应用PRIMER软件进行浮游植物群落结构的多元统计分析

根据青岛近海进行的一次添加营养盐的围隔实验数据,采用专业软件PRIMER中的多元统计分析方法,进行了浮游植物群落结构的变化研究。结果表明,虽然由于本实验中浮游植物的生长受到Si的限制,硅藻类群不占明显的优势,使得优势种随时间的变化不明显,没有出现明显的群落演替,但用PRIMER软件的聚类分析(Clustering)和多维尺度转换排序(MDS)分析表明,添加营养盐后浮游植物群落结构有较明显的变化,同时,PRIMER软件的相异系数(ANOSIM)分析也表明,在同时添加N、P的围隔内,浮游植物群落结构变化较只添加P的围隔中的变化显著。这显示了PRIMER软件在分析浮游植物群落结构变化和评价水域富营养化中的应用前景。

多元统计软件PRIMER在张网渔具种类选择性研究中的运用

本文根据2014年秋季青岛斋堂岛海域双桩竖杆张网的网囊网目选择性试验数据,运用PRIMER软件中的相似性分析、非参数多维标度分析和相似性百分比分析模块分析了张网渔具种类选择性。试验采用平行作业法,以传统网具(2a=16mm的菱形目网囊)作为对照网,设计了网目尺寸(2a)为30和35mm的菱形目网囊(以30D和35D表示)和网目尺寸(2a)为20、30和35mm的方形目网囊(以20S、30S和35S表示),进行选择性实验。研究显示:(1)35D、30S和35S渔获组成与对照网存在显著性差异,其余各网之间差异不显著。(2)改良后的试验网具能有效释放舒氏海龙;除20S组外,其余各试验网对方氏云鳚亦有较好的释放效果。(3)方形目网囊较相同尺寸的菱形目网囊具有更好的种类选择性;对相同网目形状的试验网囊进行比较,种类选择性随网目尺寸的增大而增大。(4)综合分析不同网具的渔获物组成和规格,在斋堂岛海域秋季小黄鱼、带鱼渔汛期,推荐使用30mm方形目网囊;方氏云鳚渔汛期,推荐使用20mm方形目网囊进行捕捞作业。研究结果表明,运用多元统计软件PRIMER进行张网渔具种类选择性分析虽然存在待完善之处,但该方法为本领域研究提供了有益参考。

DNA Extraction and ISSR Primer Screening of Camellia chekiangoleosa

[Objective] The aim was to screen out simple methods of DNA extraction and effective ISSR primers suitable to all germplasm materials of Camellia chekiangoleosa.[Method] The modified CTAB method was used in the extraction of the genomic DNA,50 ISSR primers from other Camellia plants were used in the ISSR-PCR amplification for 20 germplasm materials from 10 populations in Fujian,Zhejiang,Jiangxi and Anhui Province.[Result] Pure DNA could be obtained rapidly by using the improved CTAB method,and the 20 selected effective primers had rich polymorphism,clear bands and good repeatability.337 DNA bands were obtained,of which 281 bands were polymorphic,accounting for 83.4% of the total amplified bands.And 16.85 bands could be amplified with a primer,averagely.[Conclusion] The selected 20 primers could be effectively applied to ISSR analysis of the germplasm resources of C.chekiangoleosa in Zhejiang.

利用软件Primer Premier 5.0进行PCR引物设计的研究

目的 运用PrimerPremier 5 0软件设计PCR引物 ,并且检验所设计引物的PCR扩增效率和特异性。方法 运用PrimerPremier 5 0软件设计 16对猪和犬的PCR扩增引物 ,通过PCR扩增后进行琼脂糖凝胶电泳检测实验结果。结果 在所设计的 16对引物中有 8对PCR扩增特异性好且效率高 ,成功率 5 0 %。但是 ,其中早期设计引物 12对只有 4对成功 ,后期设计引物 4对全部成功。结论 从引物设计的过程中可以看到一种趋势 -后期引物设计的成功率远远高于早期。这表明我们在引物设计方面正在逐步成熟 。

Designing Primers for H5 and H7 Subtypes of Avian Influenza Virus and Multiplex RT-PCR Amplification

[Objective] The research aimed to design primers that are suitable for detecting H5 and H7 subtypes of avian influenza virus （AIV） ; [Method] DNAStar was used to analyze the homology of the sequences of H5 and H7 subtypes of AIV accessed in GenBank, and design primers（ by Primer Premier 5.0） on high homologous region of these sequences, and then amplified by RT-PCR. [Result] The multiplex RT-PCR amplification, agarose gel electrophoresis and sequencing results showed that the self-designed primers are successful for detecting AIV. [Conclusion] It is feasible to rapidly diagnose AIV through this method.

利用MPprimer设计引物并优化扩增条件以提高多重PCR效率的实验研究

多重PCR技术广泛应用于多个研究领域,其中引物设计及扩增条件是提高多重PCR实验效率的关键因素.为探讨优化多重PCR实验的方法,以小鼠5个看家基因为研究对象,使用实验室新近开发的MPprimer程序设计多重PCR引物,并通过改变多种反应条件来优化多重PCR实验.结果表明,MPprimer程序能够设计出理想的多重PCR引物,并且通过对退火温度及延伸时间进行优化,可显著提高多重PCR实验效率,对于提高基因表达的规模化检测能力具有积极的促进作用.

Screening of SSR Core Primers for Purity Identification of Pepper(Capsicum) Hybrids

[Objective] This study aimed to screen a set of SSR core primers suitable for purity identification of pepper （Capsicum） hybrids. [Method] DNA fingerprint of 100 pepper hybrids was analyzed using 17 SSR primers. [Result] According to the polymorphism and heterozygosity, Hpms1-214, Es395 and Hpmsl-5 were determined as three preferred core primers for purity identification of pepper hybrids. By using these three preferred core primers, 97 pepper hybrids （accounting for 97%） had heterozygous band pattern with at least one primer. Es330, Es363, Epms923, Es120 and Es64 were determined as candidate core primers for purity identification of pepper hybrids. Specific primers of 14 varieties were obtained, which could be used to further screen parent-complementary primers of each pepper hybrid. [Con- clusion] This study laid the foundation for constructing standard DNA fingerprints for purity identification of pepper hybrids.

Primer Screening on Germplasm Resources of Mallotus oblongiolus by ISSR Molecular Marker

[ Objective] To provide effective primers for the rapid and accurate ISSR analysis of the germplasm materials of Mallotus oblongiolus （Miq.） Muello-Arg.. [Method] The modified CTAB method was used in the extraction of the genomic DNA. 99 ISSR primers were used in the ISSR-PCR amplification for 20 germplasm materials from 10 populations in Hainan Island, so that some primers, which were suitable to all gerplasm materials of M. oblongiolu, could be selected. [ Result] 15 effective primers with characteristics of rich polymorphism, clear bands, and good repeatability were selected from 99 test primers. The 15 primers selected were used in the ISSR-PCR amplification for 66 germplasm materials of M. oblongiolus. From all of which the abundant and distinct DNA fingerprintings could be obtained. 286 DNA bands were obtained, and of which 231 bands were polymorphic, which amounted to 80.77% of the total bands amplified. And 19.1 bands could be obtained with each primer, averagely. [ Conclusion] The 15 primers selected could be effectively applied to ISSR analysis of the germplasm resources of M. oblongiolus.

大型多元统计软件PRIMER的方法原理及其在底栖群落生态学中的应用

本文阐述大型多元统计分析软件 PRIMER的原理及其在底栖生态学中的应用。PRIMER是处理生态和环境多元数据的重要数值分析工具 ,本文从实用的角度对其中包括的技术方法和相应的子程序如 :等级聚类 (CLUSTER)、非度量多维标度 (MDS)、主分量分析 (PCA)、相似性分析检验(ANOSIM)、相似性百分比分析 (SIMPER)、生物 -环境连接的逐步分析和相关检验 (BIOENV/BVSTEP)等作了较为系统的介绍。

Application of Abnormal Touchdown PCR with High Degeneracy Primer in Amplification of Large-Family Genes

[Objective] The aim was to explore the special methods for amplification of large-family genes by using primers with high degeneracy.[Method] By using the primers with high degeneracy,conventional PCR,conventional touchdown PCR and the optimized abnormal touchdown PCR were respectively carried out to amplify the genomic DNA of Cyprinus carpio.[Result] Only one evident electrophoretic band and a few Sox genes were obtained by using normal PCR;no obvious electrophoretic band but dispersive product was obtained by normal touchdown PCR;ideal result was obtained by the abnormal touchdown PCR that three evident electrophoretic bands and much more Sox genes were amplified.[Conclusion] The research provided theoretical basis for the optimization and selection of PCR amplification conditions of the large-family genes.

利用Primer Premier 5.0进行引物设计

目前，PCR引物设计大都通过计算机软件进行。本文详细的介绍了怎样利用“Primer Premier5．0”软件进行PCR引物设计。

Primer Extension-DHPLC检测线粒体DNA 12SrRNA常见耳聋相关突变方法的建立~△

目的建立针对线粒体DNA 12SrRNA基因常见耳聋相关突变的Primer Extension-DHPLC(PE-DHPLC)基因诊断新方法。方法将包含线粒体DNA 12SrRNA 961insC、T1095C、C1494T、A1555G 4种不同突变的4例外周血DNA样本和4例正常对照标本经测序验证,PCR扩增靶序列,设计延伸引物,延伸后得到线粒体DNA 12SrRNA基因961insC、T1095C、C1494T、A1555G 4个常见突变的特异性延伸片段,用全变性高效液相色谱分析延伸片段混合物,分离图谱鉴定被检样本的基因型。结果 4例包含线粒体DNA 12SrRNA基因4种耳聋相关突变的DNA样本均扩增出包含4种突变的975 bp特征性条带,PE-DHPLC检测结果显示突变者有引物峰和突变峰两个峰,完全可以鉴别出4种突变,而4例正常对照标本仅显示单个引物峰。结论本实验建立了Primer Extension-DHPLC的线粒体相关耳聋突变分析新方法,可用于耳聋的基因诊断。

3'-terminus shifted bases degeneracy primers increasing sensitivity of polymerase chain reaction

目的:通过使用3’末端碱基游移混合引物,减少引物末端错配,提高多变区基因片段的PCR检测阳性率。方法:在HBV DNA P基因区设计一对检测阳性率相对较高的引物(原型引物),在原型引物序列基础上将两根引物的3’末端分别截去1个或2个碱基,设计出两对参数与原型引物近似的突变引物。分别单独用原型引物和原型引物与突变引物组成的混合引物,在相同条件下对27份HBV DNA阳性标本行PCR,比较二者阳性率。用混合引物扩增4例拉米呋丁治疗无效患者血清HBV DNA,将扩增产物测序并评价3’末端错配对PCR的影响。结果:原型引物和混合引物检测阳性率分别为70.4％(19/27)和85.2％(23/27),两者差异非常显著(P<0.05)。引物3’末端错配能导致PCR假阴性。结论:扩增保守性相对较差的基因片段,采用3’末端单个或多个碱基截短的引物,构成3’末端碱基游移混合引物,可以避免因3’末端错配产生的PCR假阴性,提高检测阳性率。

False Positives Caused by Single Primer in DDRT Analysis of Soybean

[Objective] The aim was to explore the reasons of false positives in Different Display Reverse Transcription（DDRT）analysis.[Method] Soybean varieties ＂Jilin 30＂ and ＂Tongnong 13＂ were used as materials to carry out analysis on false positives in DDRT analysis.[Result] An important origin of false positives appeared in DDRT analysis was the non-specific amplification caused by the combination of single primer and cDNA.The parallel PCR test of single primer should be set so as to verify whether the obtained fragments were the false positives or the PCR productions combined with single primer.[Conclusion] This study had provided basis for improving the success rate of DDRT experiment.

Cricket Grb-AST_7 Gene Concatemer Constructed by "Self Template-primer" PCR

[Objective] Grb-AST7 concatemer including 17 copies of Grb-AST7 was constructed by ＂self template-primer＂ PCR. [Method] Two primers of which were synthesized based on the amino acid sequence of Gryllus bimaculatus’ Grb-AST7. [Result] Through splicing, a length of 570 bp, containing 17 copies of the Grb-AST7 gene repeats were obtained. Appropriate primer splicing conditions were as follows：splice time 20 min, 25 μl PCR system, containing 2 μl template. [Conclusion] The results laid foundation for the future studies on Grb-AST7 gene expression and bio-activity analysis.

Enlightenment from The Three Character Primer on Building a Democratic and Harmonious Family Education Means

At present,there are unharmonious factors existing in Chinese family education,which particular manifest in the means of family education.The Three Character Primer,as an epitome of the essence of Chinese traditional culture,can provide enlightenment on building a democratic and harmonious family education means.

Development of Walnut EST-SSR Markers and Primer Design

[Objective] This research aimed to develop walnut EST-SSR markers and design corresponding primers.[Method] 5213 EST sequences of walnut（Juglans regia Linn.） public online in NCBI were used for character analysis with bioinformatics methods,and primers were designed for the selected EST sequences by using Primer 3.0 software.[Result] 207 SSRs were obtained from the EST sequences,including 188 non-redundant sequences,the detection rate was 3.97% with an average distribution distance of 21.12 kb.Totally 92 types of repeat motifs were involved,which were mainly composed of dinucleotide and trinucleotide,accounting for 31.40% and 35.27% of the total number of repeat motifs,respectively.30 pairs of primers were initially selected from the 50 randomly-selected SSR primers by PCR amplification.[Conclusion] This research would lay foundations for the development of EST-SSR molecular markers in walnut and design of the targeted EST-SSR primers by mining and analyzing the SSR sites in walnut EST sequences.

Optimization of Multiplex PCR and Multiplex Gel Electrophoresis in Sunflower SSR Analysis Using Infrared Fluorescence and Tailed Primers

In an effort to simplify the procedure and to reduce the cost of fluorescence SSR analysis, the conditions of the multiplex PCR and the multiplex gel electrophoresis were optimized in the genetic analysis of sunflower (Helianthus annuus L.) inbred lines. Results indicated that factors for a successful multiplex PCR assay were related to the cycling touchdown annealing temperature, the balance of primer concentration at the various loci, the concentration of PCR buffer and the Taq DNA polymerase. Based on the optimization, a tailed primer strategy was outlined, and the effective ways were proposed to overcome the troubleshootings commonly encountered in the multiplex PCR and the multiplex gel electrophoresis.

Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome

AIM:To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome.METHODS:A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral,esophageal,and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species.Candidate primers evaluated were from the European rRNA database.To assess the effect of sequence length on accuracy of classification,16S rRNA genes of various lengths were created by trimming the full length sequences.Sequences spanning various hypervariable regions were selected to simulate the amplicons that would be obtained using possible primer pairs.The sequences were compared with full length 16S rRNA genes for accuracy in taxonomic classification using online software at the Ribosomal Database Project (RDP).The universality of the primer set was evaluated using the RDP 16S rRNA database which is comprised of 433 306 16S rRNA genes,represented by 36 phyla.RESULTS:Truncation to 100 nucleotides(nt)downstream from the position corresponding to base 28 in the Escherichia coli 16S rRNA gene caused misclassification of 87(39.7%)of the 219 sequences,compared with misclassification of only 29(13.2%)sequences with truncation to 350 nt.Among 350-nt sequence reads within various regions of the 16S rRNA gene,the reverse read of an amplicon generated using the 343F/798R primers had the least(8.2%)effect on classification.In comparison,truncation to 900 nt mimicking single pass Sanger reads misclassified 5.0%of the 219 sequences.The 343F/798R amplicon accurately assigned 91.8%of the 219 sequences at the species level.Weighted by abundance of the species in the esophageal dataset,the 343F/798R amplicon yielded similar classification accuracy without a significant loss in species coverage(92%).Modification of the 343F/798R primers to 347F/803R increased their universality among foregut species.Assuming that a typicalpolymerase chain reaction can tolerate 2 mismatches between a primer and a template,the modified 347F and 803R primers should be able to anneal 98%and 99.6%of all 16S rRNA genes in the RDP database.CONCLUSION:347F/803R is the most suitable pair of primers for classification of foregut 16S rRNA genes but also possess universality suitable for analyses of other complex microbiomes.

Tetra-primer ARMS PCR检测丙型肝炎患者IL-28B rs12979860基因多态性

目的评价四引物扩增受阻突变体系聚合酶链反应(Tetra-primer ARMS PCR)检测IL-28Brs12979860基因多态性在丙型肝炎患者抗病毒治疗中的应用。方法选取2015年5月至2016年7月就诊的慢性丙型肝炎患者275例,提取外周血DNA,建立Tetra-primer ARMS PCR检测体系,与Sanger测序法对比验证,检测该地区丙型肝炎患者IL-28Brs12979860基因多态性分布情况。结果 Tetra-primer ARMS PCR检测体系成功建立,与Sanger测序法的符合率100%,rs12979860基因的C/C型、C/T型和T/T型的分布频率分别为86.5%、12.0%、1.5%。在C/C型组与C/T或T/T型组中,年龄分布、性别分布、HCV基因1b型比例差异均无统计学意义(P>0.05);患者肝硬化比例和持续病毒学应答比例比较差异有统计学意义(P<0.05),在C/T或T/T组中肝硬化比例较高,在C/C组中持续病毒学应答比例较高。结论该研究建立的Tetra-primer ARMS PCR技术是一种操作简单、成本低廉、快速有效的基因多态性检测方法,对于丙型肝炎患者IL-28Brs12979860基因多态性基因分型具有很好的临床应用价值。