event-specific PCR method has become the key method for genetically modified organism (GMO) identification and quantification. This study reveals the 3' flanking sequence of the exogenous integration of Oxy-235 canola employing thermal asymmetric interlaced PCR (TAIL-PCR). On the basis of the revealed 3' flanking sequence, PCR primers and TaqMan probe were designed and qualitative and quantitative PCR assays were established for Oxy-235 canola. The specificity and limits of detection (LOD) and quantification (LOQ) of these two PCR assays were validated to as low as 0.1% for the relative LOD of qualitative PCR assay; the absolute LOD and LOQ were low to 10 and 20 copies of canola genomic DNA in quantitative PCR assay, respectively. Furthermore, ideal quantified results were obtained in the practical canola sample detection. All of the results indicate that the developed qualitative and quantitative PCR methods based on the revealed 3' integration flanking sequence are suitable for GM canola Oxy-235 identification and quantification. 相似文献
12.
AS-PCR技术检测鸡EX-FABP基因型方法的建立 总被引：5，自引：0，他引：5
摘要： 鸡(Gallus gallus )胞外脂肪酸结合蛋白(extracelluar fatty acid binding protein, EX-FABP)基因型与鸡腹脂量的积累密切相关。尝试了用等位基因特异性PCR(allele-Specific PCR, AS-PCR) 技术检测EX-FABP基因型的方法，确定了检测的参数和方案, 对AS-PCR检验单碱基突变的方法和策略进行了讨论,并在此基础上对该技术进行了改进，建立了等位基因特异性片段长度差异PCR（allele-specific and length-different PCR, ASLD PCR）技术。 相似文献
13.
Iida M Yamashiro S Yamakawa H Hayakawa K Kuribara H Kodama T Furui S Akiyama H Maitani T Hino A 《Journal of agricultural and food chemistry》2005,53(16):6294-6300
Qualitative and quantitative Polymerase Chain Reaction (PCR) systems aimed at the specific detection and quantification of common wheat DNA are described. Many countries have issued regulations to label foods that include genetically modified organisms (GMOs). PCR technology is widely recognized as a reliable and useful technique for the qualitative and quantitative detection of GMOs. Detection methods are needed to amplify a target GM gene, and the amplified results should be compared with those of the corresponding taxon-specific reference gene to obtain reliable results. This paper describes the development of a specific DNA sequence in the waxy-D1 gene for common wheat (Triticum aestivum L.) and the design of a specific primer pair and TaqMan probe on the waxy-D1 gene for PCR analysis. The primers amplified a product (Wx012) of 102 bp. It is indicated that the Wx012 DNA sequence is specific to common wheat, showing homogeneity in qualitative PCR results and very similar quantification accuracy along 19 distantly related common wheat varieties. In Southern blot and real-time PCR analyses, this sequence showed either a single or a low number of copy genes. In addition, by qualitative and quantitative PCR using wx012 primers and a wx012-T probe, the limits of detection of the common wheat genome were found to be about 15 copies, and the reproducibility was reliable. In consequence, the PCR system using wx012 primers and wx012-T probe is considered to be suitable for use as a common wheat-specific taxon-specific reference gene in DNA analyses, including GMO tests. 相似文献
14.
三种对虾病毒多重实时荧光PCR检测方法的建立 总被引：1，自引：0，他引：1
根据基因库中白斑综合征病毒（WSSV）、传染性皮下及造血器官坏死病毒（IHHNV）和桃拉综合征病毒（TSV）的基因序列,设计了WSSV 、IHHNV和TSV的三对特异性引物和三条用不同荧光基团标记的TaqMan探针。对反应条件和试剂浓度进行优化,建立了能够同时检测WSSV 、IHHNV和TSV的三重实时荧光PCR方法。该方法特异性好,对WSSV 、IHHNV和TSV的检测敏感性分别达到20000、20和20000个模板拷贝数;此外抗干扰能力强,对WSSV 、IHHNV和TSV不同模板浓度进行组合,仍可有效地同时检测这三个病毒。对保存的45份经常规PCR检测仅为WSSV 、IHHNV和TSV阳性的样品进行二重实时荧光PCR检测,结果都为阳性,其中2份为WSSV和IHHNV混合感染。本研究建立的三重实时荧光PCR方法用于WSSV、IHHNV和TSV的检测具有特异、敏感、快速、定量等优点。 相似文献
15.
加强对进口饲料中牛羊源成分的检测是防止疯牛病和痒病传播的一个重要措施。根据已发表的牛和羊特异性基因及引物序列，分别设计了1条牛和羊特异性semi-nested PCR引物，并采用semi-nested PCR技术对饲料中的牛和羊成分进行了扩增检测。结果表明，semi-nested PCR能够扩增得到247 bp的牛特异性基因条带和214 bp的羊特异性基因条带，其对饲料中牛或羊源性成分的检测灵敏度可达到0.00001%～0.0001%，比普通PCR检测灵敏度要高出103倍；对牛或羊成分DNA的检测灵敏度可以达到10-6～10-5 ng，比普通PCR检测灵敏度要高出105倍以上。该技术具有快速、灵敏和结果稳定的特点，是检测饲料中痕量牛羊源成分的一种有效方法。 相似文献
16.
微囊藻毒素（Microcystin,MC）的产生受微囊藻毒素合成酶基因簇（microcystin biosynthesis gene,mcy）调控,常用PCR扩增mcy基因检测产毒微囊藻。采集江苏省5大淡水湖泊——太湖、滆湖、高宝-邵伯湖、洪泽湖和骆马湖的水样,测定水体营养盐浓度和叶绿素a浓度,根据叶绿素a浓度计算5个湖泊的富营养化指数（Trophic state index,TSI）,同时应用单一和多重PCR扩增mcy基因。结果表明,太湖和滆湖处于富营养和超富营养化水平,洪泽湖和骆马湖处于中营养和富营养化水平,高宝-邵伯湖处于寡营养水平。太湖、滆湖、洪泽湖和骆马湖的所有水样均检出mcy基因,4个湖泊水体受到微囊藻毒素的潜在威胁,高宝-邵伯湖没有检测出mcy基因的存在,尚未受微囊藻毒素的污染。 相似文献
17.
Nakamura S Haraguchi K Mitani N Ohtsubo K 《Journal of agricultural and food chemistry》2007,55(25):10388-10395
Template DNAs were extracted from wine and purified for use as samples for PCR to differentiate grape cultivars. It has been pointed out that the authentication of grape material by PCR using wine as a material is very difficult. The problems are (1) decomposition of DNAs during fermentation; (2) contamination of DNAs from microorganisms such as yeast; (3) interference of DNA extraction by polysaccharides and polypeptides in the beverages; and (4) coexistence of PCR inhibitors, such as polyphenols. For this study was developed a novel preparation method of template DNA from wine to differentiate grape cultivars using PCR by (1) lyophilizing and pulverizing the fermented beverage to concentrate the DNAs; (2) decomposition of polysaccharides and proteins so as not to inhibit DNA extraction using heat-resistant amylase and proteinase K without DNA damage by endogenous DNase; and (3) separation of the template DNAs for PCR from PCR inhibitors, such as polyphenols, by purification using 70% EtOH extraction and isopropyl alcohol precipitation. To prevent the amplification of microorganisms' DNAs during PCR, suitable PCR primers closely related to the specific plant DNAs, such as chloroplast DNA and mitochondrial DNA, were selected. The sequences of the amplified DNAs by PCR were ascertained to be the same as those of grape materials. 相似文献
18.
Sensitive PCR analysis of animal tissue samples for fragments of endogenous and transgenic plant DNA
Nemeth A Wurz A Artim L Charlton S Dana G Glenn K Hunst P Jennings J Shilito R Song P 《Journal of agricultural and food chemistry》2004,52(20):6129-6135
An optimized DNA extraction protocol for animal tissues coupled with sensitive PCR methods was used to determine whether trace levels of feed-derived DNA fragments, plant and/or transgenic, are detectable in animal tissue samples including dairy milk and samples of muscle (meat) from chickens, swine, and beef steers. Assays were developed to detect DNA fragments of both the high copy number chloroplast-encoded maize rubisco gene (rbcL) and single copy nuclear-encoded transgenic elements (p35S and a MON 810-specific gene fragment). The specificities of the two rbcL PCR assays and two transgenic DNA PCR assays were established by testing against a range of conventional plant species and genetically modified maize crops. The sensitivities of the two rbcL PCR assays (resulting in 173 and 500 bp amplicons) were similar, detecting as little as 0.08 and 0.02 genomic equivalents, respectively. The sensitivities of the p35S and MON 810 PCR assays were approximately 5 and 10 genomic equivalents for 123 bp and 149 bp amplicons, respectively, which were considerably less than the sensitivity of the rbcL assays in terms of plant cell equivalents, but approximately similar when the higher numbers of copies of the chloroplast genome per cell are taken into account. The 173 bp rbcL assay detected the target plant chloroplast DNA fragment in 5%, 15%, and 53% of the muscle samples from beef steers, broiler chickens, and swine, respectively, and in 86% of the milk samples from dairy cows. Reanalysis of new aliquots of 31 of the pork samples that were positive in the 173 bp rbcL PCR showed that 58% of these samples were reproducibly positive in this same PCR assay. The 500 bp rbcL assay detected DNA fragments in 43% of the swine muscle samples and 79% of the milk samples. By comparison, no statistically significant detections of transgenic DNA fragments by the p35S PCR assay occurred with any of these animal tissue samples. 相似文献
19.
Platteau C De Loose M De Meulenaer B Taverniers I 《Journal of agricultural and food chemistry》2011,59(21):11395-11402
Hazelnuts (Corylus avellana) are used widely in the food industry, especially in confectionery, where they are used raw, roasted, or in a processed formulation (e.g., praline paste and hazelnut oil). Hazelnuts contain multiple allergenic proteins, which can induce an allergic reaction associated with symptoms ranging from mild irritation to life-threatening anaphylactic shock. To date, immunochemical (e.g., ELISA or dipstick) and PCR-based analyses are the only methods available that can be applied as routine tests. The aim of this study is to make a comparative evaluation of the effectiveness of ELISA and real-time PCR in detecting and correctly quantifying hazelnut in food model systems. To this end, the performances of two commercial ELISAs were compared to those of two commercial and one in-house-developed real-time PCR assays. The results showed that although ELISA seemed to be more sensitive compared to real-time PCR, both detection techniques suffered from matrix effects and lacked robustness with regard to food processing. As these impacts were highly variable among the different evaluated assays (both ELISA and real-time PCR), no firm conclusion can be made as to which technique is suited best to detect hazelnut in (processed) food products. In this regard, the current lack of appropriate DNA calibrators to quantify an allergenic ingredient by means of real-time PCR is highlighted. 相似文献
20.
藏猪氟烷基因PCR-RFLP和序列多态性分析 总被引：1，自引：0，他引：1
本文应用PCR方法,体外扩增了94头藏猪的氟烷基因18149～18760位之间612bp的片段（包含完整的外显子17和cDNA C1843→T1843突变位点）,采用HhaⅠ内切酶对扩增产物进行了酶切多态性分析,发现94头藏猪个体都呈现氟烷阳性。并对4个藏猪个体的氟烷基因的612bp片段进行序列测定,测定结果与GenBank中Brening公布的序列和帅素容测定的6个中国地方猪种氟烷基因序列的相同区段进行比较分析,结果表明,本文所测藏猪序列共发现2个变异位点,都发生在内含子区段,皆为转换型突变;在内含子16内有1个位点存在转换型杂合子。在4个藏猪个体中未发现cDNA C1843→T1843突变位点。 相似文献