A Study of the Impact of the Dissemination of Ancient Chinese Primer Classic in the United States

Carrying the roots of Chinese civilization to its origins,the Chinese Primer classic has received considerable attention in the international community’s cultural diffusion.The spread of the Chinese Primer class studies in the United States,aided by missionaries such as E.C.Bridgeman,Samuel Wells Williams,and William Alexander Parsons Martin,sinologists such as Witter Bynner and David Hinton,has been characterized by unidirectionality,permeability,and pluralism.

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。

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.

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.

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.

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.

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.

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 。

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.

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.

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.

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.

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.

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.

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。

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.

Effects of DNA extraction and universal primers on 16S rRNA gene-based DGGE analysis of a bacterial community from fish farming water

Among many reports investigating microbial diversity from environmental samples with denaturing gradient gel electrophoresis (DGGE), limited attention has been given to the effects of universal primers and DNA extraction on the outcome of DGGE analysis. In this study, these effects were tested with 16S rRNA gene-based DGGE on a bacterial community from farming water samples. The results indicate that the number of discernable bands in the DGGE fingerprint differed with the primer pairs used; the bands produced by 63f/518r, 341f/926r and 933f/1387r primer pairs were obviously fewer than those by 968f/1401r. Also, we found that each DNA extraction method resulted in different community profiles, reflected by the number and intensity of bands in the DGGE fingerprint. Furthermore, the main bands (theoretically representing dominant bacteria) differed with the extraction methods applied. It is therefore believed that the effects of universal primers and DNA extraction should be given more attention and carefully chosen before performing an investigation into a new environment with DGGE.

Development of Genomic Microsatellite Multiplex PCR Using Dye-Labeled Universal Primer and Its Validation in Pedigree Analysis of Pacific Oyster(Crassostrea gigas)

There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical labels. Genetic traceability technique depending on DNA-based tracking system can overcome this problem. Genealogy information is essential for genetic traceability, and microsatellite DNA marker is a good choice for pedigree analysis. As increasing genotyping throughput of microsatellites, microsatellite multiplex PCR has become a fast and cost-effective technique. As a commercially important cultured aquatic species, Pacific oyster Crassostrea gigas has the highest global production. The objective of this study was to develop microsatellite multiplex PCR panels with dye-labeled universal primer for pedigree analysis in C. gigas, and these multiplex PCRs were validated using 12 full-sib families with known pedigrees. Here we developed six informative multiplex PCRs using 18 genomic microsatellites in C. gigas. Each multiplex panel contained a single universal primer M13(-21) used as a tail on each locus-specific forward primer and a single universal primer M13(-21) labeled with fluorophores. The polymorphisms of the markers were moderate, with an average of 10.3 alleles per locus and average polymorphic information content of 0.740. The observed heterozygosity per locus ranged from 0.492 to 0.822. Cervus simulations revealed that the six panels would still be of great value when massive families were analysed. Pedigree analysis of real offspring demonstrated that 100% of the offspring were unambiguously allocated to their parents when two multiplex PCRs were used. The six sets of multiplex PCRs can be an important tool for tracing cultured individuals, population genetic analysis, and selective breeding program in C. gigas.

Development of Species-Specific PCR Primers and Sensitive Detection of the Tylenchulus semipenetrans in China

Tylenchulus semipenetrans is the most economically important and widespread nematode pest of citrus in China.rDNA-ITS of 14 populations of T.semipenetrans which were collected from different citrus groves or Chinese fir（Cunninghamia lanceolata） plantations in China were amplified and sequenced.The species-specific primers were designed for the first time to diagnosis T.semipenetrans based on the sequences of rDNA-ITS regions of geographic population above.The primers were sensitive to amplify the expected band size（297 bp） from DNA template of a single second-stage juvenile（J2） or different life stages of T.semipenetrans.No specific band was amplified from 15 non-target nematode species which were commonly found in citrus groves.Specificity and reliability of the primers were validated by further PCR amplification of 16 extra populations of T.semipenetrans collected from 4 provinces of China.The primers successfully detected a single J2 of T.semipenetrans within a whole nematode community comprising a large numbers of non-target nematode.The developed diagnostic technique can be used for accurate identification of T.semipenetrans and also as a decision tool for nematode management for citrus or Chinese fir in China.