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Sample records for nested primers specific

  1. Design and application of specific 16S rDNA-targeted primers for assessing endophytic diversity in Dendrobium officinale using nested PCR-DGGE.

    PubMed

    Yu, Jie; Zhou, Xiao-Feng; Yang, Sui-Juan; Liu, Wen-Hong; Hu, Xiu-Fang

    2013-11-01

    Novel specific 16S rDNA-targeted primers were successfully designed and applied to the characterization of endophytic diversity in Dendrobium officinale. Using the popular universal bacterial primers 27f/1492r, the fragments of chloroplast and mitochondrion 16S/18S rDNA were amplified from D. officinale. They shared high nucleotide identity with the chloroplast 16S rDNAs (99-100 %) and with the mitochondrion 18S rDNAs (93-100 %) from various plants, respectively, and both shared 73-86 % identities with the bacterial 16S rDNA sequences in GenBank. The current bacterial universal primers, including 27f/1492r, match well with the chloroplast and mitochondrion 16S/18S rDNAs, which accordingly renders these primers not useful for endophytic diversity analysis. Novel 16S rDNA-targeted primers fM1 (5'-CCGCGTGNRBGAHGAAGGYYYT-3') and rC5 (5'-TAATCCTGTTTGCTCC CCAC-3') were designed, which show good specificity compared to the 16S/18S rDNAs of D. officinale, and perfect universality within bacteria except for Cyanobacteria. The primers fM1/rC5, together with 515f-GC/rC5, which overlaps the whole V4 region of 16S rDNA, were subjected to nested polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) to analyze the diversity of endophytic bacteria in D. officinale from three different sources in China. The results showed diversities in roots and stems of the plants from all three locations. Altogether, 29 bands were identified as bacteria, with the dominant group being Proteobacteria and the dominant genus being Burkholderia, some of which commonly has the function of nitrogen fixation and thus may play potentially important roles in D. officinale. Therefore, the nested PCR-DGGE method based on the novel primers provides a good alternative for investigating the communities and roles of endophytes in D. officinale.

  2. URPD: a specific product primer design tool

    PubMed Central

    2012-01-01

    Background Polymerase chain reaction (PCR) plays an important role in molecular biology. Primer design fundamentally determines its results. Here, we present a currently available software that is not located in analyzing large sequence but used for a rather straight-forward way of visualizing the primer design process for infrequent users. Findings URPD (yoUR Primer Design), a web-based specific product primer design tool, combines the NCBI Reference Sequences (RefSeq), UCSC In-Silico PCR, memetic algorithm (MA) and genetic algorithm (GA) primer design methods to obtain specific primer sets. A friendly user interface is accomplished by built-in parameter settings. The incorporated smooth pipeline operations effectively guide both occasional and advanced users. URPD contains an automated process, which produces feasible primer pairs that satisfy the specific needs of the experimental design with practical PCR amplifications. Visual virtual gel electrophoresis and in silico PCR provide a simulated PCR environment. The comparison of Practical gel electrophoresis comparison to virtual gel electrophoresis facilitates and verifies the PCR experiment. Wet-laboratory validation proved that the system provides feasible primers. Conclusions URPD is a user-friendly tool that provides specific primer design results. The pipeline design path makes it easy to operate for beginners. URPD also provides a high throughput primer design function. Moreover, the advanced parameter settings assist sophisticated researchers in performing experiential PCR. Several novel functions, such as a nucleotide accession number template sequence input, local and global specificity estimation, primer pair redesign, user-interactive sequence scale selection, and virtual and practical PCR gel electrophoresis discrepancies have been developed and integrated into URPD. The URPD program is implemented in JAVA and freely available at http://bio.kuas.edu.tw/urpd/. PMID:22713312

  3. Homolog-specific PCR primer design for profiling splice variants

    PubMed Central

    Srivastava, Gyan Prakash; Hanumappa, Mamatha; Kushwaha, Garima; Nguyen, Henry T.; Xu, Dong

    2011-01-01

    To study functional diversity of proteins encoded from a single gene, it is important to distinguish the expression levels among the alternatively spliced variants. A variant-specific primer pair is required to amplify each alternatively spliced variant individually. For this purpose, we developed a new feature, homolog-specific primer design (HSPD), in our high-throughput primer and probe design software tool, PRIMEGENS-v2. The algorithm uses a de novo approach to design primers without any prior information of splice variants or close homologs for an input query sequence. It not only designs primer pairs but also finds potential isoforms and homologs of the input sequence. Efficiency of this algorithm was tested for several gene families in soybean. A total of 187 primer pairs were tested under five different abiotic stress conditions with three replications at three time points. Results indicate a high success rate of primer design. Some primer pairs designed were able to amplify all splice variants of a gene. Furthermore, by utilizing combinations within the same multiplex pool, we were able to uniquely amplify a specific variant or duplicate gene. Our method can also be used to design PCR primers to specifically amplify homologs in the same gene family. PRIMEGENS-v2 is available at: http://primegens.org. PMID:21415011

  4. Genus-Specific Primers for Study of Fusarium Communities in Field Samples.

    PubMed

    Karlsson, Ida; Edel-Hermann, Véronique; Gautheron, Nadine; Durling, Mikael Brandström; Kolseth, Anna-Karin; Steinberg, Christian; Persson, Paula; Friberg, Hanna

    2015-10-30

    Fusarium is a large and diverse genus of fungi of great agricultural and economic importance, containing many plant pathogens and mycotoxin producers. To date, high-throughput sequencing of Fusarium communities has been limited by the lack of genus-specific primers targeting regions with high discriminatory power at the species level. In the present study, we evaluated two Fusarium-specific primer pairs targeting translation elongation factor 1 (TEF1). We also present the new primer pair Fa+7/Ra+6. Mock Fusarium communities reflecting phylogenetic diversity were used to evaluate the accuracy of the primers in reflecting the relative abundance of the species. TEF1 amplicons were subjected to 454 high-throughput sequencing to characterize Fusarium communities. Field samples from soil and wheat kernels were included to test the method on more-complex material. For kernel samples, a single PCR was sufficient, while for soil samples, nested PCR was necessary. The newly developed primer pairs Fa+7/Ra+6 and Fa/Ra accurately reflected Fusarium species composition in mock DNA communities. In field samples, 47 Fusarium operational taxonomic units were identified, with the highest Fusarium diversity in soil. The Fusarium community in soil was dominated by members of the Fusarium incarnatum-Fusarium equiseti species complex, contradicting findings in previous studies. The method was successfully applied to analyze Fusarium communities in soil and plant material and can facilitate further studies of Fusarium ecology.

  5. Genus-Specific Primers for Study of Fusarium Communities in Field Samples

    PubMed Central

    Edel-Hermann, Véronique; Gautheron, Nadine; Durling, Mikael Brandström; Kolseth, Anna-Karin; Steinberg, Christian; Persson, Paula; Friberg, Hanna

    2015-01-01

    Fusarium is a large and diverse genus of fungi of great agricultural and economic importance, containing many plant pathogens and mycotoxin producers. To date, high-throughput sequencing of Fusarium communities has been limited by the lack of genus-specific primers targeting regions with high discriminatory power at the species level. In the present study, we evaluated two Fusarium-specific primer pairs targeting translation elongation factor 1 (TEF1). We also present the new primer pair Fa+7/Ra+6. Mock Fusarium communities reflecting phylogenetic diversity were used to evaluate the accuracy of the primers in reflecting the relative abundance of the species. TEF1 amplicons were subjected to 454 high-throughput sequencing to characterize Fusarium communities. Field samples from soil and wheat kernels were included to test the method on more-complex material. For kernel samples, a single PCR was sufficient, while for soil samples, nested PCR was necessary. The newly developed primer pairs Fa+7/Ra+6 and Fa/Ra accurately reflected Fusarium species composition in mock DNA communities. In field samples, 47 Fusarium operational taxonomic units were identified, with the highest Fusarium diversity in soil. The Fusarium community in soil was dominated by members of the Fusarium incarnatum-Fusarium equiseti species complex, contradicting findings in previous studies. The method was successfully applied to analyze Fusarium communities in soil and plant material and can facilitate further studies of Fusarium ecology. PMID:26519387

  6. Specific Polymerase Chain Reaction Primers for the Detection of Plasmodiophora brassicae in Soil and Water.

    PubMed

    Faggian, R; Bulman, S R; Lawrie, A C; Porter, I J

    1999-05-01

    ABSTRACT The development of specific oligonucleotide primers for Plasmodiophora brassicae has led to a nested polymerase chain reaction (PCR) detection method for P. brassicae in soil and water. Initially, the PCR was used to amplify a section of the rDNA repeat. The PCR products were sequenced and the data used to design primers that were directed at the ribosomal RNA genes and internal transcribed spacer regions. Specificity was tested against more than 40 common soil organisms, host plants, and spore suspension contaminants, as well as P. brassicae isolates from around Australia and the world. Sensitivity was determined to be 0.1 fentograms (fg; 10(-15) g) for pure template and as low as 1,000 spores per g of potting mix. In soil, P. brassicae was detected in all soils where the inoculum was sufficient to result in clubroot symptoms. Also outlined is a simple method of DNA extraction from soil. PMID:18944752

  7. Fusion primer and nested integrated PCR (FPNI-PCR): a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning

    PubMed Central

    2011-01-01

    Background The advent of genomics-based technologies has revolutionized many fields of biological enquiry. However, chromosome walking or flanking sequence cloning is still a necessary and important procedure to determining gene structure. Such methods are used to identify T-DNA insertion sites and so are especially relevant for organisms where large T-DNA insertion libraries have been created, such as rice and Arabidopsis. The currently available methods for flanking sequence cloning, including the popular TAIL-PCR technique, are relatively laborious and slow. Results Here, we report a simple and effective fusion primer and nested integrated PCR method (FPNI-PCR) for the identification and cloning of unknown genomic regions flanked known sequences. In brief, a set of universal primers was designed that consisted of various 15-16 base arbitrary degenerate oligonucleotides. These arbitrary degenerate primers were fused to the 3' end of an adaptor oligonucleotide which provided a known sequence without degenerate nucleotides, thereby forming the fusion primers (FPs). These fusion primers are employed in the first step of an integrated nested PCR strategy which defines the overall FPNI-PCR protocol. In order to demonstrate the efficacy of this novel strategy, we have successfully used it to isolate multiple genomic sequences namely, 21 orthologs of genes in various species of Rosaceace, 4 MYB genes of Rosa rugosa, 3 promoters of transcription factors of Petunia hybrida, and 4 flanking sequences of T-DNA insertion sites in transgenic tobacco lines and 6 specific genes from sequenced genome of rice and Arabidopsis. Conclusions The successful amplification of target products through FPNI-PCR verified that this novel strategy is an effective, low cost and simple procedure. Furthermore, FPNI-PCR represents a more sensitive, rapid and accurate technique than the established TAIL-PCR and hiTAIL-PCR procedures. PMID:22093809

  8. Sequence-specific DNA primer effects on telomerase polymerization activity.

    PubMed Central

    Lee, M S; Blackburn, E H

    1993-01-01

    The ribonucleoprotein enzyme telomerase synthesizes one strand of telomeric DNA by copying a template sequence within the RNA moiety of the enzyme. Kinetic studies of this polymerization reaction were used to analyze the mechanism and properties of the telomerase from Tetrahymena thermophila. This enzyme synthesizes TTGGGG repeats, the telomeric DNA sequence of this species, by elongating a DNA primer whose 3' end base pairs with the template-forming domain of the RNA. The enzyme was found to act nonprocessively with short (10- to 12-nucleotide) primers but to become processive as TTGGGG repeats were added. Variation of the 5' sequences of short primers with a common 3' end identified sequence-specific effects which are distinct from those involving base pairing of the 3' end of the primer with the RNA template and which can markedly induce enzyme activity by increasing the catalytic rate of the telomerase polymerization reaction. These results identify an additional mechanistic basis for telomere and DNA end recognition by telomerase in vivo. Images PMID:8413255

  9. A Primer for Analyzing Nested Data: Multilevel Modeling in SPSS Using an Example from a REL Study. REL 2015-046

    ERIC Educational Resources Information Center

    O'Dwyer, Laura M.; Parker, Caroline E.

    2014-01-01

    Analyzing data that possess some form of nesting is often challenging for applied researchers or district staff who are involved in or in charge of conducting data analyses. This report provides a description of the challenges for analyzing nested data and provides a primer of how multilevel regression modeling may be used to resolve these…

  10. GSP: A web-based platform for designing genome-specific primers in polyploids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The sequences among subgenomes in a polyploid species have high similarity. This makes difficult to design genome-specific primers for sequence analysis. We present a web-based platform named GSP for designing genome-specific primers to distinguish subgenome sequences in the polyploid genome backgr...

  11. Cross-kingdom amplification using Bacteria-specific primers: Complications for studies of coral microbial ecology

    USGS Publications Warehouse

    Galkiewicz, J.P.; Kellogg, C.A.

    2008-01-01

    PCR amplification of pure bacterial DNA is vital to the study of bacterial interactions with corals. Commonly used Bacteria-specific primers 8F and 27F paired with the universal primer 1492R amplify both eukaryotic and prokaryotic rRNA genes. An alternative primer set, 63F/1542R, is suggested to resolve this problem. Copyright ?? 2008, American Society for Microbiology. All Rights Reserved.

  12. MFEprimer-2.0: a fast thermodynamics-based program for checking PCR primer specificity.

    PubMed

    Qu, Wubin; Zhou, Yang; Zhang, Yanchun; Lu, Yiming; Wang, Xiaolei; Zhao, Dongsheng; Yang, Yi; Zhang, Chenggang

    2012-07-01

    Evaluating the specificity of polymerase chain reaction (PCR) primers is an essential step in PCR primer design. The MFEprimer-2.0 server allows users to check primer specificity against genomic DNA and messenger RNA/complementary DNA sequence databases quickly and easily. MFEprimer-2.0 uses a k-mer index algorithm to accelerate the search process for primer binding sites and uses thermodynamics to evaluate binding stability between each primer and its DNA template. Several important characteristics, such as the sequence, melting temperature and size of each amplicon, either specific or non-specific, are reported on the results page. Based on these characteristics and the user-friendly output, users can readily draw conclusions about the specificity of PCR primers. Analyses for degenerate primers and multiple PCR primers are also supported in MFEprimer-2.0. In addition, the databases supported by MFEprimer-2.0 are comprehensive, and custom databases can also be supported on request. The MFEprimer-2.0 server does not require a login and is freely available at http://biocompute.bmi.ac.cn/CZlab/MFEprimer-2.0. More over, the MFEprimer-2.0 command-line version and local server version are open source and can be downloaded at https://github.com/quwubin/MFEprimer/wiki/Manual/.

  13. A Molecular Approach to Nested RT-PCR Using a New Set of Primers for the Detection of the Human Immunodeficiency Virus Protease Gene

    PubMed Central

    Zarei, Mohammad; Ravanshad, Mehrdad; Bagban, Ashraf; Fallahi, Shahab

    2016-01-01

    Background The human immunodeficiency virus (HIV-1) is the etiologic agent of AIDS. The disease can be transmitted via blood in the window period prior to the development of antibodies to the disease. Thus, an appropriate method for the detection of HIV-1 during this window period is very important. Objectives This descriptive study proposes a sensitive, efficient, inexpensive, and easy method to detect HIV-1. Patients and Methods In this study 25 serum samples of patients under treatment and also 10 positive and 10 negative control samples were studied. Twenty-five blood samples were obtained from HIV-1-infected individuals who were receiving treatment at the acquired immune deficiency syndrome (AIDS) research center of Imam Khomeini hospital in Tehran. The identification of HIV-1-positive samples was done by using reverse transcription to produce copy deoxyribonucleic acid (cDNA) and then optimizing the nested polymerase chain reaction (PCR) method. Two pairs of primers were then designed specifically for the protease gene fragment of the nested real time-PCR (RT-PCR) samples. Electrophoresis was used to examine the PCR products. The results were analyzed using statistical tests, including Fisher’s exact test, and SPSS17 software. Results The 325 bp band of the protease gene was observed in all the positive control samples and in none of the negative control samples. The proposed method correctly identified HIV-1 in 23 of the 25 samples. Conclusions These results suggest that, in comparison with viral cultures, antibody detection by enzyme linked immunosorbent assay (ELISAs), and conventional PCR methods, the proposed method has high sensitivity and specificity for the detection of HIV-1. PMID:27679699

  14. A Molecular Approach to Nested RT-PCR Using a New Set of Primers for the Detection of the Human Immunodeficiency Virus Protease Gene

    PubMed Central

    Zarei, Mohammad; Ravanshad, Mehrdad; Bagban, Ashraf; Fallahi, Shahab

    2016-01-01

    Background The human immunodeficiency virus (HIV-1) is the etiologic agent of AIDS. The disease can be transmitted via blood in the window period prior to the development of antibodies to the disease. Thus, an appropriate method for the detection of HIV-1 during this window period is very important. Objectives This descriptive study proposes a sensitive, efficient, inexpensive, and easy method to detect HIV-1. Patients and Methods In this study 25 serum samples of patients under treatment and also 10 positive and 10 negative control samples were studied. Twenty-five blood samples were obtained from HIV-1-infected individuals who were receiving treatment at the acquired immune deficiency syndrome (AIDS) research center of Imam Khomeini hospital in Tehran. The identification of HIV-1-positive samples was done by using reverse transcription to produce copy deoxyribonucleic acid (cDNA) and then optimizing the nested polymerase chain reaction (PCR) method. Two pairs of primers were then designed specifically for the protease gene fragment of the nested real time-PCR (RT-PCR) samples. Electrophoresis was used to examine the PCR products. The results were analyzed using statistical tests, including Fisher’s exact test, and SPSS17 software. Results The 325 bp band of the protease gene was observed in all the positive control samples and in none of the negative control samples. The proposed method correctly identified HIV-1 in 23 of the 25 samples. Conclusions These results suggest that, in comparison with viral cultures, antibody detection by enzyme linked immunosorbent assay (ELISAs), and conventional PCR methods, the proposed method has high sensitivity and specificity for the detection of HIV-1.

  15. Microarrays for high-throughput genotyping of MICA alleles using allele-specific primer extension.

    PubMed

    Baek, I C; Jang, J-P; Choi, H-B; Choi, E-J; Ko, W-Y; Kim, T-G

    2013-10-01

    The role of major histocompatibility complex (MHC) class I chain-related gene A (MICA), a ligand of NKG2D, has been defined in human diseases by its allele associations with various autoimmune diseases, hematopoietic stem cell transplantation (HSCT) and cancer. This study describes a practical system to develop MICA genotyping by allele-specific primer extension (ASPE) on microarrays. From the results of 20 control primers, strict and reliable cut-off values of more than 30,000 mean fluorescence intensity (MFI) as positive and less than 3000 MFI as negative, were applied to select high-quality specific extension primers. Among 55 allele-specific primers, 44 primers could be initially selected as optimal primer. Through adjusting the length, six primers were improved. The other failed five primers were corrected by refractory modification. MICA genotypes by ASPE on microarrays showed the same results as those by nucleotide sequencing. On the basis of these results, ASPE on microarrays may provide high-throughput genotyping for MICA alleles for population studies, disease-gene associations and HSCT.

  16. Construction of Specific Primers for Rapid Detection of South African Exportable Vegetable Macergens.

    PubMed

    Aremu, Bukola Rhoda; Babalola, Olubukola Oluranti

    2015-10-01

    Macergens are bacteria causing great damages to the parenchymatous tissues of vegetable both on the field and in transit. To effectively and rapidly investigate the diversity and distribution of these macergens, four specific primers were designed by retrieving 16S rDNA sequences of pectolytic bacteria from GenBank through the National Center for Biotechnology Information (NCBI). These were aligned using ClusterW via BioEdit and primers were designed using Primer3Plus platform. The size and primer location of each species and PCR product size were accurately defined. For specificity enhancement, DNA template of known macergens (Pectobacterium chrysanthermi) and fresh healthy vegetable were used. These primers yielded expected size of approximately 1100 bp product only when tested with known macergens and no amplicon with fresh healthy vegetable was detected. Rapid detection of macergens in rotten vegetable samples was then carried out using these primers. Nucleotide sequences of macergens identified were deposited into the GenBank and were assigned accession numbers. Hence, with these specific primers, macergens can be identified with minimal quantities of the vegetable tissues using molecular techniques, for future use of the quarantine section of the Agricultural Department of the country for quick and rapid detection of macergens before exportation. PMID:26437427

  17. Construction of Specific Primers for Rapid Detection of South African Exportable Vegetable Macergens

    PubMed Central

    Aremu, Bukola Rhoda; Babalola, Olubukola Oluranti

    2015-01-01

    Macergens are bacteria causing great damages to the parenchymatous tissues of vegetable both on the field and in transit. To effectively and rapidly investigate the diversity and distribution of these macergens, four specific primers were designed by retrieving 16S rDNA sequences of pectolytic bacteria from GenBank through the National Center for Biotechnology Information (NCBI). These were aligned using ClusterW via BioEdit and primers were designed using Primer3Plus platform. The size and primer location of each species and PCR product size were accurately defined. For specificity enhancement, DNA template of known macergens (Pectobacterium chrysanthermi) and fresh healthy vegetable were used. These primers yielded expected size of approximately 1100 bp product only when tested with known macergens and no amplicon with fresh healthy vegetable was detected. Rapid detection of macergens in rotten vegetable samples was then carried out using these primers. Nucleotide sequences of macergens identified were deposited into the GenBank and were assigned accession numbers. Hence, with these specific primers, macergens can be identified with minimal quantities of the vegetable tissues using molecular techniques, for future use of the quarantine section of the Agricultural Department of the country for quick and rapid detection of macergens before exportation. PMID:26437427

  18. Construction of Specific Primers for Rapid Detection of South African Exportable Vegetable Macergens.

    PubMed

    Aremu, Bukola Rhoda; Babalola, Olubukola Oluranti

    2015-09-30

    Macergens are bacteria causing great damages to the parenchymatous tissues of vegetable both on the field and in transit. To effectively and rapidly investigate the diversity and distribution of these macergens, four specific primers were designed by retrieving 16S rDNA sequences of pectolytic bacteria from GenBank through the National Center for Biotechnology Information (NCBI). These were aligned using ClusterW via BioEdit and primers were designed using Primer3Plus platform. The size and primer location of each species and PCR product size were accurately defined. For specificity enhancement, DNA template of known macergens (Pectobacterium chrysanthermi) and fresh healthy vegetable were used. These primers yielded expected size of approximately 1100 bp product only when tested with known macergens and no amplicon with fresh healthy vegetable was detected. Rapid detection of macergens in rotten vegetable samples was then carried out using these primers. Nucleotide sequences of macergens identified were deposited into the GenBank and were assigned accession numbers. Hence, with these specific primers, macergens can be identified with minimal quantities of the vegetable tissues using molecular techniques, for future use of the quarantine section of the Agricultural Department of the country for quick and rapid detection of macergens before exportation.

  19. primers4clades: a web server that uses phylogenetic trees to design lineage-specific PCR primers for metagenomic and diversity studies

    PubMed Central

    Contreras-Moreira, Bruno; Sachman-Ruiz, Bernardo; Figueroa-Palacios, Iraís; Vinuesa, Pablo

    2009-01-01

    Primers4clades is an easy-to-use web server that implements a fully automatic PCR primer design pipeline for cross-species amplification of novel sequences from metagenomic DNA, or from uncharacterized organisms, belonging to user-specified phylogenetic clades or taxa. The server takes a set of non-aligned protein coding genes, with or without introns, aligns them and computes a neighbor-joining tree, which is displayed on screen for easy selection of species or sequence clusters to design lineage-specific PCR primers. Primers4clades implements an extended CODEHOP primer design strategy based on both DNA and protein multiple sequence alignments. It evaluates several thermodynamic properties of the oligonucleotide pairs, and computes the phylogenetic information content of the predicted amplicon sets from Shimodaira–Hasegawa-like branch support values of maximum likelihood phylogenies. A non-redundant set of primer formulations is returned, ranked according to their thermodynamic properties. An amplicon distribution map provides a convenient overview of the coverage of the target locus. Altogether these features greatly help the user in making an informed choice between alternative primer pair formulations. Primers4clades is available at two mirror sites: http://maya.ccg.unam.mx/primers4clades/and http://floresta.eead.csic.es/primers4clades/. Three demo data sets and a comprehensive documentation/tutorial page are provided for easy testing of the server's capabilities and interface. PMID:19465390

  20. primers4clades: a web server that uses phylogenetic trees to design lineage-specific PCR primers for metagenomic and diversity studies.

    PubMed

    Contreras-Moreira, Bruno; Sachman-Ruiz, Bernardo; Figueroa-Palacios, Iraís; Vinuesa, Pablo

    2009-07-01

    Primers4clades is an easy-to-use web server that implements a fully automatic PCR primer design pipeline for cross-species amplification of novel sequences from metagenomic DNA, or from uncharacterized organisms, belonging to user-specified phylogenetic clades or taxa. The server takes a set of non-aligned protein coding genes, with or without introns, aligns them and computes a neighbor-joining tree, which is displayed on screen for easy selection of species or sequence clusters to design lineage-specific PCR primers. Primers4clades implements an extended CODEHOP primer design strategy based on both DNA and protein multiple sequence alignments. It evaluates several thermodynamic properties of the oligonucleotide pairs, and computes the phylogenetic information content of the predicted amplicon sets from Shimodaira-Hasegawa-like branch support values of maximum likelihood phylogenies. A non-redundant set of primer formulations is returned, ranked according to their thermodynamic properties. An amplicon distribution map provides a convenient overview of the coverage of the target locus. Altogether these features greatly help the user in making an informed choice between alternative primer pair formulations. Primers4clades is available at two mirror sites: http://maya.ccg.unam.mx/primers4clades/and http://floresta.eead.csic.es/primers4clades/. Three demo data sets and a comprehensive documentation/tutorial page are provided for easy testing of the server's capabilities and interface.

  1. Soil clone library analyses to evaluate specificity and selectivity of PCR primers targeting fungal 18S rDNA for denaturing-gradient gel electrophoresis (DGGE).

    PubMed

    Takada Hoshino, Yuko; Morimoto, Sho

    2010-01-01

    We evaluated the fungal specificity and detection bias of four fungal 18S rRNA gene (18S rDNA) primer sets for denaturing-gradient gel electrophoresis (DGGE). We constructed and compared clone libraries amplified from upland and paddy field soils with each primer set (1, NS1/GCFung; 2, FF390/FR1-GC; 3, NS1/FR1-GC; and 4, NS1/EF3 for the first PCR and NS1/FR1-GC for the second PCR). Primer set 4 (for nested PCR) showed the highest specificity for fungi but biased specific sequences. Sets 1, 2, and 3 (for single PCR) amplified non-fungal eukaryotic sequences (from 7 to 16% for upland soil and from 20 to 31% for paddy field soil) and produced libraries with similar distributions of fungal 18S rDNA sequences at both the phylum and the class level. Set 2 tended to amplify more diverse fungal sequences, maintaining higher specificity for fungi. In addition, clone analyses revealed differences among primer sets in the frequency of chimeras. In upland field soil, the libraries amplified with primer sets 3 and 4, which targeted long fragments, contained many chimeric 18S rDNA sequences (18% and 48%, respectively), while the libraries obtained with sets 1 and 2, which targeted short fragments, contained fewer chimeras (5% and 10%, respectively).

  2. Indexed PCR Primers Induce Template-Specific Bias in Large-Scale DNA Sequencing Studies

    PubMed Central

    O’Donnell, James L.; Kelly, Ryan P.; Lowell, Natalie C.; Port, Jesse A.

    2016-01-01

    Massively parallel sequencing is rapidly emerging as an efficient way to quantify biodiversity at all levels, from genetic variation and expression to ecological community assemblage. However, the number of reads produced per sequencing run far exceeds the number required per sample for many applications, compelling researchers to sequence multiple samples per run in order to maximize efficiency. For studies that include a PCR step, this can be accomplished using primers that include an index sequence allowing sample origin to be determined after sequencing. The use of indexed primers assumes they behave no differently than standard primers; however, we found that indexed primers cause substantial template sequence-specific bias, resulting in radically different profiles of the same environmental sample. Likely the outcome of differential amplification efficiency due to primer-template mismatch, two indexed primer sets spuriously change the inferred sequence abundance from the same DNA extraction by up to 77.1%. We demonstrate that a double PCR approach alleviates these effects in applications where indexed primers are necessary. PMID:26950069

  3. Time-specific variation in passerine nest survival: new insights for old questions

    USGS Publications Warehouse

    Grant, T.A.; Shaffer, T.L.; Madden, E.M.; Pietz, P.J.

    2005-01-01

    Nest survival likely varies with nest age and date, but until recently researchers had only limited tools to efficiently address those sources of variability. Beginning with Mayfield (1961), many researchers have averaged survival rates within time-specific categories (e.g. egg and nestling stages; early and late nesting dates). However, Mayfield's estimator assumes constant survival within categories, and violations of that assumption can lead to biased estimates. We used the logistic-exposure method to examine nest survival as a function of nest age and date in Clay-colored Sparrows (Spizella pallida) and Vesper Sparrows (Pooecetes gramineus) breeding in north-central North Dakota. Daily survival rates increased during egg laying, decreased during incubation to a low shortly after hatch, and then increased during brood rearing in both species. Variation in survival with nest age suggests that traditional categorical averaging using Mayfield's or similar methods would have been inappropriate for this study; similar variation may bias results of other studies. Nest survival also varied with date. For both species, survival was high during the peak of nest initiations in late May and early June and declined throughout the remainder of the nesting season. Models of nest survival that incorporate time-specific information may provide insights that are unavailable from averaged data.

  4. Nested methylation-specific polymerase chain reaction cancer detection method

    DOEpatents

    Belinsky, Steven A.; Palmisano, William A.

    2007-05-08

    A molecular marker-based method for monitoring and detecting cancer in humans. Aberrant methylation of gene promoters is a marker for cancer risk in humans. A two-stage, or "nested" polymerase chain reaction method is disclosed for detecting methylated DNA sequences at sufficiently high levels of sensitivity to permit cancer screening in biological fluid samples, such as sputum, obtained non-invasively. The method is for detecting the aberrant methylation of the p16 gene, O 6-methylguanine-DNA methyltransferase gene, Death-associated protein kinase gene, RAS-associated family 1 gene, or other gene promoters. The method offers a potentially powerful approach to population-based screening for the detection of lung and other cancers.

  5. Nesting behaviour influences species-specific gas exchange across avian eggshells

    PubMed Central

    Portugal, Steven J.; Maurer, Golo; Thomas, Gavin H.; Hauber, Mark E.; Grim, Tomáš; Cassey, Phillip

    2014-01-01

    Carefully controlled gas exchange across the eggshell is essential for the development of the avian embryo. Water vapour conductance (GH2O) across the shell, typically measured as mass loss during incubation, has been demonstrated to optimally ensure the healthy development of the embryo while avoiding desiccation. Accordingly, eggs exposed to sub-optimal gas exchange have reduced hatching success. We tested the association between eggshell GH2O and putative life-history correlates of adult birds, ecological nest parameters and physical characteristics of the egg itself to investigate how variation in GH2O has evolved to maintain optimal water loss across a diverse set of nest environments. We measured gas exchange through eggshell fragments in 151 British breeding bird species and fitted phylogenetically controlled, general linear models to test the relationship between GH2O and potential predictor parameters of each species. Of our 17 life-history traits, only two were retained in the final model: wet-incubating parent and nest type. Eggs of species where the parent habitually returned to the nest with wet plumage had significantly higher GH2O than those of parents that returned to the nest with dry plumage. Eggs of species nesting in ground burrows, cliffs and arboreal cups had significantly higher GH2O than those of species nesting on the ground in open nests or cups, in tree cavities and in shallow arboreal nests. Phylogenetic signal (measured as Pagel's λ) was intermediate in magnitude, suggesting that differences observed in the GH2O are dependent upon a combination of shared ancestry and species-specific life history and ecological traits. Although these data are correlational by nature, they are consistent with the hypothesis that parents constrained to return to the nest with wet plumage will increase the humidity of the nest environment, and the eggs of these species have evolved a higher GH2O to overcome this constraint and still achieve optimal water

  6. Novel primers and PCR protocols for the specific detection and quantification of Sphingobium suberifaciens in situ

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pathogen causing corky root on lettuce, Sphingobium suberifaciens, is recalcitrant to standard epidemiological methods. Primers were selected from 16S rDNA sequences useful for the specific detection and quantification of S. suberifaciens. Conventional (PCR) and quantitative (qPCR) PCR protocols...

  7. GSP: a web-based platform for designing genome-specific primers in polyploids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The primary goal of this research was to develop a web-based platform named GSP for designing genome-specific primers to distinguish subgenome sequences in the polyploid genome background. GSP uses BLAST to extract homeologous sequences of the subgenomes in the existing databases, performed a multip...

  8. Development of Species-specific Primers for Rapid Detection of Phellinus linteus and P. baumii

    PubMed Central

    Kim, Mun-Ok; Kim, Gi-Young; Nam, Byung-Hyouk; Jin, Cheng-Yun; Lee, Ki-Won; Park, Jae-Min; Lee, Sang-Joon

    2005-01-01

    Genus Phellinus taxonomically belongs to Aphyllophorales and some species of this genus have been used as a medicinal ingredients and Indian folk medicines. Especially, P. linteus and morphological-related species are well-known medicinal fungi that have various biological activities such as humoral and cell-mediated, anti-mutagenic, and anti-cancer activities. However, little is known about the rapid detection for complex Phellinus species. Therefore, this study was carried out to develop specific primers for the rapid detection of P. linteus and other related species. Designing the species-specific primers was done based on internal transcribed spacer sequence data. Each primer set detected specifically P. linteus (PL2/PL5R) and P. baumii (PB1/PB4R). These primer sets could be useful for the rapid detection of specific-species among unidentified Phellinus species. Moreover, restriction fragment length polymorphism analysis of the ITS region with HaeIII was also useful for clarifying the relationship between each 5 Phellinus species. PMID:24049482

  9. Living archaeology: artefacts of specific nest site fidelity in wild chimpanzees.

    PubMed

    Stewart, F A; Piel, A K; McGrew, W C

    2011-10-01

    Savanna chimpanzees are known to re-use areas of the landscape for sleep, and patterns of chimpanzee sleeping site re-use are proposed as a referential model for early hominin archaeological site formation. We recorded the prevalence of deformed but healed branches and remnants of dead branches found around fresh nests at the savanna site of Issa in Ugalla, Tanzania. These old nest scars were found in 79% of 112 beds. We also randomly selected potential nesting locations for a subset of 32 beds within the same trees, and found nest scars in only 19% of these "control" locations. We then monitored 275 nests for up to 19 months for decay, regeneration of new branches, and re-use. Of these 275 nest locations, 24% were re-used within the first nine months of monitoring, and most re-use occurred when the nest had already decayed and was not easily visible from the ground. After 18 months, the proportion of specific nest positions re-used increased to 48%. This fidelity is likely a result of the creation of ideally-shaped support structures and supple new growth for mattress material with successive use of nest locations. We propose that specific nest site re-use may not be a direct product of environmental determination, but a result of "niche construction" through formation of good building sites within trees. Environmental modification through construction behaviour may have influenced both chimpanzee and early hominin ranging, and thus leaves behind recognisable patterns of artefact deposition across the landscape.

  10. Improved group-specific primers based on the full SILVA 16S rRNA gene reference database.

    PubMed

    Pfeiffer, Stefan; Pastar, Milica; Mitter, Birgit; Lippert, Kathrin; Hackl, Evelyn; Lojan, Paul; Oswald, Andreas; Sessitsch, Angela

    2014-08-01

    Quantitative PCR (qPCR) and community fingerprinting methods, such as the Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis,are well-suited techniques for the examination of microbial community structures. The use of phylum and class-specific primers can provide enhanced sensitivity and phylogenetic resolution as compared with domain-specific primers. To date, several phylum- and class-specific primers targeting the 16S ribosomal RNA gene have been published. However, many of these primers exhibit low discriminatory power against non-target bacteria in PCR. In this study, we evaluated the precision of certain published primers in silico and via specific PCR. We designed new qPCR and T-RFLP primer pairs (for the classes Alphaproteobacteria and Betaproteobacteria, and the phyla Bacteroidetes, Firmicutes and Actinobacteria) by combining the sequence information from a public dataset (SILVA SSU Ref 102 NR) with manual primer design. We evaluated the primer pairs via PCR using isolates of the above-mentioned groups and via screening of clone libraries from environmental soil samples and human faecal samples. As observed through theoretical and practical evaluation, the primers developed in this study showed a higher level of precision than previously published primers, thus allowing a deeper insight into microbial community dynamics. PMID:25229098

  11. Specificity of Methylation Assays in Cancer Research: A Guideline for Designing Primers and Probes

    PubMed Central

    Barekati, Zeinab; Radpour, Ramin; Kohler, Corina; Zhong, Xiao Yan

    2010-01-01

    DNA methylation is an epigenetic regulation mechanism of genomic function, and aberrant methylation pattern has been found to be a common event in many diseases and human cancers. A large number of cancer studies have been focused on identification of methylation changes as biomarkers (i.e., breast cancer). However, still clinical use of them is very limited because of lack of specificity and sensitivity for diagnostic test. This highlights the critical need for specific primer and probe design to avoid false-positive detection of methylation profiling. The guideline and online web tools that are introduced in this paper might help to perform a successful experiment and to develop specific diagnosis biomarkers by designing right primer pair and probe prior to experimental step. PMID:20798774

  12. Bee species-specific nesting material attracts a generalist parasitoid: implications for co-occurring bees in nest box enhancements.

    PubMed

    Macivor, J Scott; Salehi, Baharak

    2014-08-01

    Artificial nests (e.g., nest boxes) for bees are increasingly being used to contribute to nesting habitat enhancement for bees that use preexisting cavities to provision brood. They usually incorporate additional nesting materials that vary by species. Cavity-nesting bees are susceptible to brood parasitoids that recognize their host(s) using visual and chemical cues. Understanding the range of cues that attract parasitoids to bee nests, including human-made analogues, is important if we wish to control parasitism and increase the potential value of artificial nests as habitat-enhancement strategies. In this study, we investigated the cues associated with the orientation of the generalist brood parasitoid Monodontomerus obscurus Westwood (Hymenoptera: Torymidae) to the nests of a common cavity-nesting resin bee Megachile campanulae (Robertson) (Megachilidae). The parasitoids were reared from previously infested M. campanulae brood cells and placed into choice trials where they were presented with pairs of different nest material cues. Among different materials tested, we found that Mo. obscurus was most attracted to fresh resin collected directly from Pinus strobus trees followed by previously used resin collected from the bee nest. The parasitoid also attacked other bee species in the same nest boxes, including those that do not use resin for nesting. Our findings suggest that M. campanulae could act as a magnet, drawing parasites away from other bee hosts co-occurring in nest boxes, or, as an attractant of Mo. obscurus to nest boxes, increasing attacks on co-occurring host bee species, potentially undermining bee diversity enhancement initiatives. PMID:24959997

  13. SP-Designer: a user-friendly program for designing species-specific primer pairs from DNA sequence alignments.

    PubMed

    Villard, Pierre; Malausa, Thibaut

    2013-07-01

    SP-Designer is an open-source program providing a user-friendly tool for the design of specific PCR primer pairs from a DNA sequence alignment containing sequences from various taxa. SP-Designer selects PCR primer pairs for the amplification of DNA from a target species on the basis of several criteria: (i) primer specificity, as assessed by interspecific sequence polymorphism in the annealing regions, (ii) the biochemical characteristics of the primers and (iii) the intended PCR conditions. SP-Designer generates tables, detailing the primer pair and PCR characteristics, and a FASTA file locating the primer sequences in the original sequence alignment. SP-Designer is Windows-compatible and freely available from http://www2.sophia.inra.fr/urih/sophia_mart/sp_designer/info_sp_designer.php.

  14. Development of species-specific primers for rapid identification of Debaryomyces hansenii.

    PubMed

    Wrent, Petra; Rivas, Eva-María; Gil de Prado, Elena; Peinado, José M; de Silóniz, María-Isabel

    2015-01-16

    In this work, we developed a specific PCR assay for Debaryomyces hansenii strains that uses a putative homologous PAD1 region (729 bp) present in this yeast species as a target. The amplification of this sequence with the D. hansenii specific primer pair (DhPADF/DhPADR) was found to be a rapid, specific and an affordable method enabling identification of D. hansenii from other yeast strains. Primers were tested in almost 100 strains, 49 strains from Type Culture Collection belonging to the genus Debaryomyces and to other yeast species commonly found in foods or related genera. These primers were able to discriminate between closely related species of Debaryomyces, such as Debaryomyces fabryi and Debaryomyces subglobosus, with a 100% detection rate for D. hansenii. Also, the method was tested in 45 strains from different foods. Results confirmed the specificity of the PCR method and detected two earlier misidentifications of D. hansenii strains obtained by RFLP analysis of the 5.8S ITS rDNA region. Subsequently we confirmed by sequencing the D1/D2 domain of 26S rDNA that these strains belonged to D. fabryi. We call attention in this work to the fact that the RFLPs of the 5.8S ITS rDNA profiles of D. hansenii, D. fabryi and D. subglobosus are the same and this technique will thus lead to incorrect identifications. PMID:25462930

  15. Specific oligonucleotide primers for detection of lecithinase-positive Bacillus spp. by PCR.

    PubMed Central

    Schraft, H; Griffiths, M W

    1995-01-01

    An assay based on the PCR has been developed to facilitate detection and identification of Bacillus cereus in foods. Three primers for the PCR have been designed within the sequence for cereolysin AB, a cytolytic determinant that encodes lecithin-hydrolyzing and hemolytic activities of B. cereus. With the PCR and hybridization, the specificity of the primers was tested with 39 isolates of the B. cereus group, with 17 other Bacillus spp., and with 21 non-Bacillus strains. Results demonstrate a high specificity of the three oligonucleotides for isolates of the B. cereus group. With a combined PCR-hybridization assay, the detection limit for B. cereus in artificially contaminated milk was 1 CFU/ml of milk. PMID:7887632

  16. Modeling and estimation of stage-specific daily survival probabilities of nests

    USGS Publications Warehouse

    Stanley, T.R.

    2000-01-01

    In studies of avian nesting success, it is often of interest to estimate stage-specific daily survival probabilities of nests. When data can be partitioned by nesting stage (e.g., incubation stage, nestling stage), piecewise application of the Mayfield method or Johnsona??s method is appropriate. However, when the data contain nests where the transition from one stage to the next occurred during the interval between visits, piecewise approaches are inappropriate. In this paper, I present a model that allows joint estimation of stage-specific daily survival probabilities even when the time of transition between stages is unknown. The model allows interval lengths between visits to nests to vary, and the exact time of failure of nests does not need to be known. The performance of the model at various sample sizes and interval lengths between visits was investigated using Monte Carlo simulations, and it was found that the model performed quite well: bias was small and confidence-interval coverage was at the nominal 95% rate. A SAS program for obtaining maximum likelihood estimates of parameters, and their standard errors, is provided in the Appendix.

  17. In silico vs in vitro analysis of primer specificity for the detection of Gardnerella vaginalis, Atopobium vaginae and Lactobacillus spp.

    PubMed Central

    2012-01-01

    Background Bacterial vaginosis (BV) is a common pathology of women in reproductive age that can lead to serious health complications, and is associated with shifts in the normal microflora from predominance of Lactobacillus spp. to a proliferation of other anaerobes such as G. vaginalis and A vaginae, which can be detected by PCR. The optimal PCR pathogen detection assay relies mainly on the specificity and sensitivity of the primers used. Findings Here we demonstrate that in silico analytical testing of primer specificity is not a synonym to in vitro analytical specificity by testing a range of published and newly designed primers with both techniques for the detection of BV-associated microorganisms. Conclusions By testing primer in vitro specificity with a sufficient range of bacterial strains, we were able to design primers with higher specificity and sensitivity. Also by comparing the results obtained for the newly designed primers with other previously published primers, we confirmed that in silico analysis is not sufficient to predict in vitro specificity. As such care must be taken when choosing the primers for a detection assay. PMID:23153093

  18. Isolation of Fungal Pathogens to an Edible Mushroom, Pleurotus eryngii, and Development of Specific ITS Primers.

    PubMed

    Kim, Sang-Woo; Kim, Sinil; Lee, Hyun-Jun; Park, Ju-Wan; Ro, Hyeon-Su

    2013-12-01

    Fungal pathogens have caused severe damage to the commercial production of Pleurotus eryngii, the king oyster mushroom, by reducing production yield, causing deterioration of commercial value, and shortening shelf-life. Four strains of pathogenic fungi, including Trichoderma koningiopsis DC3, Phomopsis sp. MP4, Mucor circinelloides MP5, and Cladosporium bruhnei MP6, were isolated from the bottle culture of diseased P. eryngii. A species-specific primer set was designed for each fungus from the ITS1-5.8S rDNA-ITS2 sequences. PCR using the ITS primer set yielded a unique DNA band for each fungus without any cross-reaction, proving the validity of our method in detection of mushroom fungal pathogens. PMID:24493949

  19. Nest Success and Cause-Specific Nest Failure of Grassland Passerines Breeding in Prairie Grazed by Livestock

    EPA Science Inventory

    This manuscript describes two years of field research on ground-nesting songbird species at Zumwalt Prairie Reserve, northeastern Oregon, USA. Cattle-grazing has long been suspected in declines of ground-nesting songbirds in grazed grassland, primarily due to increased trampling...

  20. PrimerMapper: high throughput primer design and graphical assembly for PCR and SNP detection

    PubMed Central

    O’Halloran, Damien M.

    2016-01-01

    Primer design represents a widely employed gambit in diverse molecular applications including PCR, sequencing, and probe hybridization. Variations of PCR, including primer walking, allele-specific PCR, and nested PCR provide specialized validation and detection protocols for molecular analyses that often require screening large numbers of DNA fragments. In these cases, automated sequence retrieval and processing become important features, and furthermore, a graphic that provides the user with a visual guide to the distribution of designed primers across targets is most helpful in quickly ascertaining primer coverage. To this end, I describe here, PrimerMapper, which provides a comprehensive graphical user interface that designs robust primers from any number of inputted sequences while providing the user with both, graphical maps of primer distribution for each inputted sequence, and also a global assembled map of all inputted sequences with designed primers. PrimerMapper also enables the visualization of graphical maps within a browser and allows the user to draw new primers directly onto the webpage. Other features of PrimerMapper include allele-specific design features for SNP genotyping, a remote BLAST window to NCBI databases, and remote sequence retrieval from GenBank and dbSNP. PrimerMapper is hosted at GitHub and freely available without restriction. PMID:26853558

  1. PrimerMapper: high throughput primer design and graphical assembly for PCR and SNP detection.

    PubMed

    O'Halloran, Damien M

    2016-01-01

    Primer design represents a widely employed gambit in diverse molecular applications including PCR, sequencing, and probe hybridization. Variations of PCR, including primer walking, allele-specific PCR, and nested PCR provide specialized validation and detection protocols for molecular analyses that often require screening large numbers of DNA fragments. In these cases, automated sequence retrieval and processing become important features, and furthermore, a graphic that provides the user with a visual guide to the distribution of designed primers across targets is most helpful in quickly ascertaining primer coverage. To this end, I describe here, PrimerMapper, which provides a comprehensive graphical user interface that designs robust primers from any number of inputted sequences while providing the user with both, graphical maps of primer distribution for each inputted sequence, and also a global assembled map of all inputted sequences with designed primers. PrimerMapper also enables the visualization of graphical maps within a browser and allows the user to draw new primers directly onto the webpage. Other features of PrimerMapper include allele-specific design features for SNP genotyping, a remote BLAST window to NCBI databases, and remote sequence retrieval from GenBank and dbSNP. PrimerMapper is hosted at GitHub and freely available without restriction. PMID:26853558

  2. PrimerMapper: high throughput primer design and graphical assembly for PCR and SNP detection.

    PubMed

    O'Halloran, Damien M

    2016-01-01

    Primer design represents a widely employed gambit in diverse molecular applications including PCR, sequencing, and probe hybridization. Variations of PCR, including primer walking, allele-specific PCR, and nested PCR provide specialized validation and detection protocols for molecular analyses that often require screening large numbers of DNA fragments. In these cases, automated sequence retrieval and processing become important features, and furthermore, a graphic that provides the user with a visual guide to the distribution of designed primers across targets is most helpful in quickly ascertaining primer coverage. To this end, I describe here, PrimerMapper, which provides a comprehensive graphical user interface that designs robust primers from any number of inputted sequences while providing the user with both, graphical maps of primer distribution for each inputted sequence, and also a global assembled map of all inputted sequences with designed primers. PrimerMapper also enables the visualization of graphical maps within a browser and allows the user to draw new primers directly onto the webpage. Other features of PrimerMapper include allele-specific design features for SNP genotyping, a remote BLAST window to NCBI databases, and remote sequence retrieval from GenBank and dbSNP. PrimerMapper is hosted at GitHub and freely available without restriction.

  3. Specific Detection of Xanthomonas axonopodis pv. dieffenbachiae in Anthurium (Anthurium andreanum) Tissues by Nested PCR†

    PubMed Central

    Robène-Soustrade, Isabelle; Laurent, Philippe; Gagnevin, Lionel; Jouen, Emmanuel; Pruvost, Olivier

    2006-01-01

    Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires a sensitive and reliable diagnostic tool. A nested PCR test was developed from a sequence-characterized amplified region marker identified by randomly amplified polymorphic DNA PCR for the detection of X. axonopodis pv. dieffenbachiae. Serological and pathogenicity tests were performed concurrently with the nested PCR test with a large collection of X. axonopodis pv. dieffenbachiae strains that were isolated worldwide and are pathogenic to anthurium and/or other aroids. The internal primer pair directed amplification of the expected product (785 bp) for all 70 X. axonopodis pv. dieffenbachiae strains pathogenic to anthurium tested and for isolates originating from syngonium and not pathogenic to anthurium. This finding is consistent with previous studies which indicated that there is a high level of relatedness between strains from anthurium and strains from syngonium. Strains originating from the two host genera can be distinguished by restriction analysis of the amplification product. No amplification product was obtained with 98 strains of unrelated phytopathogenic bacteria or saprophytic bacteria from the anthurium phyllosphere, except for a weak signal obtained for one X. axonopodis pv. allii strain. Nevertheless, restriction enzyme analysis permitted the two pathovars to be distinguished. The detection threshold obtained with pure cultures or plant extracts (103 CFU ml−1) allowed detection of the pathogen from symptomless contaminated plants. This test could be a useful diagnostic tool for screening propagation stock plant material and for monitoring international movement of X. axonopodis pv. dieffenbachiae. PMID:16461651

  4. Design of phylum-specific hybrid primers for DNA barcoding: addressing the need for efficient COI amplification in the Echinodermata.

    PubMed

    Hoareau, T B; Boissin, E

    2010-11-01

    Recent research has shown the usefulness of the Folmer region of the cytochrome oxidase I (COI) as a genetic barcode to assist in species delimitation of echinoderms. However, amplification of COI is often challenging in echinoderms (low success or pseudogenes). We present a method that allows the design of phylum-specific hybrid primers, and use this to develop COI primers for the Echinodermata. We aligned COI sequences from 310 echinoderm species and designed all possible primers along the consensus sequence with two methods (standard degenerate and hybrid). We found much lower degeneracy for hybrid primers (4-fold degeneracy) than for standard degenerate primers (≥48-fold degeneracy). We then designed the most conserved hybrid primers to amplify a >500-bp region within COI. These primers successfully amplified this gene region in all tested taxa (123 species across all echinoderm classes). Sequencing of 30 species among these confirmed both the quality of the sequences (>500 bp, no pseudogenes) and their utility as a DNA barcode. This method should be useful for developing primers for other mitochondrial genes and other phyla. The method will also be of interest for the development of future projects involving both community-based genetic assessments on macroorganisms and biodiversity assessment of environmental samples using high-throughput sequencing.

  5. Approach to analyze the diversity of myxobacteria in soil by semi-nested PCR-denaturing gradient gel electrophoresis (DGGE) based on taxon-specific gene.

    PubMed

    Li, Baiyuan; Yao, Qing; Zhu, Honghui

    2014-01-01

    The genotypic diversity of insoluble macromolecules degraded myxobacteria, provided an opportunity to discover new bacterial resources and find new ecological functions. In this study, we developed a semi-nested-PCR-denaturing gradient gel electrophoresis (DGGE) strategy to determine the presence and genotypic diversity of myxobacteria in soil. After two rounds of PCR with myxobacteria-specific primers, an 194 bp fragment of mglA, a key gene involved in gliding motility, suitable for DGGE was obtained. A large number of bands were observed in DGGE patterns, indicating diverse myxobacteria inhabiting in soils. Furthermore, sequencing and BLAST revealed that most of the bands belonged to the myxobacteria-group, and only three of the twenty-eight bands belonged to other group, i.e., Deinococcus maricopensis. The results verified that myxobacterial strains with discrepant sequence compositions of gene mglA could be discriminated by DGGE with myxobacteria-specific primers. Collectively, the developed semi-nested-PCR-DGGE strategy is a useful tool for studying the diversity of myxobacteria.

  6. PCR amplification of the hrcV gene through specific primers for detecting Pseudomonas syringae pathovars.

    PubMed

    Vaseghi, Akbar; Bakhshinejad, Babak; Safaie, Naser; Parchin, Reza Ashrafi; Sadeghizadeh, Majid

    2014-02-01

    Pseudomonas syringae pathovars are important pathogens among phytopathogenic bacteria causing a variety of diseases in plants. These pathogens can rapidly disseminate in a large area leading to infection and destruction of plants. To prevent the incidence of the bacteria, appropriate detection methods should be employed. Routinely serological tests, being time-consuming and costly, are exploited to detect these pathogens in plants, soil, water and other resources. Over the recent years, DNA-based detection approaches which are stable, rapid, specific and reliable have been developed and sequence analysis of various genes are widely utilized to identify different strains of P. syringe. However, the greatest limitation of these genes is inability to detect numerous pathovars of P. syringae. Herein, by using bioinformatic analysis, we found the hrcV gene located at pathogenicity islands of bacterial genome with the potential of being used as a new marker for phylogenetic detection of numerous pathovars of P. syringae. Following design of specific primers to hrcV, we amplified a 440 bp fragment. Of 13 assayed pathovars, 11 were detected. Also, through experimental procedures and bioinformatic analysis it was revealed that the designed primers have the capacity to detect 19 pathovars. Our findings suggest that hrcV could be used as a gene with the merit of detecting more pathovars of P. syringae in comparison with other genes used frequently for detection purposes.

  7. Exploration of Deinococcus-Thermus molecular diversity by novel group-specific PCR primers.

    PubMed

    Theodorakopoulos, Nicolas; Bachar, Dipankar; Christen, Richard; Alain, Karine; Chapon, Virginie

    2013-10-01

    The deeply branching Deinococcus-Thermus lineage is recognized as one of the most extremophilic phylum of bacteria. In previous studies, the presence of Deinococcus-related bacteria in the hot arid Tunisian desert of Tataouine was demonstrated through combined molecular and culture-based approaches. Similarly, Thermus-related bacteria have been detected in Tunisian geothermal springs. The present work was conducted to explore the molecular diversity within the Deinococcus-Thermus phylum in these extreme environments. A set of specific primers was designed in silico on the basis of 16S rRNA gene sequences, validated for the specific detection of reference strains, and used for the polymerase chain reaction (PCR) amplification of metagenomic DNA retrieved from the Tataouine desert sand and Tunisian hot spring water samples. These analyses have revealed the presence of previously undescribed Deinococcus-Thermus bacterial sequences within these extreme environments. The primers designed in this study thus represent a powerful tool for the rapid detection of Deinococcus-Thermus in environmental samples and could also be applicable to clarify the biogeography of the Deinococcus-Thermus phylum.

  8. Exploration of Deinococcus-Thermus molecular diversity by novel group-specific PCR primers

    PubMed Central

    Theodorakopoulos, Nicolas; Bachar, Dipankar; Christen, Richard; Alain, Karine; Chapon, Virginie

    2013-01-01

    The deeply branching Deinococcus-Thermus lineage is recognized as one of the most extremophilic phylum of bacteria. In previous studies, the presence of Deinococcus-related bacteria in the hot arid Tunisian desert of Tataouine was demonstrated through combined molecular and culture-based approaches. Similarly, Thermus-related bacteria have been detected in Tunisian geothermal springs. The present work was conducted to explore the molecular diversity within the Deinococcus-Thermus phylum in these extreme environments. A set of specific primers was designed in silico on the basis of 16S rRNA gene sequences, validated for the specific detection of reference strains, and used for the polymerase chain reaction (PCR) amplification of metagenomic DNA retrieved from the Tataouine desert sand and Tunisian hot spring water samples. These analyses have revealed the presence of previously undescribed Deinococcus-Thermus bacterial sequences within these extreme environments. The primers designed in this study thus represent a powerful tool for the rapid detection of Deinococcus-Thermus in environmental samples and could also be applicable to clarify the biogeography of the Deinococcus-Thermus phylum. PMID:23996915

  9. Mining of novel species-specific primers for PCR detection of Listeria monocytogenes based on genomic approach.

    PubMed

    Tao, Tingting; Chen, Qiming; Bie, Xiaomei; Lu, Fengxia; Lu, Zhaoxin

    2015-12-01

    Listeria monocytogenes in contaminated food is considered as a serious health threat for consumers due to its high mortality rate. The objective of this study was to obtain novel species-specific target-genes and primers for the molecular detection of L. monocytogenes using a comparative genomic approach. By comparative analysis of L. monocytogenes and non-L. monocytogenes genome sequences in the GenBank database with BLAST program, 26 specific target sequences were used as candidates and the primers were designed for L. monocytogenes species-specificity verification by using PCR assay. Finally, the three genes LMOf2365_0970, LMOf2365_2721 and mpl were identified to have L. monocytogenes species-specificity and be unique as detection targets for diagnostic application. The species-specific primer Lm8 of gene LMOf2365_0970, Lm13 of gene LMOf2365_2721 and Lm20 of gene mpl showed better specificity and sensitivity than the primers described previously. The PCR detection limits of the three specific primer sets were 430, 43, 4.3 fg/μL for genomic DNA, and 5 × 10(3), 50, 5 cfu/mL for pure culture of L. monocytogenes. There was no interference in specificity of detecting L. monocytogenes by co-culture with other foodborne pathogens in high concentration. Moreover, after 6-8 h of enrichment, L. monocytogenes in the artificially contaminated milk samples at an inoculum dose of 38 cfu/10 mL milk could be detected successfully with the studied three primers. Therefore, the three specific genes and primers can be applied to establish a novel rapid and accurate method for detecting L. monocytogenes in food materials. PMID:26354019

  10. A novel one cycle allele specific primer extension--molecular beacon displacement method for DNA point mutation detection with improved specificity.

    PubMed

    Li, Xiaomin; Huang, Yong; Guan, Yuan; Zhao, Meiping; Li, Yuanzong

    2007-02-12

    We report here a new method for the real-time detection of DNA point mutations with molecular beacon as the fluorescence tracer and 3' (exo-) Bst DNA polymerase large fragment as the polymerase. The method is based on the mechanism of allele specific primer extension-strand displacement (ASPE-SD). To improve the specificity of the method only one cycle of the allele specific polymerase chain reaction (PCR) was used that could largely eliminate the non-specific reactions between the primers and template of the "wrong" genotype. At first, the primer and molecular beacon both hybridize to the DNA template, and the molecular beacon emits intensive fluorescence. The role of 3' exonuclease excision of Bst DNA polymerase large fragment is utilized for primer extension. When 3'-termini matches its corresponding template, the primer would efficiently extend and replace the molecular beacon that would simultaneously return to its closed form leading to the quenching of the fluorescence. However, when 3'-termini of the primer mismatches its corresponding template primer extension and molecular beacon displacement would not happen and fluorescence of the hybridized molecular beacon holds the line without fluorescence quenching. This approach was fully demonstrated in synthetic template systems and applied to detect point mutation at codon 259, a possible point mutation site in exon 7 of p53 gene, obtained from human genomic DNA samples with unambiguous differentiation power.

  11. Identification of Mycobacterium bovis in bovine clinical samples by PCR species-specific primers.

    PubMed Central

    Romero, R E; Garzón, D L; Mejía, G A; Monroy, W; Patarroyo, M E; Murillo, L A

    1999-01-01

    Tuberculosis, caused by Mycobacterium bovis is emerging as the most important disease affecting cattle. Furthermore, it results in a major public health problem when transmitted to humans. Due to its difficult and non-specific diagnosis, M. bovis has been declared to be one of the etiologic agents causing significant economic loss in the cattle industry. Our group evaluated a more rapid and specific method, based on a new polymerase chain reaction species-specific primers, which amplifies a 470-base pair fragment of the M. bovis genome. A total of 275 milk-producing cows were studied by intradermal tuberculin test (ITT) which gave 184 positive and 91 negative cases. From them, 50 animals were taken from a cattle ranch free of tuberculosis. Three different samples were collected from each animal (blood, nasal mucus, and milk). Positive results were obtained from 26 animals by PCR (11.4%), 1 by bacteriological culturing (0.4%) and 1 by bacilloscopy (0.4%). This finding suggests, as in previous reports, that ITT, normally used for bovine tuberculosis detection, has the inconvenience of having a broad range of specificity and sensitivity, and the PCR technique is a more specific and sensitive test to detect infection associated with M. bovis. Therefore, we propose this PCR assay as a useful tool in the epidemiological characterization of infected animals in areas considered to be at high risk of transmission. Images Figure 1. PMID:10369566

  12. Development of Species-Specific Primers for Agronomical Thrips and Multiplex Assay for Quarantine Identification of Western Flower Thrips.

    PubMed

    Yeh, W B; Tseng, M J; Chang, N T; Wu, S Y; Tsai, Y S

    2014-10-01

    While morphological identification of thrips species has been difficult because of their minute size and a lack of easily recognizable characteristics, molecular identification based on the development of specific molecular markers can be easily and reliably carried out. Among the known molecular markers, the nuclear internal transcribed spacer (ITS) exhibits distinguishable variations among thrips species. In this study, sequences of ITS2 region of 10 agriculturally important thrips were established to design species-specific primers for polymerase chain reaction (PCR). ITS2 sequence variations within these species were far less than those among species, indicating the suitability of this marker for species-specific primers design. These primers, though with one or two sporadically variable positions, showed a good efficacy within species. The specificity of these primers, examined on thrips species belonging to 15 genera, proved satisfactory. Furthermore, a multiplex PCR was used successfully for identifying Frankliniella occidentalis (Pergande), an insect pest monitored for quarantine purpose, and three additional thrips also commonly found in imported agricultural products and field samples, i.e., Thrips tabaci Lindeman, Thrips hawaiiensis (Morgan), and Frankliniella intonsa (Trybom). This study has demonstrated that specific primers and multiplex PCR based on ITS2 are reliable, convenient, and diagnostic tool to discriminate thrips species of quarantine and agricultural importance.

  13. Development of Prevotella nigrescens-specific PCR primers based on the nucleotide sequence of a Pn23 DNA probe.

    PubMed

    Kim, Min Jung; Lee, Young-Seok; Park, Jae-Yoon; Kook, Joong-Ki

    2011-02-01

    A previous study reported the cloning of a putative Prevotella nigrescens-specific DNA probe, Pn23, using random shotgun method. The present study evaluated the species-specificity of Pn23 for P. nigrescens using the clinical strains of Prevotella intermedia and P. nigrescens to develop P. nigrescens-specific polymerase chain reaction (PCR) primers. Southern blot analysis showed that the DNA probe, Pn23, detected only the genomic DNA of P. nigrescens strains. PCR showed that the two sets of PCR primers, Pn23-F1/Pn23-R1 and Pn23-F2/Pn23-R2, had species-specificity for P. nigrescens. Interestingly, the two sets of PCR primers, Pn23-F6/Pn23-R6 and Pn23-F7/Pn23-R7, had strain-specificity for P. nigrescens ATCC 33563. The detection limits of the four primer sets were 40 or 4 pg of the purified genomic DNA of P. nigrescens ATCC 33563. These results suggest that the DNA probe, Pn23, and the two sets of PCR primers, Pn23-F1/Pn23-R1 and Pn23-F2/Pn23-R2, can be useful for the detection of P. nigrescens in the molecular epidemiological studies of oral infectious diseases. PMID:21184839

  14. A species-specific primer pair for distinguishing between Paramisgurnus dabryanus and Misgurnus anguillicaudatus based on mitochondrial DNA polymorphisms.

    PubMed

    Liu, Yongfu; Hou, Jilun; Wang, Guixing; Zhang, Xiaoyan; Liu, Haijin

    2016-07-01

    Paramisgurnus dabryanus and Misgurnus anguillicaudatus (family Cobitidae) are loaches with high morphological similarity. In this study, we designed primers to distinguish between Paramisgurnus dabryanus and Misgurnus anguillicaudatus based on the length differences in the mitochondrial COXII to tRNA(Lys) gene region. Samples of P. dabryanus and M. anguillicaudatus from different geographical locations were collected and amplified to verify primer specificity. The results of electrophoresis revealed the successful amplification of all P. dabryanus and M. anguillicaudatus DNA samples, which had distinct, specific-specific sizes (214 bp for P. dabryanus and 285 bp for M. anguillicaudatus). In conclusion, the new primers provide fast, reliable, and accurate identification between P. dabryanus and M. anguillicaudatus.

  15. Rapid plant identification using species- and group-specific primers targeting chloroplast DNA.

    PubMed

    Wallinger, Corinna; Juen, Anita; Staudacher, Karin; Schallhart, Nikolaus; Mitterrutzner, Evi; Steiner, Eva-Maria; Thalinger, Bettina; Traugott, Michael

    2012-01-01

    Plant identification is challenging when no morphologically assignable parts are available. There is a lack of broadly applicable methods for identifying plants in this situation, for example when roots grow in mixture and for decayed or semi-digested plant material. These difficulties have also impeded the progress made in ecological disciplines such as soil- and trophic ecology. Here, a PCR-based approach is presented which allows identifying a variety of plant taxa commonly occurring in Central European agricultural land. Based on the trnT-F cpDNA region, PCR assays were developed to identify two plant families (Poaceae and Apiaceae), the genera Trifolium and Plantago, and nine plant species: Achillea millefolium, Fagopyrum esculentum, Lolium perenne, Lupinus angustifolius, Phaseolus coccineus, Sinapis alba, Taraxacum officinale, Triticum aestivum, and Zea mays. These assays allowed identification of plants based on size-specific amplicons ranging from 116 bp to 381 bp. Their specificity and sensitivity was consistently high, enabling the detection of small amounts of plant DNA, for example, in decaying plant material and in the intestine or faeces of herbivores. To increase the efficacy of identifying plant species from large number of samples, specific primers were combined in multiplex PCRs, allowing screening for multiple species within a single reaction. The molecular assays outlined here will be applicable manifold, such as for root- and leaf litter identification, botanical trace evidence, and the analysis of herbivory. PMID:22253728

  16. Rapid Plant Identification Using Species- and Group-Specific Primers Targeting Chloroplast DNA

    PubMed Central

    Staudacher, Karin; Schallhart, Nikolaus; Mitterrutzner, Evi; Steiner, Eva-Maria; Thalinger, Bettina; Traugott, Michael

    2012-01-01

    Plant identification is challenging when no morphologically assignable parts are available. There is a lack of broadly applicable methods for identifying plants in this situation, for example when roots grow in mixture and for decayed or semi-digested plant material. These difficulties have also impeded the progress made in ecological disciplines such as soil- and trophic ecology. Here, a PCR-based approach is presented which allows identifying a variety of plant taxa commonly occurring in Central European agricultural land. Based on the trnT-F cpDNA region, PCR assays were developed to identify two plant families (Poaceae and Apiaceae), the genera Trifolium and Plantago, and nine plant species: Achillea millefolium, Fagopyrum esculentum, Lolium perenne, Lupinus angustifolius, Phaseolus coccineus, Sinapis alba, Taraxacum officinale, Triticum aestivum, and Zea mays. These assays allowed identification of plants based on size-specific amplicons ranging from 116 bp to 381 bp. Their specificity and sensitivity was consistently high, enabling the detection of small amounts of plant DNA, for example, in decaying plant material and in the intestine or faeces of herbivores. To increase the efficacy of identifying plant species from large number of samples, specific primers were combined in multiplex PCRs, allowing screening for multiple species within a single reaction. The molecular assays outlined here will be applicable manifold, such as for root- and leaf litter identification, botanical trace evidence, and the analysis of herbivory. PMID:22253728

  17. Time-specific patterns of nest survival for ducks and passerines breeding in North Dakota

    USGS Publications Warehouse

    Shaffer, Terry L.; Grant, Todd A.

    2012-01-01

    In many bird species, survival can vary with the age of the nest, with the date a nest was initiated, or among years within the same nesting area. A literature review showed that patterns of survival vary in relation to nest age and date and are often contradictory. Inconsistencies could be a result of temporal variation in the environment or life-history differences among species. We examined patterns of nest survival in relation to nest age, date, and year for several duck and passerine species nesting at a single location in North Dakota during 1998–2003. We predicted that if environment shaped nest survival patterns, then temporal patterns in survival might be similar among three species of upland nesting ducks, and also among three species of grassland passerines nesting at the same site. We expected that survival patterns would differ between ducks and passerines because of relatively disparate life histories and differences in predators that prey on their nests. Nest survival was rarely constant among years, seasonally, or with age of the nest for species that we studied. As predicted, the pattern of survival was similar among duck species, driven mainly by differences in nest survival associated with nest initiation date. The pattern of survival also was similar among passerine species, but nest survival was more influenced by nest age than by date. Our findings suggest that some but not all variation in temporal patterns of nest survival in grassland birds reported in the literature can be explained on the basis of temporal environmental variation. Because patterns of survival were dissimilar among ducks and passerines, it is likely that mechanisms such as predation or brood parasitism have variable influences on productivity of ducks and passerines nesting in the same area. Our results indicate that biologists and managers should not assume that temporal environmental variations, especially factors that affect nest survival, act similarly on all

  18. Characterization of the 18S rRNA gene for designing universal eukaryote specific primers.

    PubMed

    Hadziavdic, Kenan; Lekang, Katrine; Lanzen, Anders; Jonassen, Inge; Thompson, Eric M; Troedsson, Christofer

    2014-01-01

    High throughput sequencing technology has great promise for biodiversity studies. However, an underlying assumption is that the primers used in these studies are universal for the prokaryotic or eukaryotic groups of interest. Full primer universality is difficult or impossible to achieve and studies using different primer sets make biodiversity comparisons problematic. The aim of this study was to design and optimize universal eukaryotic primers that could be used as a standard in future biodiversity studies. Using the alignment of all eukaryotic sequences from the publicly available SILVA database, we generated a full characterization of variable versus conserved regions in the 18S rRNA gene. All variable regions within this gene were analyzed and our results suggested that the V2, V4 and V9 regions were best suited for biodiversity assessments. Previously published universal eukaryotic primers as well as a number of self-designed primers were mapped to the alignment. Primer selection will depend on sequencing technology used, and this study focused on the 454 pyrosequencing GS FLX Titanium platform. The results generated a primer pair yielding theoretical matches to 80% of the eukaryotic and 0% of the prokaryotic sequences in the SILVA database. An empirical test of marine sediments using the AmpliconNoise pipeline for analysis of the high throughput sequencing data yielded amplification of sequences for 71% of all eukaryotic phyla with no isolation of prokaryotic sequences. To our knowledge this is the first characterization of the complete 18S rRNA gene using all eukaryotes present in the SILVA database, providing a robust test for universal eukaryotic primers. Since both in silico and empirical tests using high throughput sequencing retained high inclusion of eukaryotic phyla and exclusion of prokaryotes, we conclude that these primers are well suited for assessing eukaryote diversity, and can be used as a standard in biodiversity studies.

  19. Characterization of the 18S rRNA Gene for Designing Universal Eukaryote Specific Primers

    PubMed Central

    Hadziavdic, Kenan; Lekang, Katrine; Lanzen, Anders; Jonassen, Inge; Thompson, Eric M.; Troedsson, Christofer

    2014-01-01

    High throughput sequencing technology has great promise for biodiversity studies. However, an underlying assumption is that the primers used in these studies are universal for the prokaryotic or eukaryotic groups of interest. Full primer universality is difficult or impossible to achieve and studies using different primer sets make biodiversity comparisons problematic. The aim of this study was to design and optimize universal eukaryotic primers that could be used as a standard in future biodiversity studies. Using the alignment of all eukaryotic sequences from the publicly available SILVA database, we generated a full characterization of variable versus conserved regions in the 18S rRNA gene. All variable regions within this gene were analyzed and our results suggested that the V2, V4 and V9 regions were best suited for biodiversity assessments. Previously published universal eukaryotic primers as well as a number of self-designed primers were mapped to the alignment. Primer selection will depend on sequencing technology used, and this study focused on the 454 pyrosequencing GS FLX Titanium platform. The results generated a primer pair yielding theoretical matches to 80% of the eukaryotic and 0% of the prokaryotic sequences in the SILVA database. An empirical test of marine sediments using the AmpliconNoise pipeline for analysis of the high throughput sequencing data yielded amplification of sequences for 71% of all eukaryotic phyla with no isolation of prokaryotic sequences. To our knowledge this is the first characterization of the complete 18S rRNA gene using all eukaryotes present in the SILVA database, providing a robust test for universal eukaryotic primers. Since both in silico and empirical tests using high throughput sequencing retained high inclusion of eukaryotic phyla and exclusion of prokaryotes, we conclude that these primers are well suited for assessing eukaryote diversity, and can be used as a standard in biodiversity studies. PMID:24516555

  20. Microarrays for Genotyping Human Group A Rotavirus by Multiplex Capture and Type-Specific Primer Extension

    PubMed Central

    Lovmar, Lovisa; Fock, Caroline; Espinoza, Felix; Bucardo, Filemon; Syvänen, Ann-Christine; Bondeson, Kåre

    2003-01-01

    Human group A rotavirus (HRV) is the major cause of severe gastroenteritis in infants worldwide. HRV shares the feature of a high degree of genetic diversity with many other RNA viruses, and therefore, genotyping of this organism is more complicated than genotyping of more stable DNA viruses. We describe a novel microarray-based method that allows high-throughput genotyping of RNA viruses with a high degree of polymorphism by multiplex capture and type-specific extension on microarrays. Denatured reverse transcription (RT)-PCR products derived from two outer capsid genes of clinical isolates of HRV were hybridized to immobilized capture oligonucleotides representing the most commonly occurring P and G genotypes on a microarray. Specific primer extension of the type-specific capture oligonucleotides was applied to incorporate the fluorescent nucleotide analogue cyanine 5-labeled dUTP as a detectable label. Laser scanning and fluorescence detection of the microarrays was followed by visual or computer-assisted interpretation of the fluorescence patterns generated on the microarrays. Initially, the method detected HRV in all 40 samples and correctly determined both the G and the P genotypes of 35 of the 40 strains analyzed. After modification by inclusion of additional capture oligonucleotides specific for the initially unassigned genotypes, all genotypes could be correctly defined. The results of genotyping with the microarray fully agreed with the results obtained by nucleotide sequence analysis and sequence-specific multiplex RT-PCR. Owing to its robustness, simplicity, and general utility, the microarray-based method may gain wide applicability for the genotyping of microorganisms, including highly variable RNA and DNA viruses. PMID:14605152

  1. Microarrays for genotyping human group a rotavirus by multiplex capture and type-specific primer extension.

    PubMed

    Lovmar, Lovisa; Fock, Caroline; Espinoza, Felix; Bucardo, Filemon; Syvänen, Ann-Christine; Bondeson, Kåre

    2003-11-01

    Human group A rotavirus (HRV) is the major cause of severe gastroenteritis in infants worldwide. HRV shares the feature of a high degree of genetic diversity with many other RNA viruses, and therefore, genotyping of this organism is more complicated than genotyping of more stable DNA viruses. We describe a novel microarray-based method that allows high-throughput genotyping of RNA viruses with a high degree of polymorphism by multiplex capture and type-specific extension on microarrays. Denatured reverse transcription (RT)-PCR products derived from two outer capsid genes of clinical isolates of HRV were hybridized to immobilized capture oligonucleotides representing the most commonly occurring P and G genotypes on a microarray. Specific primer extension of the type-specific capture oligonucleotides was applied to incorporate the fluorescent nucleotide analogue cyanine 5-labeled dUTP as a detectable label. Laser scanning and fluorescence detection of the microarrays was followed by visual or computer-assisted interpretation of the fluorescence patterns generated on the microarrays. Initially, the method detected HRV in all 40 samples and correctly determined both the G and the P genotypes of 35 of the 40 strains analyzed. After modification by inclusion of additional capture oligonucleotides specific for the initially unassigned genotypes, all genotypes could be correctly defined. The results of genotyping with the microarray fully agreed with the results obtained by nucleotide sequence analysis and sequence-specific multiplex RT-PCR. Owing to its robustness, simplicity, and general utility, the microarray-based method may gain wide applicability for the genotyping of microorganisms, including highly variable RNA and DNA viruses.

  2. Robust Detection of Rare Species Using Environmental DNA: The Importance of Primer Specificity

    PubMed Central

    Wilcox, Taylor M.; McKelvey, Kevin S.; Young, Michael K.; Jane, Stephen F.; Lowe, Winsor H.; Whiteley, Andrew R.; Schwartz, Michael K.

    2013-01-01

    Environmental DNA (eDNA) is being rapidly adopted as a tool to detect rare animals. Quantitative PCR (qPCR) using probe-based chemistries may represent a particularly powerful tool because of the method’s sensitivity, specificity, and potential to quantify target DNA. However, there has been little work understanding the performance of these assays in the presence of closely related, sympatric taxa. If related species cause any cross-amplification or interference, false positives and negatives may be generated. These errors can be disastrous if false positives lead to overestimate the abundance of an endangered species or if false negatives prevent detection of an invasive species. In this study we test factors that influence the specificity and sensitivity of TaqMan MGB assays using co-occurring, closely related brook trout (Salvelinus fontinalis) and bull trout (S. confluentus) as a case study. We found qPCR to be substantially more sensitive than traditional PCR, with a high probability of detection at concentrations as low as 0.5 target copies/µl. We also found that number and placement of base pair mismatches between the Taqman MGB assay and non-target templates was important to target specificity, and that specificity was most influenced by base pair mismatches in the primers, rather than in the probe. We found that insufficient specificity can result in both false positive and false negative results, particularly in the presence of abundant related species. Our results highlight the utility of qPCR as a highly sensitive eDNA tool, and underscore the importance of careful assay design. PMID:23555689

  3. Development of Prevotella intermedia-specific PCR primers based on the nucleotide sequences of a DNA probe Pig27.

    PubMed

    Kim, Min Jung; Hwang, Kyung Hwan; Lee, Young-Seok; Park, Jae-Yoon; Kook, Joong-Ki

    2011-03-01

    The aim of this study was to develop Prevotella intermedia-specific PCR primers based on the P. intermedia-specific DNA probe. The P. intermedia-specific DNA probe was screened by inverted dot blot hybridization and confirmed by Southern blot hybridization. The nucleotide sequences of the species-specific DNA probes were determined using a chain termination method. Southern blot analysis showed that the DNA probe, Pig27, detected only the genomic DNA of P. intermedia strains. PCR showed that the PCR primers, Pin-F1/Pin-R1, had species-specificity for P. intermedia. The detection limits of the PCR primer sets were 0.4pg of the purified genomic DNA of P. intermedia ATCC 49046. These results suggest that the PCR primers, Pin-F1/Pin-R1, could be useful in the detection of P. intermedia as well as in the development of a PCR kit in epidemiological studies related to periodontal diseases. PMID:21192988

  4. A novel PCR technique using Alu-specific primers to identify unknown flanking sequences from the human genome

    SciTech Connect

    Minami, M.; Poussin, K.; Brechot, C.; Paterlini, P.

    1995-09-20

    The rapid and reproducible identification of new cellular DNA sequences is difficult to achieve with the currently available procedures. Here we describe a novel approach based on the polymerase chain reaction (PCR) using a primer specific to the known sequence and another directed to a human Alu repeat. To avoid undesirable amplifications between Alu sequences, primers are constructed with dUTPs and destroyed by uracil DNA glycosylase treatment after 10 initial cycles of amplification. Only desirable fragments are then further amplified with specific primers to the known region and to a tag sequence introduced in the Alu-specific primer. Using this protocol, we have successfully indentified cellular sequences flanking integrated hepatitis B virus DNA from the human genome of three hepatoma tissues. The method enables a direct specific amplification without any ligation or nonspecific annealing steps as required by previous PCR-based protocols. This rapid and straightforward approach will be a powerful tool for the study of viral integration sites, but is also widely applicable to other studies of the human genome. 39 refs., 4 figs.

  5. Metabarcoding Analysis of Phytophthora Diversity Using Genus-Specific Primers and 454 Pyrosequencing.

    PubMed

    Prigigallo, Maria I; Abdelfattah, Ahmed; Cacciola, Santa O; Faedda, Roberto; Sanzani, Simona M; Cooke, David E L; Schena, L

    2016-03-01

    A metabarcoding method based on genus-specific primers and 454 pyrosequencing was utilized to investigate the genetic diversity of Phytophthora spp. in soil and root samples of potted plants, from eight nurseries. Pyrosequencing enabled the detection of 25 Phytophthora phylotypes distributed in seven different clades and provided a much higher resolution than a corresponding cloning/Sanger sequencing approach. Eleven of these phylotypes, including P. cactorum, P. citricola s.str., P. palmivora, P. palmivora-like, P. megasperma or P. gonapodyides, P. ramorum, and five putative new Phytophthora species phylogenetically related to clades 1, 2, 4, 6, and 7 were detected only with the 454 pyrosequencing approach. We also found an additional 18 novel records of a phylotype in a particular nursery that were not detected with cloning/Sanger sequencing. Several aspects confirmed the reliability of the method: (i) many identical sequence types were identified independently in different nurseries, (ii) most sequence types identified with 454 pyrosequencing were identical to those from the cloning/Sanger sequencing approach and/or perfectly matched GenBank deposited sequences, and (iii) the divergence noted between sequence types of putative new Phytophthora species and all other detected sequences was sufficient to rule out sequencing errors. The proposed method represents a powerful tool to study Phytophthora diversity providing that particular attention is paid to the analysis of 454 pyrosequencing raw read sequences and to the identification of sequence types.

  6. Metabarcoding Analysis of Phytophthora Diversity Using Genus-Specific Primers and 454 Pyrosequencing.

    PubMed

    Prigigallo, Maria I; Abdelfattah, Ahmed; Cacciola, Santa O; Faedda, Roberto; Sanzani, Simona M; Cooke, David E L; Schena, L

    2016-03-01

    A metabarcoding method based on genus-specific primers and 454 pyrosequencing was utilized to investigate the genetic diversity of Phytophthora spp. in soil and root samples of potted plants, from eight nurseries. Pyrosequencing enabled the detection of 25 Phytophthora phylotypes distributed in seven different clades and provided a much higher resolution than a corresponding cloning/Sanger sequencing approach. Eleven of these phylotypes, including P. cactorum, P. citricola s.str., P. palmivora, P. palmivora-like, P. megasperma or P. gonapodyides, P. ramorum, and five putative new Phytophthora species phylogenetically related to clades 1, 2, 4, 6, and 7 were detected only with the 454 pyrosequencing approach. We also found an additional 18 novel records of a phylotype in a particular nursery that were not detected with cloning/Sanger sequencing. Several aspects confirmed the reliability of the method: (i) many identical sequence types were identified independently in different nurseries, (ii) most sequence types identified with 454 pyrosequencing were identical to those from the cloning/Sanger sequencing approach and/or perfectly matched GenBank deposited sequences, and (iii) the divergence noted between sequence types of putative new Phytophthora species and all other detected sequences was sufficient to rule out sequencing errors. The proposed method represents a powerful tool to study Phytophthora diversity providing that particular attention is paid to the analysis of 454 pyrosequencing raw read sequences and to the identification of sequence types. PMID:26574783

  7. FUNGAL-SPECIFIC PCR PRIMERS DEVELOPED FOR ANALYSIS OF THE ITS REGION OF ENVIRONMENTAL DNA EXTRACTS

    EPA Science Inventory

    Background The Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) are highly variable sequences of great importance in distinguishing fungal species by PCR analysis. Previously published PCR primers available for amplifying these sequences from environmenta...

  8. Sex determination in cattle based on simultaneous amplification of a new male-specific DNA sequence and an autosomal locus using the same primers.

    PubMed

    Weikard, R; Kühn, C; Brunner, R M; Roschlau, D; Pitra, C; Laurent, P; Schwerin, M

    2001-09-01

    A PCR-based method for sex determination of bovine DNA samples and embryo biopsies is presented. Using only one primer pair both the male-specific sequence FBNY (127 bp) and a sex-independent control PCR-fragment, the microsatellite marker FBN17 (136-140 bp) are generated in the same PCR reaction. Synteny mapping assigned the male-specific sequence to bovine chromosome Y (BTA Y), whereas FBN17 was mapped to bovine chromosome 2. Localisation of FBNY on BTA Y was confirmed by fluorescence in hybridisation of two BAC clones containing the male-specific sequence. There was no amplification of the male-specific target sequence FBNY in sheep, pig, goat, mice, man, and several wild species of the tribe Bovini. The bovine male-specific fragment was detected in dilutions containing as little as 10 pg genomic DNA and in blastomeres from embryo biopsies. The PCR assay presented here does require neither restriction endonuclease digestion of the PCR product nor additional nested PCR steps. Owing to the advantage of parallel amplification of the autosomal locus FBN17 no additional control fragment is necessary to detect PCR failure. The results of sex determination in embryo biopsies using FBNY were in agreement with the outcome from a reference assay used in commercial breeding programs. PMID:11550263

  9. HIV-1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA.

    PubMed Central

    Barat, C; Lullien, V; Schatz, O; Keith, G; Nugeyre, M T; Grüninger-Leitch, F; Barré-Sinoussi, F; LeGrice, S F; Darlix, J L

    1989-01-01

    The virion cores of the replication competent type 1 human immunodeficiency virus (HIV-1), a retrovirus, contain and RNA genome associated with nucleocapsid (NC) and reverse transcriptase (RT p66/p51) molecules. In vitro reconstructions of these complexes with purified components show that NC is required for efficient annealing of the primer tRNALys,3. In the absence of NC, HIV-1 RT is unable to retrotranscribe the viral RNA template from the tRNA primer. We demonstrate that the HIV-1 RT p66/p51 specifically binds to its cognate primer tRNALys,3 even in the presence of a 100-fold molar excess of other tRNAs. Cross-linking analysis of this interaction locates the contact site to a region within the heavily modified anti-codon domain of tRNALys,3. Images PMID:2479543

  10. Evaluation of PCR primer selectivity and phylogenetic specificity by using amplification of 16S rRNA genes from betaproteobacterial ammonia-oxidizing bacteria in environmental samples.

    PubMed

    Junier, Pilar; Kim, Ok-Sun; Hadas, Ora; Imhoff, Johannes F; Witzel, Karl-Paul

    2008-08-01

    The effect of primer specificity for studying the diversity of ammonia-oxidizing betaproteobacteria (betaAOB) was evaluated. betaAOB represent a group of phylogenetically related organisms for which the 16S rRNA gene approach is especially suitable. We used experimental comparisons of primer performance with water samples, together with an in silico analysis of published sequences and a literature review of clone libraries made with four specific PCR primers for the betaAOB 16S rRNA gene. With four aquatic samples, the primers NitA/NitB produced the highest frequency of ammonia-oxidizing-bacterium-like sequences compared to clone libraries with products amplified with the primer combinations betaAMOf/betaAMOr, betaAMOf/Nso1255g, and NitA/Nso1225g. Both the experimental examination of ammonia-oxidizing-bacterium-specific 16S rRNA gene primers and the literature search showed that neither specificity nor sensitivity of primer combinations can be evaluated reliably only by sequence comparison. Apparently, the combination of sequence comparison and experimental data is the best approach to detect possible biases of PCR primers. Although this study focused on betaAOB, the results presented here more generally exemplify the importance of primer selection and potential primer bias when analyzing microbial communities in environmental samples.

  11. Diversity of Methane-Cycling Archaea in Hydrothermal Sediment Investigated by General and Group-Specific PCR Primers

    PubMed Central

    Teske, Andreas P.

    2014-01-01

    The zonation of anaerobic methane-cycling Archaea in hydrothermal sediment of Guaymas Basin was studied by general primer pairs (mcrI, ME1/ME2, mcrIRD) targeting the alpha subunit of methyl coenzyme M reductase gene (mcrA) and by new group-specific mcrA and 16S rRNA gene primer pairs. The mcrIRD primer pair outperformed the other general mcrA primer pairs in detection sensitivity and phylogenetic coverage. Methanotrophic ANME-1 Archaea were the only group detected with group-specific primers only. The detection of 14 mcrA lineages surpasses the diversity previously found in this location. Most phylotypes have high sequence similarities to hydrogenotrophs, methylotrophs, and anaerobic methanotrophs previously detected at Guaymas Basin or at hydrothermal vents, cold seeps, and oil reservoirs worldwide. Additionally, five mcrA phylotypes belonging to newly defined lineages are detected. Two of these belong to deeply branching new orders, while the others are new species or genera of Methanopyraceae and Methermicoccaceae. Downcore diversity decreases from all groups detected in the upper 6 cm (∼2 to 40°C, sulfate measurable to 4 cm) to only two groups below 6 cm (>40°C). Despite the presence of hyperthermophilic genera (Methanopyrus, Methanocaldococcus) in cooler surface strata, no genes were detected below 10 cm (≥60°C). While mcrA-based and 16S rRNA gene-based community compositions are generally congruent, the deeply branching mcrA cannot be assigned to specific 16S rRNA gene lineages. Our study indicates that even among well-studied metabolic groups and in previously characterized model environments, major evolutionary branches are overlooked. Detecting these groups by improved molecular biological methods is a crucial first step toward understanding their roles in nature. PMID:25527539

  12. Searching for Beta-Haemolysin hlb Gene in Staphylococcus pseudintermedius with Species-Specific Primers.

    PubMed

    Kmieciak, Wioletta; Szewczyk, Eligia M; Ciszewski, Marcin

    2016-07-01

    The paper presents an analysis of 51 Staphylococcus pseudintermedius clinically isolated strains from humans and from animals. Staphylococcus pseudintermedius strains' ability to produce β-haemolysin was evaluated with phenotypic methods (hot-cold effect, reverse CAMP test). In order to determine the hlb gene presence (coding for β-haemolysin) in a genomic DNA, PCR reactions were conducted with two different pairs of primers: one described in the literature for Staphylococcus aureus and recommended for analysing SIG group staphylococci and newly designed one in CLC Main Workbench software. Only reactions with newly designed primers resulted in product amplification, the presence of which was fully compatible with the results of phenotypic β-haemolysin test. Negative results for S. aureus and S. intermedius reference ATCC strains suggest that after further analysis the fragment of hlb gene amplified with primers described in this study might be included in the process of S. pseudintermedius strains identification.

  13. Searching for Beta-Haemolysin hlb Gene in Staphylococcus pseudintermedius with Species-Specific Primers.

    PubMed

    Kmieciak, Wioletta; Szewczyk, Eligia M; Ciszewski, Marcin

    2016-07-01

    The paper presents an analysis of 51 Staphylococcus pseudintermedius clinically isolated strains from humans and from animals. Staphylococcus pseudintermedius strains' ability to produce β-haemolysin was evaluated with phenotypic methods (hot-cold effect, reverse CAMP test). In order to determine the hlb gene presence (coding for β-haemolysin) in a genomic DNA, PCR reactions were conducted with two different pairs of primers: one described in the literature for Staphylococcus aureus and recommended for analysing SIG group staphylococci and newly designed one in CLC Main Workbench software. Only reactions with newly designed primers resulted in product amplification, the presence of which was fully compatible with the results of phenotypic β-haemolysin test. Negative results for S. aureus and S. intermedius reference ATCC strains suggest that after further analysis the fragment of hlb gene amplified with primers described in this study might be included in the process of S. pseudintermedius strains identification. PMID:27086303

  14. Age-specific productivity and nest site characteristics of Cooper's hawks (Accipiter cooperii)

    USGS Publications Warehouse

    Moore, K.R.; Henny, C.J.

    1984-01-01

    Nesting Cooper's Hawks (Accipiter cooperii) were studied in northeastern Oregon. Second-year (SY) males did not breed, but 22 percent of the breeding females were SY's. Mean clutch size (P = 0.012) and mean number of young fledged per pair that laid eggs (P < 0.10) were lower for SY females than for adult (after second year [ASY}) females; however, an equal percentage of the eggs (excluding a collected sample egg) yielded fledged young for each age class. Stepwise discriminant analysis suggested differences in structural characteristics of the nest site habitat for ASY and SY females, i.e., SY female nest sites were associated with younger successional stages than ASY female nest sites. Nests of both age groups were built in trees with high crown volume, but ASY females utilized mistletoe (Arceuthobium sp.) for nest structures more frequently (P < 0.01) than SY females.

  15. Primer pairs for the specific environmental detection and T-RFLP analysis of the ubiquitous flagellate taxa Chrysophyceae and Kinetoplastea.

    PubMed

    Glaser, Karin; Kuppardt, Anke; Krohn, Sandra; Heidtmann, Anett; Harms, Hauke; Chatzinotas, Antonis

    2014-05-01

    Bacterivorous protists play a key role in microbial soil food webs, however due to the lack of specific PCR protocols targeting selected protist taxa, knowledge on the diversity and dynamics of these groups is scarce. We developed specific PCR primers in combination with a T-RFLP protocol for the cultivation-independent analysis of two important taxa of bacterivorous flagellates, the Chrysophyceae and Kinetoplastea, in soil samples. Sequence analysis of clone libraries originating from two soils in temperate regions demonstrated the specificity of the respective primer pairs. Clone sequences affiliating to the Chrysophyceae mainly clustered within the clade C2, which has been known so far for its presence mainly in cold climatic regions, whereas Kinetoplastea sequences were mainly related to the Neobodonid clade. Based on an in silico restriction analysis of database sequence entries, suitable restriction enzymes for a T-RFLP approach were selected. This in silico approach revealed the necessity to use a combination of two restriction enzymes for T-RFLP analysis of the Chrysophyceae. Soil T-RFLP profiles reflected all T-RFs of the clone library sequences obtained from the same soils and allowed to distinguish flagellate communities from different sites. We propose to use these primer pairs for PCR detection and rapid fingerprint screening in environmental samples and envisage their use also for quantitative PCR or next generation sequencing approaches.

  16. Diversity of methane-cycling archaea in hydrothermal sediment investigated by general and group-specific PCR primers.

    PubMed

    Lever, Mark A; Teske, Andreas P

    2015-02-01

    The zonation of anaerobic methane-cycling Archaea in hydrothermal sediment of Guaymas Basin was studied by general primerpairs (mcrI, ME1/ME2, mcrIRD) targeting the alpha subunit of methyl coenzyme M reductase gene (mcrA) and by new group specific mcrA and 16S rRNA gene primer pairs. The mcrIRD primer pair outperformed the other general mcrA primer pairs indetection sensitivity and phylogenetic coverage. Methanotrophic ANME-1 Archaea were the only group detected with group specific primers only. The detection of 14 mcrA lineages surpasses the diversity previously found in this location. Most phylotypes have high sequence similarities to hydrogenotrophs, methylotrophs, and anaerobic methanotrophs previously detected at Guaymas Basin or at hydrothermal vents, cold seeps, and oil reservoirs worldwide. Additionally, five mcrA phylotypes belonging to newly defined lineages are detected. Two of these belong to deeply branching new orders, while the others are new species or genera of Methanopyraceae and Methermicoccaceae. Downcore diversity decreases from all groups detected in the upper 6 cm(2 to 40 °C, sulfate measurable to 4 cm) to only two groups below 6 cm (>40 °C). Despite the presence of hyperthermophilic genera (Methanopyrus, Methanocaldococcus) in cooler surface strata, no genes were detected below 10 cm (>60 °C). While mcrAbased and 16S rRNA gene-based community compositions are generally congruent, the deeply branching mcrA cannot be assigned to specific 16S rRNA gene lineages. Our study indicates that even among well-studied metabolic groups and in previously characterized model environments, major evolutionary branches are overlooked. Detecting these groups by improved molecular biological methods is a crucial first step toward understanding their roles in nature.

  17. Development of species-specific PCR primers and polyphasic characterization of Lactobacillus sanfranciscensis isolated from Korean sourdough.

    PubMed

    Lee, Hyeongrho; Baek, Hyunwook; Lim, Sae Bom; Hur, Jin Soo; Shim, Sangmin; Shin, So-Yeon; Han, Nam Soo; Seo, Jin-Ho

    2015-05-01

    Lactobacillus sanfranciscensis is a bacterium used in sourdough that provides desirable properties such as better flavor and texture to the sourdough bread. Here, the intra-species diversity of L. sanfranciscensis strains isolated from Korean sourdough was studied using genotypic (multiplex-RAPD-PCR: multiplex-Randomly Amplified Polymorphic DNA-polymerase chain reaction) and phenotypic (VITEK2 Compact system) analyses. For this, a novel species-specific set of PCR primers was developed to identify L. sanfranciscensis using the recently published genome database. The primers were able to detect L. sanfranciscensis isolated from Korean sourdough with 100% accuracy. Genotyping and phenotyping analyses at the strain level demonstrated that Korean sourdough possesses various biotypes of L. sanfranciscensis strains. These strains were clustered into 5 subtypes (genotyping) or 7 subtypes (phenotyping). In summary, this strategy to construct novel primers reduced the chance of cross amplification and was able to identify the desired strain. The various strains isolated in this study can be used to develop a sourdough starter after the analysis of their fermentation characteristics.

  18. A multiplex set of species-specific primers for rapid identification of members of the genus Saccharomyces.

    PubMed

    Muir, Alastair; Harrison, Elizabeth; Wheals, Alan

    2011-11-01

    The Saccharomyces genus (previously Saccharomyces sensu stricto) formally comprises Saccharomyces arboricola, Saccharomyces bayanus, Saccharomyces cariocanus, Saccharomyces cerevisiae, Saccharomyces kudriavzevii, Saccharomyces mikatae, Saccharomyces paradoxus and Saccharomyces pastorianus. Species-specific primer pairs that produce a single band of known and different product size have been developed for each member of the clade with the exception of S. pastorianus, which is a polyphyletic allopolyploid hybrid only found in lager breweries, and for which signature sequences could not be reliably created. Saccharomyces cariocanus is now regarded as an American variant of S. paradoxus, and accordingly a single primer pair that recognizes both species was developed. A different orthologous and essential housekeeping gene was used to detect each species, potentially avoiding competition between PCR primers and overlap between amplicons. In multiplex format, two or more different species could be identified in a single reaction; double and triple hybrids could not always be correctly identified. Forty-two unidentified yeasts from sugar cane juice fermentations were correctly identified as S. cerevisiae. A colony PCR method was developed that is rapid, robust, inexpensive and capable of automation, requires no mycological expertise on the part of the user and is thus useful for large-scale preliminary screens.

  19. A multiplex set of species-specific primers for rapid identification of members of the genus Saccharomyces.

    PubMed

    Muir, Alastair; Harrison, Elizabeth; Wheals, Alan

    2011-11-01

    The Saccharomyces genus (previously Saccharomyces sensu stricto) formally comprises Saccharomyces arboricola, Saccharomyces bayanus, Saccharomyces cariocanus, Saccharomyces cerevisiae, Saccharomyces kudriavzevii, Saccharomyces mikatae, Saccharomyces paradoxus and Saccharomyces pastorianus. Species-specific primer pairs that produce a single band of known and different product size have been developed for each member of the clade with the exception of S. pastorianus, which is a polyphyletic allopolyploid hybrid only found in lager breweries, and for which signature sequences could not be reliably created. Saccharomyces cariocanus is now regarded as an American variant of S. paradoxus, and accordingly a single primer pair that recognizes both species was developed. A different orthologous and essential housekeeping gene was used to detect each species, potentially avoiding competition between PCR primers and overlap between amplicons. In multiplex format, two or more different species could be identified in a single reaction; double and triple hybrids could not always be correctly identified. Forty-two unidentified yeasts from sugar cane juice fermentations were correctly identified as S. cerevisiae. A colony PCR method was developed that is rapid, robust, inexpensive and capable of automation, requires no mycological expertise on the part of the user and is thus useful for large-scale preliminary screens. PMID:22093682

  20. Evaluation of human immunodeficiency virus type 1 reverse transcriptase primer tRNA binding by fluorescence spectroscopy: specificity and comparison to primer/template binding.

    PubMed

    Thrall, S H; Reinstein, J; Wöhrl, B M; Goody, R S

    1996-04-01

    A host cell-derived tRNA3Lys molecule is utilized by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) to prime DNA synthesis from the viral RNA genome. We performed fluorescence titration experiments to characterize the interaction between RT and its natural primer, tRNA3Lys, and to address RT's putative role in the required and specific packaging of tRNA3Lys into the budding virus. Titration of RT with tRNA3Lys resulted in a 30% maximal quenching of RT tryptophan fluorescence, from which a dissociation constant (Kd) of 57.6 +/- 7.5 nM was derived. Titration of RT with Escherichia coli tRNA2Glu, E. coli tRNA2Tyr, E. coli tRNALys, yeast tRNAPhe, or in vitro-synthesized human tRNA3Lys (no base modifications) resulted in similar fluorescence changes and Kd values as obtained for the natural tRNA3Lys. The specific interaction between RT and tRNA3Lys during viral assembly suggested by previous in vivo studies is therefore not present in the fully processed, in vitro form of RT. Other factors during viral assembly must therefore cooperate in the packaging of tRNA3Lys. The nonspecific and ionic strength dependent RT-tRNA interaction detected in the present studies suggests that the overall shape and charges of tRNA constitute recognition features for RT binding. The fluorescence of the wyebutine base contained on the anticodon loop of yeast tRNAPhe was found to increase upon RT binding, supporting speculation that RT interacts with the anticodon loop of tRNA. The individual tRNAs also displaced a fluorescent DNA primer/template (p/t) substrate from RT, indicating overlapping tRNA and p/t binding sites. Cubic fit evaluation of the displacement titrations allowed further assessment of the affinities of the two competing ligands. The presence of both overlapping and separate p/t and tRNA binding regions on RT was tested by examination of the affinity of a possible RT bisubstrate type inhibitor, containing motifs proposed to be essential for both t

  1. New primer for specific amplification of the CAG repeat in Huntington disease alleles

    SciTech Connect

    Bond, C.E.; Hodes, M.E.

    1994-09-01

    Huntington disease is an autosomal dominant neurodegenerative disorder caused by an expansion of a CAG trinucleotide repeat near the 5{prime} end of the gene for Huntington disease (IT15). The CAG repeat is flanked by a variable-length CCG repeat that is included in the amplification product obtained with most currently used primer sets and PCR protocols. Inclusion of this adjacent CCG repeat complicates the accurate assessment of CAG repeat length and interferes with the genotype determination of those individuals carrying alleles in the intermediate range between normal and expanded sized. Due to the GC-rich nature of this region, attempts at designing a protocol for amplification of only the CAG repeat have proved unreliable and difficult to execute. We report here the development of a compatible primer set and PCR protocol that yields consistent amplification of the CAG-repeat region. PCR products can be visualized in ethidium bromide-stained agarose gels for rapid screening or in 6% polyacrylamide gels for determination of exact repeat length. This assay produces bands that can be sized accurately, while eliminating most nonspecific products. Fifty-five specimens examined showed consistency with another well-known method, but one that amplifies the CCG repeats as well. The results we obtained also matched the known carrier status of the donors.

  2. Linear-After-The-Exponential (LATE)-PCR: primer design criteria for high yields of specific single-stranded DNA and improved real-time detection.

    PubMed

    Pierce, Kenneth E; Sanchez, J Aquiles; Rice, John E; Wangh, Lawrence J

    2005-06-14

    Traditional asymmetric PCR uses conventional PCR primers at unequal concentrations to generate single-stranded DNA. This method, however, is difficult to optimize, often inefficient, and tends to promote nonspecific amplification. An alternative approach, Linear-After-The-Exponential (LATE)-PCR, solves these problems by using primer pairs deliberately designed for use at unequal concentrations. The present report systematically examines the primer design parameters that affect the exponential and linear phases of LATE-PCR amplification. In particular, we investigated how altering the concentration-adjusted melting temperature (Tm) of the limiting primer (TmL) relative to that of the excess primer (TmX) affects both amplification efficiency and specificity during the exponential phase of LATE-PCR. The highest reaction efficiency and specificity were observed when TmL - TmX 5 degrees C. We also investigated how altering TmX relative to the higher Tm of the double-stranded amplicon (TmA) affects the rate and extent of linear amplification. Excess primers with TmX closer to TmA yielded higher rates of linear amplification and stronger signals from a hybridization probe. These design criteria maximize the yield of specific single-stranded DNA products and make LATE-PCR more robust and easier to implement. The conclusions were validated by using primer pairs that amplify sequences within the cystic fibrosis transmembrane regulator (CFTR) gene, mutations of which are responsible for cystic fibrosis.

  3. Prey choice by carabid beetles feeding on an earthworm community analysed using species- and lineage-specific PCR primers.

    PubMed

    King, R Andrew; Vaughan, Ian P; Bell, James R; Bohan, David A; Symondson, William O C

    2010-04-01

    The carabid beetle Pterostichus melanarius is a major natural enemy of pests, such as aphids and slugs in agricultural systems. Earthworms are a dominant non-pest component of the diet of P. melanarius which help sustain the beetles during periods when the pest population is low or absent. In this study we wanted to test whether this predator exercises prey choice among different earthworm species or ecological groups. High levels of genetic diversity within morphological species of earthworm necessitated the development of primers that were specific not just to species but lineages and sub-lineages within species as well. Gut samples from beetles were analysed using multiplex-PCR and fluorescent-labelled primers. Calibratory feeding trials were undertaken to calculate median detection times for prey DNA following ingestion. Extensive testing demonstrated that the primers were species-specific, that detection periods were negatively related to amplicon size and that meal size had a highly significant effect on detection periods. Monte Carlo simulations showed that, in general, worms were being predated in proportion to their densities in the field with little evidence of prey choice, other than probable avoidance of the larger, deep-living species. There was no evidence that epigeic species were being taken preferentially in comparison with endogeic species. There was also no evidence that defensive secretions by Allolobophora chlorotica reduced predation pressure on this species by P. melanarius. We concluded that any management system that increases earthworm densities generally, regardless of component species, is likely to be optimal for increasing numbers of this beneficial beetle predator. PMID:20345680

  4. Retrospective study of central nervous system lesions and association with Parelaphostrongylus species by histology and specific nested polymerase chain reaction in domestic camelids and wild ungulates.

    PubMed

    Dobey, Carrie L; Grunenwald, Caroline; Newman, Shelley J; Muller, Lisa; Gerhold, Richard W

    2014-11-01

    Formalin-fixed, paraffin-embedded tissues from elk (Cervus elaphus), goats, and camelids with case histories and lesions suggestive of Parelaphostrongylus tenuis were examined by histology to characterize lesions that could aid in definitively diagnosing P. tenuis infection. Additionally, sections of paraffin-embedded tissue were used in a nested polymerase chain reaction (nPCR) using Parelaphostrongylus-specific primers to determine how PCR results corresponded with histological findings. Histological changes in brain and spinal cord consisted of linear tracks of hemorrhage; tracks or perivascular accumulations of hemosiderin-laden macrophages; acute foci of axonal degeneration and/or linear glial scars; and perivascular, parenchymal, or meningeal accumulations of eosinophils and/or lymphocytes and plasma cells. Of the 43 samples with histologic lesions consistent with neural larval migrans, 19 were PCR positive; however, only 8 were confirmed Parelaphostrongylus by DNA sequencing. Additionally, 1 goat was identified with a protostrongylid that had a 97% identity to both Parelaphostrongylus odocoilei and a protostrongylid nematode from pampas deer (Ozotoceros bezoarticus celer) from Argentina. None of the histologic lesions individually or in combination correlated statistically to positive molecular tests for the nematode. The results indicate that it is possible to extract Parelaphostrongylus DNA from formalin-fixed, paraffin-embedded tissue, but extended fixation presumably can cause DNA crosslinking. Nested PCR provides another diagnostic tool to identify the cause of neurologic disease in camelids and elk with histologic lesions consistent with neural larval migrans. Furthermore, potential novel protostrongylid DNA was detected from a goat with lesions consistent with P. tenuis infection, suggesting that other neurotropic Parelaphostrongylus species may occur locally.

  5. Development of Primers to O-Antigen Biosynthesis Genes for Specific Detection of Escherichia coli O157 by PCR

    PubMed Central

    Maurer, John J.; Schmidt, Denise; Petrosko, Patricia; Sanchez, Susan; Bolton, Lance; Lee, Margie D.

    1999-01-01

    The chemical composition of each O-antigen subunit in gram-negative bacteria is a reflection of the unique DNA sequences within each rfb operon. By characterizing DNA sequences contained with each rfb operon, a diagnostic serotype-specific probe to Escherichia coli O serotypes that are commonly associated with bacterial infections can be generated. Recently, from an E. coli O157:H7 cosmid library, O-antigen-positive cosmids were identified with O157-specific antisera. By using the cosmid DNAs as probes, several DNA fragments which were unique to E. coli O157 serotypes were identified by Southern analysis. Several of these DNA fragments were subcloned from O157-antigen-positive cosmids and served as DNA probes in Southern analysis. One DNA fragment within plasmid pDS306 which was specific for E. coli O157 serotypes was identified by Southern analysis. The DNA sequence for this plasmid revealed homology to two rfb genes, the first of which encodes a GDP-mannose dehydratase. These rfb genes were similar to O-antigen biosynthesis genes in Vibrio cholerae and Yersinia enterocolitica serotype O:8. An oligonucleotide primer pair was designed to amplify a 420-bp DNA fragment from E. coli O157 serotypes. The PCR test was specific for E. coli O157 serotypes. PCR detected as few as 10 cells with the O157-specific rfb oligonucleotide primers. Coupled with current enrichment protocols, O157 serotyping by PCR will provide a rapid, specific, and sensitive method for identifying E. coli O157. PMID:10388689

  6. Revealing the Diversity and Quantity of Peritrich Ciliates in Environmental Samples Using Specific Primer-based PCR and Quantitative PCR

    PubMed Central

    Liu, Xihan; Gong, Jun

    2012-01-01

    Peritrichs are a diverse, ecologically important ciliate group usually with a complex life cycle. To date, the community of the peritrichs has been investigated by using morphology-based methods such as living observation and silver staining. Here we show a molecular approach for characterizing the diversity and quantity of free-living peritrichs in environmental samples. We newly designed four peritrich-specific primers targeting 18S rRNA genes that allow clone library construction, screening and analysis. A quantitative real-time PCR (qPCR) assay was developed to quantify peritrichs in environmental samples by using rDNA copy number as an indicator. DNA extracted from four water samples of contrasting environmental gradients was analysed. The results showed that the peritrich community was differentiated among these samples, and that the diversity decreased with the increase of water salinity. The qPCR results are consistent with the library sequence analysis in terms of quantity variations from sample to sample. The development of peritrich-specific primers, for the first time, for conventional PCR and qPCR assays, provides useful molecular tools for revealing the diversity and quantity of peritrich ciliates in environmental samples. Also, our study illustrates the potential of these molecular tools to ecological studies of other ciliate groups in diverse environments. PMID:23100023

  7. TESTING THE SPECIFICITY OF PRIMERS TO ENVIRONMENTAL AMMONIA MONOOXYGENASE (AMOA) GENES IN GROUNDWATER TREATED WITH UREA TO PROMOTE CALCITE PRECIPITATION

    SciTech Connect

    Stephanie Freeman; David Reed; Yoshiko Fujita

    2006-12-01

    The diversity of bacterial ammonia monooxygenase (amoA) genes in DNA isolated from microorganisms in groundwater was characterized by amplification of amoA DNA using polymerase chain reaction (PCR), Restriction Fragment Length Polymorphism (RFLP) analysis, and sequencing. The amoA gene is characteristic of ammonia oxidizing bacteria (AOB). The DNA extracts were acquired from an experiment where dilute molasses and urea were sequentially introduced into a well in the Eastern Snake River Plain Aquifer (ESRPA) in Idaho to examine whether such amendments could stimulate enhanced ureolytic activity. The hydrolysis of urea into ammonium and carbonate serves as the basis for a potential remediation technique for trace metals and radionuclide contaminants that co-precipitate in calcite. The ammonium ion resulting from ureolysis can promote the growth of AOB. The goal of this work was to investigate the effectiveness of primers designed for quantitative PCR of environmental amoA genes and to evaluate the effect of the molasses and urea amendments upon the population diversity of groundwater AOB. PCR primers designed to target a portion of the amoA gene were used to amplify amoA gene sequences in the groundwater DNA extracts. Following PCR, amplified gene products were cloned and the clones were characterized by RFLP, a DNA restriction technique that can distinguish different DNA sequences, to gauge the initial diversity. Clones exhibiting unique RFLP patterns were subjected to DNA sequencing. Initial sequencing results suggest that the primers were successful at specific detection of amoA sequences and the RFLP analyses indicated that the diversity of detected amoA sequences in the ESRPA decreased with the additions of molasses and urea.

  8. Development of Nested PCR-Based Specific Markers for Detection of Peach Rosette Mosaic Virus in Plant Quarantine.

    PubMed

    Lee, S; Kim, C S; Shin, Y G; Kim, J H; Kim, Y S; Jheong, W H

    2016-03-01

    The Peach rosette mosaic virus (PRMV) is a plant pathogen of the genus Nepovirus, and has been designated as a controlled quarantine virus in Korea. In this study, a specific reverse transcription (RT)-PCR marker set, nested PCR marker set, and modified-plasmid positive control were developed to promptly and accurately diagnose PRMV at plant-quarantine sites. The final selected PRMV-specific RT-PCR marker was PRMV-N10/C70 (967 bp), and the nested PCR product of 419 bp was finally amplified. The modified-plasmid positive control, in which the SalI restriction-enzyme region (GTCGAC) was inserted, verified PRMV contamination in a comparison with the control, enabling a more accurate diagnosis. It is expected that the developed method will continuously contribute to the plant-quarantine process in Korea. PMID:26843704

  9. Use of 16S rRNA Gene-Targeted Group-Specific Primers for Real-Time PCR Analysis of Predominant Bacteria in Mouse Feces.

    PubMed

    Yang, Yun-Wen; Chen, Mang-Kun; Yang, Bing-Ya; Huang, Xian-Jie; Zhang, Xue-Rui; He, Liang-Qiang; Zhang, Jing; Hua, Zi-Chun

    2015-10-01

    Mouse models are widely used for studying gastrointestinal (GI) tract-related diseases. It is necessary and important to develop a new set of primers to monitor the mouse gut microbiota. In this study, 16S rRNA gene-targeted group-specific primers for Firmicutes, Actinobacteria, Bacteroidetes, Deferribacteres, "Candidatus Saccharibacteria," Verrucomicrobia, Tenericutes, and Proteobacteria were designed and validated for quantification of the predominant bacterial species in mouse feces by real-time PCR. After confirmation of their accuracy and specificity by high-throughput sequencing technologies, these primers were applied to quantify the changes in the fecal samples from a trinitrobenzene sulfonic acid-induced colitis mouse model. Our results showed that this approach efficiently predicted the occurrence of colitis, such as spontaneous chronic inflammatory bowel disease in transgenic mice. The set of primers developed in this study provides a simple and affordable method to monitor changes in the intestinal microbiota at the phylum level. PMID:26187967

  10. Single primer amplification reaction (SPAR) reveals inter- and intra-specific natural genetic variation in five species of Cymbidium (Orchidaceae).

    PubMed

    Sharma, Santosh Kumar; Kumaria, Suman; Tandon, Pramod; Rao, Satyawada Rama

    2011-09-01

    A total of 53 primers belonging to three SPAR methods, viz. RAPD, ISSR and DAMD, collectively produced 456 polymorphic amplicons with 96.6% polymorphism at inter-specific level in five species of Cymbidium, viz. C. aloifolium, C. mastersii, C. elegans, C. eburneum and C. tigrinum, whereas at intra-specific level, the observed polymorphism ranged from 51.2% to 77.1% among them. Three SPARs collectively revealed 25 unique species-specific amplicons; most of them were amplified with RAPD and DAMD primers besides few bands which were either missed (absent) or lost (heterozygosity). UPGMA clustering evidently distinguished the representatives of C. aloifolium and C. tigrinum, with distinct genetic distance, which may be due to their entirely different habitats as well as discrete morphological characteristics. Upon analysis of the data generated, all the three SPAR methods, either independently and/or in combination, revealed wide range of genetic variation between and within five species of Cymbidium. Comparison of matrix of individual SPAR method revealed that analysis of natural genetic variation using combination of SPAR methods, rather than an isolated approach, is highly effective. The critical analyses of the amplicon data are indicative of DAMD as the most powerful SPAR method by showing highest resolving power (Rp) followed by ISSR and RAPD. Alternatively, the total polymorphic information content was highest in case of RAPD followed by other two SPAR methods. Thus, the present investigation for the first time provides a valuable baseline data for genetic variation at inter- and intra-specific levels in horticultural Cymbidiums and also addresses conservation concerns.

  11. Development of catechol 2,3-dioxygenase-specific primers for monitoring bioremediation by competitive quantitative PCR

    SciTech Connect

    Mesarch, M.B.; Nakatsu, C.H.; Nies, L.

    2000-02-01

    Benzene, toluene, xylenes, phenol, naphthalene, and biphenyl are among a group of compounds that have at least one reported pathway for biodegradation involving catechol 2,3-dioxygenase enzymes. Thus, detection of the corresponding catechol 2,3-dioxygenase genes can serve as a basis for identifying and quantifying bacteria that have these catabolic abilities. Primes that can successfully amplify a 238-bp catechol 2,3-dioxygenase gene fragment from eight different bacteria are described. The identities of the amplicons were confirmed by hybridization with a 238-bp catechol 2,3-dioxygenase probe. The detection limit was 10{sup 2} to 10{sup 3} gene copies, which was lowered to 10{sup 0} to 10{sup 1} gene copies of hybridization. Using the dioxygenase-specific primers, an increase in catechol 2,3-dioxygenase genes was detected in petroleum-amended soils. The dioxygenase genes were enumerated by competitive quantitative PCR and a 163-bp competitor that was amplified using the same primers. Target and competitor sequences had identical amplification kinetics. Potential PCR inhibitors that could coextract with DNA, nonamplifying DNA, soil factors (humics), and soil pollutants (toluene) did not impact enumeration. Therefore, this technique can be used to accurately and reproducibly quantify catechol 2,3-dioxygenase genes in complex environments such as petroleum-contaminated soil. Direct, non-cultivation-based molecular techniques for detecting and enumerating microbial pollutant-biodegrading genes in environmental samples are powerful tools for monitoring bioremediation and developing field evidence in support of natural attenuation.

  12. Specific PCR primers directed to identify cryI and cryIII genes within a Bacillus thuringiensis strain collection.

    PubMed Central

    Cerón, J; Ortíz, A; Quintero, R; Güereca, L; Bravo, A

    1995-01-01

    In this paper we describe a PCR strategy that can be used to rapidly identify Bacillus thuringiensis strains that harbor any of the known cryI or cryIII genes. Four general PCR primers which amplify DNA fragments from the known cryI or cryIII genes were selected from conserved regions. Once a strain was identified as an organism that contains a particular type of cry gene, it could be easily characterized by performing additional PCR with specific cryI and cryIII primers selected from variable regions. The method described in this paper can be used to identify the 10 different cryI genes and the five different cryIII genes. One feature of this screening method is that each cry gene is expected to produce a PCR product having a precise molecular weight. The genes which produce PCR products having different sizes probably represent strains that harbor a potentially novel cry gene. Finally, we present evidence that novel crystal genes can be identified by the method described in this paper. PMID:8526493

  13. Isolation of a Pseudomonas solanacearum-specific DNA probe by subtraction hybridization and construction of species-specific oligonucleotide primers for sensitive detection by the polymerase chain reaction.

    PubMed Central

    Seal, S E; Jackson, L A; Daniels, M J

    1992-01-01

    A subtraction hybridization technique was employed to make a library enriched for Pseudomonas solanacearum-specific sequences. One cloned fragment, PS2096, hybridized under stringent conditions to DNA of 82 P. solanacearum strains representing all subgroups of the species. Other plant-associated bacteria, including closely related species such as Pseudomonas capacia, Pseudomonas picketti, or Pseudomonas syzygii, did not hybridize to PS2096. A minimum number of between 4 x 10(5) and 4 x 10(6) P. solanacearum cells could routinely be detected with PS2096 labelled either with [32P]dCTP or with digoxigenin-11-dUTP. To improve the sensitivity of detection, PS2096 was sequenced to allow the construction of specific oligonucleotide primers to be used for polymerase chain reaction (PCR) amplification. After 50 cycles of amplification, 5 to 116 cells, depending on the strain, could reproducibly be detected by visualization of a 148-bp PCR product on an agarose gel. A preliminary field trial in Burundi with the probe and PCR primers has confirmed that they are sensitive tools for specifically detecting low-level infections of P. solanacearum in potato tubers. Images PMID:1482193

  14. Determination of DQB1 alleles using PCR amplification and allele-specific primers.

    PubMed

    Lepage, V; Ivanova, R; Loste, M N; Mallet, C; Douay, C; Naoumova, E; Charron, D

    1995-10-01

    Molecular genotyping of HLA class II genes is commonly carried out using polymerase chain reaction (PCR) in combination with sequence-specific oligotyping (PCR-SSO) or a combination of the PCR and restriction fragment length polymorphism methods (PCR-RFLP). However, the identification of the DQB1 type by PCR-SSO and PCR-RFLP is very time-consuming which is disadvantageous for the typing of cadaveric organ donors. We have developed a DQB1 typing method using PCR in combination with allele-specific amplification (PCR-ASA), which allows the identification of the 17 most frequent alleles in one step using seven amplification mixtures. PCR allele-specific amplification HLA-DQB1 typing is easy to perform, and the results are easy to interpret in routine clinical practice. The PCR-ASA method is therefore better suited to DQB1 typing for organ transplantation than other methods.

  15. Whole blood Nested PCR and Real-time PCR amplification of Talaromyces marneffei specific DNA for diagnosis.

    PubMed

    Lu, Sha; Li, Xiqing; Calderone, Richard; Zhang, Jing; Ma, Jianchi; Cai, Wenying; Xi, Liyan

    2016-02-01

    Talaromyces marneffei is a dimorphic pathogenic fungus, which is a life-threatening invasive mycosis in the immunocompromised host. Prompt diagnosis of T. marneffei infection remains difficult although there has been progress in attempts to expedite the diagnosis of this infection. We previously demonstrated the value of nested polymerase chain reaction (PCR) to detect T. marneffei in paraffin embedded tissue samples with high sensitivity and specificity. In this study, this assay was used to detect the DNA of T. marneffei in whole blood samples. Real-time PCR assay was also evaluated to identify T. marneffei in the same samples. Twenty out of 30 whole blood samples (67%) collected from 23 patients were found positive by using the nested PCR assay, while 23/30 (77%) samples were found positive by using the real-time PCR assay. In order to express accurately the fungal loads, we used a normalized linearized plasmid as an internal control for real-time PCR. The assay results were correlated as the initial quantity (copies/μl) with fungal burden. These data indicate that combination of nested PCR and real-time PCR assay provides an attractive alternative for identification of T. marneffei DNA in whole blood samples of HIV-infected patients.

  16. Characterization of the inhabitancy of mouse intestinal bacteria (MIB) in rodents and humans by real-time PCR with group-specific primers.

    PubMed

    Kibe, Ryoko; Sakamoto, Mitsuo; Yokota, Hiroshi; Benno, Yoshimi

    2007-01-01

    Mouse intestinal bacteria (MIB) is a new operational taxonomic unit (OTU) belonging to the Bacteroides subgroup in the Cytophaga-Flavobacter-Bacteroides (CFB) phylum recently found in the intestine of mice, rats and humans. However, their characters are still unknown since they have not yet been isolated by culture. To understand their habitat characteristics in intestinal tracts, the quantification assays of MIB were established using MIB group-specific primers. The MIB population in the intestine was evaluated as a percentage of the number of 16S rRNA gene copy of MIB. A real-time PCR assay using group specific primers showed the fluctuation of MIB inhabitancy and revealed that the MIB population in the small intestine of mice was significantly lower than the large intestinal contents. Moreover, MIB was found in human feces though the number was lower than in murine. This assay using group-specific primers revealed new information about host-preference of MIB. PMID:17446674

  17. Detection of Theileria orientalis in Iran by semi-nested PCR.

    PubMed

    Ghaemi, Peyman; Hoghooghi-Rad, Nasser; Shayan, Parviz; Eckert, Brigitte

    2012-02-01

    In order to identify and differentiate Theileria orientalis in cattle which may be infected with Theileria annulata simultaneously, a semi-nested PCR was performed. Thus, 160 blood samples were collected from apparently healthy native cattle in Golestan province of northern Iran, during 2009 to 2011. The Tbs-S/Tbs-A primer set derived from the 18S rRNA encoding gene was used for first PCR amplification, and the amplified sequence weight by this primer set for Theileria sp. was 426-430 bp. Then, DNA solution from purified PCR product was used for the semi-nested PCR analysis. The first PCR product amplified using T. orientalis primer set (To-S/Tbs-A) derived from the 18SrRNA encoding gene, and this specific primer weight was 235 bp. Also, the first PCR product amplified using T. annulata primer set (Ta-S/Tbs-A) derived from the 18SrRNA encoding gene and this specific primer weight was 193 bp. Having extracted DNA of each sample, using Tbs-S/Tbs-A primer set for PCR and analyzing the PCR products on the 2% agarose gel electrophoresis, 13 out of 160 blood samples (8.12%) were positive for Theileria sp. Meanwhile, performing semi-nested PCR with T. orientalis-specific primers, 9 out of 13 blood samples (5.62%) were positive and performing semi-nested PCR with T. annulata-specific primers, 12 out of 13 blood samples (7.5%) were also positive. This molecular assay approves the presence of T. orientalis in the native cattle of northern parts of Iran for the first time. In addition, this procedure will detect the concurrent infection of T. orientalis and T. annulata in the cattle too.

  18. Kinetic characterisation of primer mismatches in allele-specific PCR: a quantitative assessment.

    PubMed

    Waterfall, Christy M; Eisenthal, Robert; Cobb, Benjamin D

    2002-12-20

    A novel method of estimating the kinetic parameters of Taq DNA polymerase during rapid cycle PCR is presented. A model was constructed using a simplified sigmoid function to represent substrate accumulation during PCR in combination with the general equation describing high substrate inhibition for Michaelis-Menten enzymes. The PCR progress curve was viewed as a series of independent reactions where initial rates were accurately measured for each cycle. Kinetic parameters were obtained for allele-specific PCR (AS-PCR) amplification to examine the effect of mismatches on amplification. A high degree of correlation was obtained providing evidence of substrate inhibition as a major cause of the plateau phase that occurs in the later cycles of PCR. PMID:12470637

  19. Kinetic characterisation of primer mismatches in allele-specific PCR: a quantitative assessment.

    PubMed

    Waterfall, Christy M; Eisenthal, Robert; Cobb, Benjamin D

    2002-12-20

    A novel method of estimating the kinetic parameters of Taq DNA polymerase during rapid cycle PCR is presented. A model was constructed using a simplified sigmoid function to represent substrate accumulation during PCR in combination with the general equation describing high substrate inhibition for Michaelis-Menten enzymes. The PCR progress curve was viewed as a series of independent reactions where initial rates were accurately measured for each cycle. Kinetic parameters were obtained for allele-specific PCR (AS-PCR) amplification to examine the effect of mismatches on amplification. A high degree of correlation was obtained providing evidence of substrate inhibition as a major cause of the plateau phase that occurs in the later cycles of PCR.

  20. Improved Selection of Internal Transcribed Spacer-Specific Primers Enables Quantitative, Ultra-High-Throughput Profiling of Fungal Communities

    PubMed Central

    Bokulich, Nicholas A.

    2013-01-01

    Ultra-high-throughput sequencing (HTS) of fungal communities has been restricted by short read lengths and primer amplification bias, slowing the adoption of newer sequencing technologies to fungal community profiling. To address these issues, we evaluated the performance of several common internal transcribed spacer (ITS) primers and designed a novel primer set and work flow for simultaneous quantification and species-level interrogation of fungal consortia. Primer comparison and validation were predicted in silico and by sequencing a “mock community” of mixed yeast species to explore the challenges of amplicon length and amplification bias for reconstructing defined yeast community structures. The amplicon size and distribution of this primer set are smaller than for all preexisting ITS primer sets, maximizing sequencing coverage of hypervariable ITS domains by very-short-amplicon, high-throughput sequencing platforms. This feature also enables the optional integration of quantitative PCR (qPCR) directly into the HTS preparatory work flow by substituting qPCR with these primers for standard PCR, yielding quantification of individual community members. The complete work flow described here, utilizing any of the qualified primer sets evaluated, can rapidly profile mixed fungal communities and capably reconstructed well-characterized beer and wine fermentation fungal communities. PMID:23377949

  1. New Design Strategy for Development of Specific Primer Sets for PCR-Based Detection of Chlorophyceae and Bacillariophyceae in Environmental Samples▿ †

    PubMed Central

    Valiente Moro, Claire; Crouzet, Olivier; Rasconi, Séréna; Thouvenot, Antoine; Coffe, Gérard; Batisson, Isabelle; Bohatier, Jacques

    2009-01-01

    Studying aquatic microalgae is essential for monitoring biodiversity and water quality. We designed new sets of 18S rRNA PCR primers for Chlorophyceae and Bacillariophyceae by using the ARB software and implementing a virtual PCR program. The results of specificity analysis showed that most of the targeted algal families were identified and nontargeted organisms, such as fungi or ciliates, were excluded. These newly developed PCR primer sets were also able to amplify microalgal rRNA genes from environmental samples with accurate specificity. These tools could be of great interest for studying freshwater microalgal ecology and for developing bioindicators of the health status of aquatic environments. PMID:19592531

  2. New design strategy for development of specific primer sets for PCR-based detection of Chlorophyceae and Bacillariophyceae in environmental samples.

    PubMed

    Moro, Claire Valiente; Crouzet, Olivier; Rasconi, Séréna; Thouvenot, Antoine; Coffe, Gérard; Batisson, Isabelle; Bohatier, Jacques

    2009-09-01

    Studying aquatic microalgae is essential for monitoring biodiversity and water quality. We designed new sets of 18S rRNA PCR primers for Chlorophyceae and Bacillariophyceae by using the ARB software and implementing a virtual PCR program. The results of specificity analysis showed that most of the targeted algal families were identified and nontargeted organisms, such as fungi or ciliates, were excluded. These newly developed PCR primer sets were also able to amplify microalgal rRNA genes from environmental samples with accurate specificity. These tools could be of great interest for studying freshwater microalgal ecology and for developing bioindicators of the health status of aquatic environments. PMID:19592531

  3. Evaluation of strain-specific primers for identification of Lactobacillus rhamnosus GG.

    PubMed

    Endo, Akihito; Aakko, Juhani; Salminen, Seppo

    2012-12-01

    Lactobacillus rhamnosus strain GG (ATCC 53103) is one of the most widely studied and commercialized probiotic strains, and thus strain-specific identification for the strain is highly valuable. In this study, two published PCR-based identification methods for strain GG, a transposase gene-targeting system and a phage-related gene-targeting system, were evaluated. The former produced amplicons from eight of the 41 strains tested and the phage-related system from five of the tested strains, including the strain GG. Fingerprinting analysis indicated that the strains LMG 18025, LMG 18030, and LMG 18038, which had an amplicon by the former system but none by the latter, were genetically distinguishable from L. rhamnosus GG at strain level. Strains LMG 23320, LMG 23325, LMG 23534, and LMG 25859 showed profiles very similar to that of the strain GG, suggesting that these strains might be identical to GG or derivative strains of it. The results here indicated that the phage-related gene-targeting system is a good tool for accurate identification of L. rhamnosus GG. This system would be able to detect both the original L. rhamnosus GG and its derivative strains.

  4. Exploring the diversity of Acinetobacter populations in river water with genus-specific primers and probes.

    PubMed

    Xin, Fangfang; Cai, Dijie; Sun, Yuhua; Guo, Dalei; Wu, Zirong; Jiang, Deming

    2014-01-01

    This study aimed to explore the diversity of river water Acinetobacter populations using culture-dependent and -independent methods. Pyrosequencing indicated that 1.5% of the total sequences from Qiandeng River water were classified as Acinetobacter. Twelve Acinetobacter strains were isolated from three different sampling sites of the Qiandeng River. Based on culture-dependent methods, A. johnsonii, A. lwoffii and A. guillouiae were the most abundantly represented Acinetobacter strains among the upper, middle and downstream populations of the river. Probing of three Acinetobacter-enriched 16S rRNA gene libraries with the Acinetobacter specific probe Act660F revealed 42 unique 16S rRNA gene sequences exhibiting a similarity of 94.9-99.9% with the known Acinetobacter strains. Among the uncultured Acinetobacter sequences, 50%, 58.3% and 68.8% of those obtained from upstream sampling site A, middle stream sampling site B and downstream sampling site C were phylogenetically located within Group I. This Group represented the most abundant strains of Acinetobacter populations in river water based on culture-independent methods. The results indicated that culture-independent methods provide more detailed information on the diversity of Acinetobacter populations than that based on culture-dependent methods. Therefore, the development of new and efficient isolation methods to identify uncultured Acinetobacter species is required.

  5. Inflammatory Bowel Disease Cause-specific Mortality: A Primer for Clinicians

    PubMed Central

    Kassam, Zain; Belga, Sara; Roifman, Idan; Hirota, Simon; Jijon, Humberto; Kaplan, Gilaad G.; Ghosh, Subrata

    2014-01-01

    Background: Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC) is perceived to harbor significant morbidity but limited excess mortality, thought to be driven by colon cancer, compared with the general population. Recent studies suggest mortality rates seem higher than previously understood, and there are emerging threats to mortality. Clinicians must be up to date and able to clearly convey the causes of mortality to arm individual patients with information to meaningfully participate in decisions regarding IBD treatment and maintenance of health. Methods: A MEDLINE search was conducted to capture all relevant articles. Keyword search included: “inflammatory bowel disease,” “Crohn's disease,” “ulcerative colitis,” and “mortality.” Results: CD and UC have slightly different causes of mortality; however, malignancy and colorectal cancer–associated mortality remains controversial in IBD. CD mortality seems to be driven by gastrointestinal disease, infection, and respiratory diseases. UC mortality was primarily attributable to gastrointestinal disease and infection. Clostridium difficile infection is an emerging cause of mortality in IBD. UC and CD patients have a marked increase in risk of thromboembolic disease. With advances in medical and surgical interventions, the exploration of treatment-associated mortality must continue to be evaluated. Conclusions: Clinicians should be aware that conventional causes of death such as malignancy do not seem to be as significant a burden as originally perceived. However, emerging threats such as infection including C. difficile are noteworthy. Although CD and UC share similar causes of death, there seems to be some differences in cause-specific mortality. PMID:25185685

  6. Formation and characterization of precise eucaryotic transcription complexes using a semisynthetic DNA template and specific oligoribonucleotide primers.

    PubMed

    Mougey, E B; Dennis, D

    1988-06-01

    An artificial template of defined sequence which supports specific in vitro initiation and elongation by yeast RNA polymerase II has been constructed. This template is a pBR322 derivative which contains a synthetic oligonucleotide inserted into the BamHI cloning site. The sequence of this oligonucleotide is such that when the plasmid is restricted with SacI the two ends obtained are identical. The addition of an oligodeoxycytidylate chain to the 3' hydroxy termini produces a DNA template, (poly dC-p+22), with the sequence: 3'(C)nTCGA-GAGTCTCCTA. . . . The underlined position denotes the beginning of the duplex region. When initiation is primed with the diribonucleotide GpC the predicted sequence of the transcript obtained is: 5'GCUCUCAGAGGAU. . . . Kinetic and product analyses indicate that a ternary complex containing a precise length of transcript can be produced which is subsequently resistant to heparin inactivation. Initiation can also be directed to a specific position dictated by a tri or tetraribonucleotide primer.

  7. Specific amplification of bacterial DNA by optimized so-called universal bacterial primers in samples rich of plant DNA.

    PubMed

    Dorn-In, Samart; Bassitta, Rupert; Schwaiger, Karin; Bauer, Johann; Hölzel, Christina S

    2015-06-01

    Universal primers targeting the bacterial 16S-rRNA-gene allow quantification of the total bacterial load in variable sample types by qPCR. However, many universal primer pairs also amplify DNA of plants or even of archaea and other eukaryotic cells. By using these primers, the total bacterial load might be misevaluated, whenever samples contain high amounts of non-target DNA. Thus, this study aimed to provide primer pairs which are suitable for quantification and identification of bacterial DNA in samples such as feed, spices and sample material from digesters. For 42 primers, mismatches to the sequence of chloroplasts and mitochondria of plants were evaluated. Six primer pairs were further analyzed with regard to the question whether they anneal to DNA of archaea, animal tissue and fungi. Subsequently they were tested with sample matrix such as plants, feed, feces, soil and environmental samples. To this purpose, the target DNA in the samples was quantified by qPCR. The PCR products of plant and feed samples were further processed for the Single Strand Conformation Polymorphism method followed by sequence analysis. The sequencing results revealed that primer pair 335F/769R amplified only bacterial DNA in samples such as plants and animal feed, in which the DNA of plants prevailed.

  8. Designing Polymerase Chain Reaction Primers Using Primer3Plus.

    PubMed

    Hung, Jui-Hung; Weng, Zhiping

    2016-01-01

    Designing oligonucleotide primers is a crucial step for successful molecular biology experiments that require the use of the polymerase chain reaction (PCR). PCR involves cycles of three steps: denaturation, annealing, and extension. During denaturation, double-stranded DNA (dsDNA) molecules (templates) are separated into single strands. During annealing, a pair of primers is annealed to the complementary regions of the single-stranded molecules. In the extension step, DNA polymerase extends the primers to produce DNA molecules that correspond to the region bracketed by the primers (the amplicons). All of these steps are temperature sensitive, and the common choice of temperatures is 94°C, 60°C, and 70°C, respectively. Poorly designed primers may lead to no amplification product or additional undesired amplified fragments. The goals of primer design include good primer specificity, high annealing efficiency, appropriate melting temperature, proper GC content, and the prevention of primer hairpins or primer dimers. PMID:27574202

  9. Nested PCR method for rapid and sensitive detection of Vibrio vulnificus in fish, sediments, and water.

    PubMed Central

    Arias, C R; Garay, E; Aznar, R

    1995-01-01

    A nested PCR for the detection of Vibrio vulnificus in fish farms was developed as an alternative to cultural methods by using universal primers flanking the V. vulnificus-specific sequences directed against 23S rRNA genes. This specific assay detected 10 fg of DNA or 12 to 120 cells in artificially inoculated samples without enrichment and within 24 h. PMID:7574657

  10. Improved DNA barcoding method for Bemisia tabaci and related Aleyrodidae: development of universal and Bemisia tabaci biotype-specific mitochondrial cytochrome c oxidase I polymerase chain reaction primers.

    PubMed

    Shatters, Robert G; Powell, Charles A; Boykin, Laura M; Liansheng, He; McKenzie, C L

    2009-04-01

    Whiteflies, heteropterans in the family Aleyrodidae, are globally distributed and severe agricultural pests. The mitochondrial cytochrome c oxidase I (mtCOI) sequence has been used extensively in whitefly phylogenetic comparisons and in biotype identification of the agriculturally important Bemisia tabaci (Gennadius) whitefly. Because of the economic importance of several whitefly genera, and the invasive nature of the B and the Q biotypes of Bemisia tabaci, mtCOI sequence data are continually generated from sampled populations worldwide. Routine phylogenetic comparisons and biotype identification is done through amplification and sequencing of an approximately 800-bp mtCOI DNA fragment. Despite its routine use, published primers for amplification of this region are often inefficient for some B. tabaci biotypes and especially across whitefly species. Through new sequence generation and comparison to available whitefly mtCOI sequence data, a set of polymerase chain reaction (PCR) amplification primers (Btab-Uni primers) were identified that are more efficient at amplifying approximately 748 bp of the approximately 800-bp fragment currently used. These universal primers amplify an mtCOI fragment from numerous B. tabaci biotypes and whitefly genera by using a single amplification profile. Furthermore, mtCOI PCR primers specific for the B, Q, and New World biotypes of B. tabaci were designed that allow rapid discrimination among these biotypes. These primers produce a 478-, 405-, and 303-bp mtCOI fragment for the B, New World, and Q biotypes, respectively. By combining these primers and using rapid PCR and electrophoretic techniques, biotype determination can be made within 3 h for up to 96 samples at a time.

  11. Protocol: a simple gel-free method for SNP genotyping using allele-specific primers in rice and other plant species

    PubMed Central

    2010-01-01

    Background Genotype analysis using multiple single nucleotide polymorphisms (SNPs) is a useful but labor-intensive or high-cost procedure in plant research. Here we describe an alternative genotyping method that is suited to multi-sample or multi-locus SNP genotyping and does not require electrophoresis or specialized equipment. Results We have developed a simple method for multi-sample or multi-locus SNP genotyping using allele-specific primers (ASP). More specifically, we (1) improved the design of allele-specific primers, (2) established a method to detect PCR products optically without electrophoresis, and (3) standardized PCR conditions for parallel genomic assay using various allele-specific primers. As an illustration of multi-sample SNP genotyping using ASP, we mapped the locus for lodging resistance in a typhoon (lrt5). Additionally, we successfully tested multi-locus ASP-PCR analysis using 96 SNPs located throughout the genomes of rice (Oryza sativa) cultivars 'Koshihikari' and 'Kasalath', and demonstrated its applicability to other diverse cultivars/subspecies, including wild rice (O. rufipogon). Conclusion Our ASP methodology allows characterization of SNPs genotypes without electrophoresis, expensive probes or specialized equipment, and is highly versatile due to the flexibility in the design of primers. The method could be established easily in any molecular biology laboratory, and is applicable to diverse organisms. PMID:20409329

  12. Isolation and cloning of the raccoon (Procyon lotor) papillomavirus type 1 by using degenerate papillomavirus-specific primers.

    PubMed

    Rector, Annabel; Van Doorslaer, Koenraad; Bertelsen, Mads; Barker, Ian K; Olberg, Rolf-Arne; Lemey, Philippe; Sundberg, John P; Van Ranst, Marc

    2005-07-01

    Partial sequences of a novel papillomavirus were amplified from a cutaneous lesion biopsy of a raccoon (Procyon lotor), by using PCR with degenerate papillomavirus-specific primers. The Procyon lotor papillomavirus type 1 (PlPV-1) DNA was amplified with long template PCR in two overlapping fragments, together encompassing the entire genome, and the complete PlPV-1 genomic sequence was determined. The PlPV-1 genome consists of 8170 bp, and contains the typical papillomaviral open reading frames, encoding five early proteins and two late capsid proteins. Besides the classical non-coding region (NCR1) between the end of L1 and the start of E6, PlPV-1 contains an additional non-coding region (NCR2) of 1065 bp between the early and late protein region, which has previously also been described for the canine oral papillomavirus (COPV) and the Felis domesticus papillomavirus (FdPV-1). Phylogenetic analysis places PlPV-1 together with COPV and FdPV-1 in a monophyletic branch which encompasses the Lambda papillomavirus genus. PMID:15958682

  13. Translation of Two Nested Genes in Bacteriophage P4 Controls Immunity-Specific Transcription Termination

    PubMed Central

    Forti, Francesca; Polo, Simona; Lane, Kirk B.; Six, Erich W.; Sironi, Gianpiero; Dehò, Gianni; Ghisotti, Daniela

    1999-01-01

    In phage P4, transcription of the left operon may occur from both the constitutive PLE promoter and the regulated PLL promoter, about 400 nucleotides upstream of PLE. A strong Rho-dependent termination site, timm, is located downstream of both promoters. When P4 immunity is expressed, transcription starting at PLE is efficiently terminated at timm, whereas transcription from PLL is immunity insensitive and reads through timm. We report the identification of two nested genes, kil and eta, located in the P4 left operon. The P4 kil gene, which encodes a 65-amino-acid polypeptide, is the first translated gene downstream of the PLE promoter, and its expression is controlled by P4 immunity. Overexpression of kil causes cell killing. This gene is the terminal part of a longer open reading frame, eta, which begins upstream of PLE. The eta gene is expressed when transcription starts from the PLL promoter. Three likely start codons predict a size between 197 and 199 amino acids for the Eta gene product. Both kil and eta overlap the timm site. By cloning kil upstream of a tRNA reporter gene, we demonstrated that translation of the kil region prevents premature transcription termination at timm. This suggests that P4 immunity might negatively control kil translation, thus enabling transcription termination at timm. Transcription starting from PLL proceeds through timm. Mutations that create nonsense codons in eta caused premature termination of transcription starting from PLL. Suppression of the nonsense mutation restored transcription readthrough at timm. Thus, termination of transcription from PLL is prevented by translation of eta. PMID:10464191

  14. Target-specific PCR primers can detect and differentiate ophiostomatoid fungi from microbial communities associated with the mountain pine beetle Dendroctonus ponderosae.

    PubMed

    Khadempour, Lily; Massoumi Alamouti, Sepideh; Hamelin, Richard; Bohlmann, Jörg; Breuil, Colette

    2010-10-01

    The aim of this study was to develop DNA probes that could identify the major fungal species associated with mountain pine beetles (MPB). The beetles are closely associated with fungal species that include ophiostomatoid fungi that can be difficult to differentiate morphologically. The most frequently isolated associates are the pine pathogens Grosmannia clavigera and Leptographium longiclavatum, the less pathogenic Ophiostoma montium, and an undescribed Ceratocystiopsis species (Cop. sp.). Because growing, isolating and extracting DNA from fungi vectored by MPB can be time and labour intensive, we designed three rDNA primer sets that specifically amplify short rDNA amplicons from O. montium, Cop. sp. and the pine Leptographium clade. We also designed two primer sets on a gene of unknown function that can differentiate G. clavigera and L. longiclavatum. We tested the primers on 76 fungal isolates that included MPB associates. The primers reliably identified their targets from DNA obtained from pure fungal cultures, pulverized beetles, beetle galleries, and tree phloem inoculated with G. clavigera. The primers will facilitate large-scale work on the ecology of the MPB-fungal-lodgepole pine ecosystem, as well as phytosanitary/quarantine sample screening.

  15. Sensitive and specific detection of miRNA using an isothermal exponential amplification method using fluorescence-labeled LNA/DNA chimera primers.

    PubMed

    Huang, Jun-Fu; Zhao, Na; Xu, Han-Qing; Xia, Han; Wei, Kun; Fu, Wei-Ling; Huang, Qing

    2016-10-01

    MicroRNAs (miRNAs) are currently considered as potential biomarkers for various human diseases. In the present study, miRNA-triggered real-time fluorescent isothermal reaction with exponential amplification (ReFIRE) with or without Thermus aquaticus MutS (Taq MutS) was developed to analyze miRNAs using DNA polymerase, a nicking endonuclease, and fluorescently labeled primers. In the absence of Taq MutS, the ReFIRE system permitted the detection of 100 ymol of targeted miRNA in 80 min. However, this system enabled limited differentiation between homologous miRNA family members. Upon addition of Taq MutS to the ReFIRE system, non-specific amplification generated from the mishybridization between primers and primer dimers or primers and the template duplex was eliminated. The addition of Taq MutS enabled the ultrasensitive detection of as little as 10 ymol of targeted miRNAs in 50 min, which corresponds to less than 10 copies of miRNAs in a total volume of 20 μl. Additionally, the assay exhibited a dynamic range of up to 12 orders of magnitude. The ReFIRE system also showed high specificity, enabling differentiation between homologous miRNA family members exhibiting only single-base differences. The sensitivity, specificity, and dynamic range associated with this system were greater than most currently available miRNA isothermal amplification assays. Moreover, when target-specific primers were labeled with different fluorescent reporters, multiplex analysis was easily performed in a single tube, permitting accurate normalization of miRNA expression. This simple, fast, ultrasensitive, highly specific, and easy-to-multiplex method could significantly contribute to research investigations pertaining to the biological roles of miRNA, as well as clinical diagnosis of various diseases that involve miRNA disruptions. Graphical Abstract The principle of ReFIRE system.

  16. Highly specific and efficient primers for in-house multiplex PCR detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum

    PubMed Central

    2014-01-01

    Background Although sophisticated methodologies are available, the use of endpoint polymerase chain reaction (PCR) to detect 16S rDNA genes remains a good approach for estimating the incidence and prevalence of specific infections and for monitoring infections. Considering the importance of the early diagnosis of sexually transmitted infections (STIs), the development of a sensitive and affordable method for identifying pathogens in clinical samples is needed. Highly specific and efficient primers for a multiplex polymerase chain reaction (m-PCR) system were designed in silico to detect the 16S rDNA genes of four bacteria that cause genital infections, and the PCR method was developed. Methods The Genosensor Probe Designer (GPD) (version 1.0a) software was initially used to design highly specific and efficient primers for in-house m-PCR. Single-locus PCR reactions were performed and standardised, and then primers for each locus in turn were added individually in subsequent amplifications until m-PCR was achieved. Amplicons of the expected size were obtained from each of the four bacterial gene fragments. Finally, the analytical specificity and limits of detection were tested. Results Because they did not amplify any product from non-STI tested species, the primers were specific. The detection limits for the Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum primer sets were 5.12 × 105, 3.9 × 103, 61.19 × 106 and 6.37 × 105 copies of a DNA template, respectively. Conclusions The methodology designed and standardised here could be applied satisfactorily for the simultaneous or individual detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum. This method is at least as efficient as other previously described methods; however, this method is more affordable for low-income countries. PMID:24997675

  17. Sensitive and specific detection of miRNA using an isothermal exponential amplification method using fluorescence-labeled LNA/DNA chimera primers.

    PubMed

    Huang, Jun-Fu; Zhao, Na; Xu, Han-Qing; Xia, Han; Wei, Kun; Fu, Wei-Ling; Huang, Qing

    2016-10-01

    MicroRNAs (miRNAs) are currently considered as potential biomarkers for various human diseases. In the present study, miRNA-triggered real-time fluorescent isothermal reaction with exponential amplification (ReFIRE) with or without Thermus aquaticus MutS (Taq MutS) was developed to analyze miRNAs using DNA polymerase, a nicking endonuclease, and fluorescently labeled primers. In the absence of Taq MutS, the ReFIRE system permitted the detection of 100 ymol of targeted miRNA in 80 min. However, this system enabled limited differentiation between homologous miRNA family members. Upon addition of Taq MutS to the ReFIRE system, non-specific amplification generated from the mishybridization between primers and primer dimers or primers and the template duplex was eliminated. The addition of Taq MutS enabled the ultrasensitive detection of as little as 10 ymol of targeted miRNAs in 50 min, which corresponds to less than 10 copies of miRNAs in a total volume of 20 μl. Additionally, the assay exhibited a dynamic range of up to 12 orders of magnitude. The ReFIRE system also showed high specificity, enabling differentiation between homologous miRNA family members exhibiting only single-base differences. The sensitivity, specificity, and dynamic range associated with this system were greater than most currently available miRNA isothermal amplification assays. Moreover, when target-specific primers were labeled with different fluorescent reporters, multiplex analysis was easily performed in a single tube, permitting accurate normalization of miRNA expression. This simple, fast, ultrasensitive, highly specific, and easy-to-multiplex method could significantly contribute to research investigations pertaining to the biological roles of miRNA, as well as clinical diagnosis of various diseases that involve miRNA disruptions. Graphical Abstract The principle of ReFIRE system. PMID:27485624

  18. Enhanced specificity of TPMT*2 genotyping using unidirectional wild-type and mutant allele-specific scorpion primers in a single tube.

    PubMed

    Chen, Dong; Yang, Zhao; Xia, Han; Huang, Jun-Fu; Zhang, Yang; Jiang, Tian-Nun; Wang, Gui-Yu; Chuai, Zheng-Ran; Fu, Wei-Ling; Huang, Qing

    2014-01-01

    Genotyping of thiopurine S-methyltransferase (TPMT) is recommended for predicting the adverse drug response of thiopurines. In the current study, a novel version of allele-specific PCR (AS-PCR), termed competitive real-time fluorescent AS-PCR (CRAS-PCR) was developed to analyze the TPMT*2 genotype in ethnic Chinese. This technique simultaneously uses wild-type and mutant allele-specific scorpion primers in a single reaction. To determine the optimal conditions for both traditional AS-PCR and CRAS-PCR, we used the Taguchi method, an engineering optimization process that balances the concentrations of all components using an orthogonal array rather than a factorial array. Instead of running up to 264 experiments with the conventional factorial method, the Taguchi method achieved the same optimization using only 16 experiments. The optimized CRAS-PCR system completely avoided non-specific amplification occurring in traditional AS-PCR and could be performed at much more relaxed reaction conditions at 1% sensitivity, similar to traditional AS-PCR. TPMT*2 genotyping of 240 clinical samples was consistent with published data. In conclusion, CRAS-PCR is a novel and robust genotyping method, and the Taguchi method is an effective tool for the optimization of molecular analysis techniques. PMID:24705376

  19. Enhanced Specificity of TPMT*2 Genotyping Using Unidirectional Wild-Type and Mutant Allele-Specific Scorpion Primers in a Single Tube

    PubMed Central

    Chen, Dong; Yang, Zhao; Xia, Han; Huang, Jun-Fu; Zhang, Yang; Jiang, Tian-Nun; Wang, Gui-Yu; Chuai, Zheng-Ran; Fu, Wei-Ling; Huang, Qing

    2014-01-01

    Genotyping of thiopurine S-methyltransferase (TPMT) is recommended for predicting the adverse drug response of thiopurines. In the current study, a novel version of allele-specific PCR (AS-PCR), termed competitive real-time fluorescent AS-PCR (CRAS-PCR) was developed to analyze the TPMT*2 genotype in ethnic Chinese. This technique simultaneously uses wild-type and mutant allele-specific scorpion primers in a single reaction. To determine the optimal conditions for both traditional AS-PCR and CRAS-PCR, we used the Taguchi method, an engineering optimization process that balances the concentrations of all components using an orthogonal array rather than a factorial array. Instead of running up to 264 experiments with the conventional factorial method, the Taguchi method achieved the same optimization using only 16 experiments. The optimized CRAS-PCR system completely avoided non-specific amplification occurring in traditional AS-PCR and could be performed at much more relaxed reaction conditions at 1% sensitivity, similar to traditional AS-PCR. TPMT*2 genotyping of 240 clinical samples was consistent with published data. In conclusion, CRAS-PCR is a novel and robust genotyping method, and the Taguchi method is an effective tool for the optimization of molecular analysis techniques. PMID:24705376

  20. BatchPrimer3: A high throughput web application for PCR and sequencing primer design

    PubMed Central

    You, Frank M; Huo, Naxin; Gu, Yong Qiang; Luo, Ming-cheng; Ma, Yaqin; Hane, Dave; Lazo, Gerard R; Dvorak, Jan; Anderson, Olin D

    2008-01-01

    Background Microsatellite (simple sequence repeat – SSR) and single nucleotide polymorphism (SNP) markers are two types of important genetic markers useful in genetic mapping and genotyping. Often, large-scale genomic research projects require high-throughput computer-assisted primer design. Numerous such web-based or standard-alone programs for PCR primer design are available but vary in quality and functionality. In particular, most programs lack batch primer design capability. Such a high-throughput software tool for designing SSR flanking primers and SNP genotyping primers is increasingly demanded. Results A new web primer design program, BatchPrimer3, is developed based on Primer3. BatchPrimer3 adopted the Primer3 core program as a major primer design engine to choose the best primer pairs. A new score-based primer picking module is incorporated into BatchPrimer3 and used to pick position-restricted primers. BatchPrimer3 v1.0 implements several types of primer designs including generic primers, SSR primers together with SSR detection, and SNP genotyping primers (including single-base extension primers, allele-specific primers, and tetra-primers for tetra-primer ARMS PCR), as well as DNA sequencing primers. DNA sequences in FASTA format can be batch read into the program. The basic information of input sequences, as a reference of parameter setting of primer design, can be obtained by pre-analysis of sequences. The input sequences can be pre-processed and masked to exclude and/or include specific regions, or set targets for different primer design purposes as in Primer3Web and primer3Plus. A tab-delimited or Excel-formatted primer output also greatly facilitates the subsequent primer-ordering process. Thousands of primers, including wheat conserved intron-flanking primers, wheat genome-specific SNP genotyping primers, and Brachypodium SSR flanking primers in several genome projects have been designed using the program and validated in several laboratories

  1. Human Platelet Antigen Alleles in 998 Taiwanese Blood Donors Determined by Sequence-Specific Primer Polymerase Chain Reaction

    PubMed Central

    Burnouf, Thierry; Chen, Jen-Wei; Lin, Liang-In

    2013-01-01

    Polymorphism of human platelet antigens (HPAs) leads to alloimmunizations and immune-mediated platelet disorders including fetal-neonatal alloimmune thrombocytopenia (FNAIT), posttransfusion purpura (PTP), and platelet transfusion refractoriness (PTR). HPA typing and knowledge of antigen frequency in a population are important in particular for the provision of HPA-matched blood components for patients with PTR. We have performed allele genotyping for HPA-1 through -6 and -15 among 998 platelet donors from 6 blood centers in Taiwan using sequence-specific primer polymerase chain reaction. The HPA allele frequency was 99.55, and 0.45% for HPA-1a and -1b; 96.49, and 3.51% for HPA-2a and -2b; 55.81, and 44.19% for HPA-3a and -3b; 99.75, and 0.25% for HPA-4a and -4b; 98.50, and 1.50% for HPA-5a and -5b; 97.75 and 2.25% for HPA-6a and -6b; 53.71 and 46.29% for HPA-15a and -15b. HPA-15b and HPA-3a, may be considered the most important, followed by HPA-2, -6, -1, -5, and -4 systems, as a cause of FNAIT, PTP, and PTR based on allele frequency. HPA-4b and HPA-5b role cannot be excluded based on their immunogenicity. A larger-scale study will now be conducted to confirm these hypotheses and to establish an apheresis donor database for the procurement of HPA-matched apheresis platelets for patients with PTR. PMID:23865077

  2. Multiplex Polymerase Chain Reaction Identification of Single Individuals of the Longidorid Nematodes Xiphinema index, X. diversicaudatum, X. vuittenezi, and X. italiae Using Specific Primers from Ribosomal Genes.

    PubMed

    Wang, Xinrong; Bosselut, Nathalie; Castagnone, Chantal; Voisin, Roger; Abad, Pierre; Esmenjaud, Daniel

    2003-02-01

    ABSTRACT The species X. index, X. diversicaudatum, X. vuittenezi, and X. italiae are established (E) or putative (P) vectors of Grapevine fanleaf virus (GFLV) (E), Arabis mosaic virus (E), Grapevine chrome mosaic virus (P), and GFLV (P) nepoviruses of grapevine, respectively. All four species are very closely related taxonomically and their low field densities make them difficult to identify from morphological and morphometrical diagnostic characters when only single or few individuals are detected. To improve diagnostic accuracy, a simple method was developed. The internal transcribed spacer 1 (ITS1) region spanning the 18S and 5.8S ribosomal genes was sequenced in one population of each species using two conserved primers from these genes. The ITS1 fragments were 1,132 bp (X. vuittenezi), 1,153 bp (X. index), 1,175 bp (X. diversicaudatum), and 1,190 bp (X. italiae), i.e., a difference of over 5% between the extremes. The sequence variability made it possible to design species-specific internal sense primers that amplified, in combination with the same antisense ITS1 primer, a single signature fragment (340 bp for X. index, 414 bp for X. italiae, 591 bp for X. vuittenezi, and 813 bp for X. diversicaudatum). Tests with DNA from a single adult or juvenile nematode confirmed the specificity of the primers from diverse isolates or populations. The primers were successfully used in a multiplex test for the reliable detection of two to four mixed species, each represented by a single individual. This multiplex-based diagnostic tool will be particularly useful for successful nematode management practices in vineyards.

  3. Preliminary level 2 specification for the nested, fixed-depth sampling system

    SciTech Connect

    BOGER, R.M.

    1999-05-26

    This revision 1 Level 2 Specification establishes the performance, design, development, and test requirements for a sampling system and for an at-tank analysis system that will support the BNFL, Inc. privatization contract in the final disposal of Hanford's high level waste (HLW) and low activity waste (LAW). The sampling system will quickly provide large volume, representative waste samples for validating the chemical, radiological, and physical properties of the tank waste without the exposure and time concerns of the baseline grab sampling method. The on-line sensors of the at-tank analysis system will provide data from which the mixing or settling status of the waste can be assessed. This revision 1 document includes functions, requirement, and specifications for the at-tank analysis system, the results of the preliminary outline design, and the FY 1998 validation testing. The sample container filling system will comply with RCRA criteria for samples with volatile organic constituents, include empty container and swipe input ports, use Hanford's Steel Pig radioactive sample package, comply with Hanford's flammable gas criteria, and have the means to recover from broken sample containers.

  4. The establishment of species-specific primers for the molecular identification of ten stored-product psocids based on ITS2 rDNA

    PubMed Central

    Zhao, Zi-Hua; Cui, Bing-Yi; Li, Zhi-Hong; Jiang, Fan; Yang, Qian-Qian; Kučerová, Zuzana; Stejskal, Václav; Opit, George; Cao, Yang; Li, Fu-Jun

    2016-01-01

    Psocids are important stored product pests found worldwide that can be spread through grain trade. Most stored-product psocids, including eggs, nymphs, and adults, are very small (~1 mm) and difficult to identify morphologically. Here, we collected 10 economically important stored-product Liposcelis spp. psocids (L. bostrychophila, L. entomophila, L. decolor, L. paeta, L. brunnea, L. corrodens, L. mendax, L. rufa, L. pearmani, and L. tricolor) from 35 geographical locations in 5 countries (China, Czech Republic, Denmark, Germany, and the United States). The ITS2 rDNA gene was extracted and sequenced. The interspecific genetic distance of the stored-product psocids was significantly higher than the intraspecific genetic distance according to the barcoding gap analysis. Ten pairs of species-specific primers based on the ITS2 rDNA were developed for psocid identification. The sensitivity estimation indicated that the species-specific primers could correctly amplify the target ITS2 gene and successfully identify psocids at 1.0 ng/mL. Additionally, these species-specific primers could quantify specificity and identify 10 stored-product psocids; this approach could also be used to accurately identify other stored-product psocids. This work provides a practical approach for the precise examination of 10 stored-product psocid species and also contributes to the development of an identification method using ITS2 rDNA. PMID:26880378

  5. Population-Specific Covariation between Immune Function and Color of Nesting Male Threespine Stickleback

    PubMed Central

    Bolnick, Daniel I.; Shim, Kum Chuan; Schmerer, Matthew; Brock, Chad D.

    2015-01-01

    Multiple biological processes can generate sexual selection on male visual signals such as color. For example, females may prefer colorful males because those males are more readily detected (perceptual bias), or because male color conveys information about male quality and associated direct or indirect benefits to females. For example, male threespine stickleback often exhibit red throat coloration, which females prefer both because red is more visible in certain environments, and red color is correlated with male immune function and parasite load. However, not all light environments favor red nuptial coloration: more tannin-stained water tends to favor the evolution of a melanic male phenotype. Do such population differences in stickleback male color, driven by divergent light environments, lead to changes in the relationship between color and immunity? Here, we show that, within stickleback populations, multiple components of male color (brightness and hue of four body parts) are correlated with multiple immune variables (ROS production, phagocytosis rates, and lymphocyte:leukocyte ratios). Some of these color-immune associations persist across stickleback populations with very different male color patterns, whereas other color-immune associations are population-specific. Overall, lakes with red males exhibit stronger color-immune covariance while melanic male populations exhibit weak if any color-immune associations. Our finding that color-immunity relationships are labile implies that any evolution of male color traits (e.g., due to female perceptual bias in a given light environment), can alter the utility of color as an indicator of male quality. PMID:26039044

  6. Conditions during adulthood affect cohort-specific reproductive success in an Arctic-nesting goose population.

    PubMed

    Weegman, Mitch D; Bearhop, Stuart; Hilton, Geoff M; Walsh, Alyn; Fox, Anthony David

    2016-01-01

    Variation in fitness between individuals in populations may be attributed to differing environmental conditions experienced among birth (or hatch) years (i.e., between cohorts). In this study, we tested whether cohort fitness could also be explained by environmental conditions experienced in years post-hatch, using 736 lifelong resighting histories of Greenland white-fronted geese (Anser albifrons flavirostris) marked in their first winter. Specifically, we tested whether variation in age at first successful reproduction, the size of the first successful brood and the proportion of successful breeders by cohort was explained by environmental conditions experienced on breeding areas in west Greenland during hatch year, those in adulthood prior to successful reproduction and those in the year of successful reproduction, using North Atlantic Oscillation indices as proxies for environmental conditions during these periods. Fifty-nine (8%) of all marked birds reproduced successfully (i.e., were observed on wintering areas with young) only once in their lifetime and 15 (2%) reproduced successfully twice or thrice. Variation in age at first successful reproduction was explained by the environmental conditions experienced during adulthood in the years prior to successful reproduction. Birds bred earliest (mean age 4) when environmental conditions were 'good' prior to the year of successful reproduction. Conversely, birds successfully reproduced at older ages (mean age 7) if they experienced adverse conditions prior to the year of successful reproduction. Hatch year conditions and an interaction between those experienced prior to and during the year of successful reproduction explained less (marginally significant) variation in age at first successful reproduction. Environmental conditions did not explain variation in the size of the first successful brood or the proportion of successful breeders. These findings show that conditions during adulthood prior to the year of

  7. Conditions during adulthood affect cohort-specific reproductive success in an Arctic-nesting goose population

    PubMed Central

    Bearhop, Stuart; Hilton, Geoff M.; Walsh, Alyn; Fox, Anthony David

    2016-01-01

    Variation in fitness between individuals in populations may be attributed to differing environmental conditions experienced among birth (or hatch) years (i.e., between cohorts). In this study, we tested whether cohort fitness could also be explained by environmental conditions experienced in years post-hatch, using 736 lifelong resighting histories of Greenland white-fronted geese (Anser albifrons flavirostris) marked in their first winter. Specifically, we tested whether variation in age at first successful reproduction, the size of the first successful brood and the proportion of successful breeders by cohort was explained by environmental conditions experienced on breeding areas in west Greenland during hatch year, those in adulthood prior to successful reproduction and those in the year of successful reproduction, using North Atlantic Oscillation indices as proxies for environmental conditions during these periods. Fifty-nine (8%) of all marked birds reproduced successfully (i.e., were observed on wintering areas with young) only once in their lifetime and 15 (2%) reproduced successfully twice or thrice. Variation in age at first successful reproduction was explained by the environmental conditions experienced during adulthood in the years prior to successful reproduction. Birds bred earliest (mean age 4) when environmental conditions were ‘good’ prior to the year of successful reproduction. Conversely, birds successfully reproduced at older ages (mean age 7) if they experienced adverse conditions prior to the year of successful reproduction. Hatch year conditions and an interaction between those experienced prior to and during the year of successful reproduction explained less (marginally significant) variation in age at first successful reproduction. Environmental conditions did not explain variation in the size of the first successful brood or the proportion of successful breeders. These findings show that conditions during adulthood prior to the year of

  8. Conditions during adulthood affect cohort-specific reproductive success in an Arctic-nesting goose population.

    PubMed

    Weegman, Mitch D; Bearhop, Stuart; Hilton, Geoff M; Walsh, Alyn; Fox, Anthony David

    2016-01-01

    Variation in fitness between individuals in populations may be attributed to differing environmental conditions experienced among birth (or hatch) years (i.e., between cohorts). In this study, we tested whether cohort fitness could also be explained by environmental conditions experienced in years post-hatch, using 736 lifelong resighting histories of Greenland white-fronted geese (Anser albifrons flavirostris) marked in their first winter. Specifically, we tested whether variation in age at first successful reproduction, the size of the first successful brood and the proportion of successful breeders by cohort was explained by environmental conditions experienced on breeding areas in west Greenland during hatch year, those in adulthood prior to successful reproduction and those in the year of successful reproduction, using North Atlantic Oscillation indices as proxies for environmental conditions during these periods. Fifty-nine (8%) of all marked birds reproduced successfully (i.e., were observed on wintering areas with young) only once in their lifetime and 15 (2%) reproduced successfully twice or thrice. Variation in age at first successful reproduction was explained by the environmental conditions experienced during adulthood in the years prior to successful reproduction. Birds bred earliest (mean age 4) when environmental conditions were 'good' prior to the year of successful reproduction. Conversely, birds successfully reproduced at older ages (mean age 7) if they experienced adverse conditions prior to the year of successful reproduction. Hatch year conditions and an interaction between those experienced prior to and during the year of successful reproduction explained less (marginally significant) variation in age at first successful reproduction. Environmental conditions did not explain variation in the size of the first successful brood or the proportion of successful breeders. These findings show that conditions during adulthood prior to the year of

  9. Specific recognition of reproductive parasite workers by nest-entrance guards in the bumble bee Bombus terrestris

    PubMed Central

    2013-01-01

    Background The impact of social parasites on their hosts’ fitness is a strong selective pressure that can lead to the evolution of adapted defence strategies. Guarding the nest to prevent the intrusion of parasites is a widespread response of host species. If absolute rejection of strangers provides the best protection against parasites, more fine-tuned strategies can prove more adaptive. Guarding is indeed costly and not all strangers constitute a real threat. That is particularly true for worker reproductive parasitism in social insects since only a fraction of non-nestmate visitors, the fertile ones, can readily engage in parasitic reproduction. Guards should thus be more restrictive towards fertile than sterile non-nestmate workers. We here tested this hypothesis by examining the reaction of nest-entrance guards towards nestmate and non-nestmate workers with varying fertility levels in the bumble bee Bombus terrestris. Because social recognition in social insects mainly relies on cuticular lipids (CLs), chemical analysis was also conducted to examine whether workers’ CLs could convey the relevant information upon which guards could base their decision. We thus aimed to determine whether an adapted defensive strategy to worker reproductive parasitism has evolved in B. terrestris colonies. Results Chemical analysis revealed that the cuticular chemical profiles of workers encode information about both their colony membership and their current fertility, therefore providing potential recognition cues for a suitable adjustment of the guards’ defensive decisions. We found that guards were similarly tolerant towards sterile non-nestmate workers than towards nestmate workers. However, as predicted, guards responded more aggressively towards fertile non-nestmates. Conclusion Our results show that B. terrestris guards discriminate non-nestmates that differ in their reproductive potential and respond more strongly to the individuals that are a greatest threat for

  10. Nesting Instincts.

    ERIC Educational Resources Information Center

    Greenman, Geri

    2003-01-01

    Describes an art project where beginning drawing students used values and chiaroscuro techniques to draw bird nests. Explains how the students observed the nest that was displayed in the art classroom. Discusses the steps involved in creating the artworks. (CMK)

  11. Novel genus-specific broad range primers for the detection of furoviruses, hordeiviruses and rymoviruses and their application in field surveys in South-East Australia.

    PubMed

    Zheng, Linda; Tang, Joe; Clover, Gerard R G; Spackman, Merrin E; Freeman, Angela J; Rodoni, Brendan C

    2015-03-01

    A number of viruses from the genera Furovirus, Hordeivirus and Rymovirus are known to infect and damage the four major temperate cereal crops, wheat, barley, sorghum and oats. Currently, there is no active testing in Australia for any of these viruses, which pose a significant biosecurity threat to the phytosanitary status of Australia's grains industry. To address this, broad spectrum PCR assays were developed to target virus species within the genera Furovirus, Hordeivirus and Rymovirus. Five sets of novel genus-specific primers were designed and tested in reverse-transcription polymerase chain reaction assays against a range of virus isolates in plant virus diagnostic laboratories in both Australia and New Zealand. Three of these assays were then chosen to screen samples in a three-year survey of cereal crops in western Victoria, Australia. Of the 8900 cereal plants screened in the survey, all were tested free of furoviruses, hordeiviruses and rymoviruses. To date, there were no published genus-specific primers available for the detection of furoviruses, hordeiviruses and rymoviruses. This study shows for the first time a broad-spectrum molecular test being used in a survey for exotic grain viruses in Australia. Results from this survey provide important evidence of the use of this method to demonstrate the absence of these viruses in Victoria, Australia. The primer pairs reported here are expected to detect a wide range of virus species within the three genera.

  12. Novel genus-specific broad range primers for the detection of furoviruses, hordeiviruses and rymoviruses and their application in field surveys in South-East Australia.

    PubMed

    Zheng, Linda; Tang, Joe; Clover, Gerard R G; Spackman, Merrin E; Freeman, Angela J; Rodoni, Brendan C

    2015-03-01

    A number of viruses from the genera Furovirus, Hordeivirus and Rymovirus are known to infect and damage the four major temperate cereal crops, wheat, barley, sorghum and oats. Currently, there is no active testing in Australia for any of these viruses, which pose a significant biosecurity threat to the phytosanitary status of Australia's grains industry. To address this, broad spectrum PCR assays were developed to target virus species within the genera Furovirus, Hordeivirus and Rymovirus. Five sets of novel genus-specific primers were designed and tested in reverse-transcription polymerase chain reaction assays against a range of virus isolates in plant virus diagnostic laboratories in both Australia and New Zealand. Three of these assays were then chosen to screen samples in a three-year survey of cereal crops in western Victoria, Australia. Of the 8900 cereal plants screened in the survey, all were tested free of furoviruses, hordeiviruses and rymoviruses. To date, there were no published genus-specific primers available for the detection of furoviruses, hordeiviruses and rymoviruses. This study shows for the first time a broad-spectrum molecular test being used in a survey for exotic grain viruses in Australia. Results from this survey provide important evidence of the use of this method to demonstrate the absence of these viruses in Victoria, Australia. The primer pairs reported here are expected to detect a wide range of virus species within the three genera. PMID:25497413

  13. Development of Primer Sets for Loop-Mediated Isothermal Amplification that Enables Rapid and Specific Detection of Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae.

    PubMed

    Wang, Deguo; Liu, Yanhong

    2015-05-26

    Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three pathogens are not available. With the 16S rRNA gene and 16S-23S rRNA intergenic spacers as targets, four sets of LAMP primers were designed for identification and differentiation of S. dysgalactiae, S. uberis and S. agalactiae. The detection limit of all four LAMP primer sets were 0.1 pg DNA template per reaction, the LAMP method with 16S rRNA gene and 16S-23S rRNA intergenic spacers as the targets can differentiate the three pathogens, which is potentially useful in epidemiological studies.

  14. Development of Primer Sets for Loop-Mediated Isothermal Amplification that Enables Rapid and Specific Detection of Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae

    PubMed Central

    Wang, Deguo; Liu, Yanhong

    2015-01-01

    Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three pathogens are not available. With the 16S rRNA gene and 16S-23S rRNA intergenic spacers as targets, four sets of LAMP primers were designed for identification and differentiation of S. dysgalactiae, S. uberis and S. agalactiae. The detection limit of all four LAMP primer sets were 0.1 pg DNA template per reaction, the LAMP method with 16S rRNA gene and 16S-23S rRNA intergenic spacers as the targets can differentiate the three pathogens, which is potentially useful in epidemiological studies. PMID:26016433

  15. Development of Primer Sets for Loop-Mediated Isothermal Amplification that Enables Rapid and Specific Detection of Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae.

    PubMed

    Wang, Deguo; Liu, Yanhong

    2015-06-01

    Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three pathogens are not available. With the 16S rRNA gene and 16S-23S rRNA intergenic spacers as targets, four sets of LAMP primers were designed for identification and differentiation of S. dysgalactiae, S. uberis and S. agalactiae. The detection limit of all four LAMP primer sets were 0.1 pg DNA template per reaction, the LAMP method with 16S rRNA gene and 16S-23S rRNA intergenic spacers as the targets can differentiate the three pathogens, which is potentially useful in epidemiological studies. PMID:26016433

  16. Development of Primer Pairs from Molecular Typing of Rabies Virus Variants Present in Mexico.

    PubMed

    Bastida-González, Fernando; Ramírez-Hernández, Dolores G; Chavira-Suárez, Erika; Lara-Padilla, Eleazar; Zárate-Segura, Paola

    2016-01-01

    Nucleoprotein (N) gene from rabies virus (RABV) is a useful sequence target for variant studies. Several specific RABV variants have been characterized in different mammalian hosts such as skunk, dog, and bats by using anti-nucleocapsid monoclonal antibodies (MAbs) via indirect fluorescent antibody (IFA) test, a technique not available in many laboratories in Mexico. In the present study, a total of 158 sequences of N gene from RABV were used to design eight pairs of primers (four external and four internal primers), for typing four different RABV variants (dog, skunk, vampire bat, and nonhematophagous bat) which are most common in Mexico. The results indicate that the primer and the typing variant from the brain samples, submitted to nested and/or real-time PCR, are in agreement in all four singleplex reactions, and the designed primer pairs are an alternative for use in specific variant RABV typing.

  17. Development of Primer Pairs from Molecular Typing of Rabies Virus Variants Present in Mexico

    PubMed Central

    Ramírez-Hernández, Dolores G.; Lara-Padilla, Eleazar; Zárate-Segura, Paola

    2016-01-01

    Nucleoprotein (N) gene from rabies virus (RABV) is a useful sequence target for variant studies. Several specific RABV variants have been characterized in different mammalian hosts such as skunk, dog, and bats by using anti-nucleocapsid monoclonal antibodies (MAbs) via indirect fluorescent antibody (IFA) test, a technique not available in many laboratories in Mexico. In the present study, a total of 158 sequences of N gene from RABV were used to design eight pairs of primers (four external and four internal primers), for typing four different RABV variants (dog, skunk, vampire bat, and nonhematophagous bat) which are most common in Mexico. The results indicate that the primer and the typing variant from the brain samples, submitted to nested and/or real-time PCR, are in agreement in all four singleplex reactions, and the designed primer pairs are an alternative for use in specific variant RABV typing. PMID:27563666

  18. Development of Primer Pairs from Molecular Typing of Rabies Virus Variants Present in Mexico.

    PubMed

    Bastida-González, Fernando; Ramírez-Hernández, Dolores G; Chavira-Suárez, Erika; Lara-Padilla, Eleazar; Zárate-Segura, Paola

    2016-01-01

    Nucleoprotein (N) gene from rabies virus (RABV) is a useful sequence target for variant studies. Several specific RABV variants have been characterized in different mammalian hosts such as skunk, dog, and bats by using anti-nucleocapsid monoclonal antibodies (MAbs) via indirect fluorescent antibody (IFA) test, a technique not available in many laboratories in Mexico. In the present study, a total of 158 sequences of N gene from RABV were used to design eight pairs of primers (four external and four internal primers), for typing four different RABV variants (dog, skunk, vampire bat, and nonhematophagous bat) which are most common in Mexico. The results indicate that the primer and the typing variant from the brain samples, submitted to nested and/or real-time PCR, are in agreement in all four singleplex reactions, and the designed primer pairs are an alternative for use in specific variant RABV typing. PMID:27563666

  19. Rapid detection and identification of Candida albicans and Torulopsis (Candida) glabrata in clinical specimens by species-specific nested PCR amplification of a cytochrome P-450 lanosterol-alpha-demethylase (L1A1) gene fragment.

    PubMed

    Burgener-Kairuz, P; Zuber, J P; Jaunin, P; Buchman, T G; Bille, J; Rossier, M

    1994-08-01

    PCR of a Candida albicans cytochrome P-450 lanosterol-alpha-demethylase (P450-L1A1) gene segment is a rapid and sensitive method of detection in clinical specimens. This enzyme is a target for azole antifungal action. In order to directly detect and identify the clinically most important species of Candida, we cloned and sequenced 1.3-kbp fragments of the cytochrome P450-L1A1 genes from Torulopsis (Candida) glabrata and from Candida krusei. These segments were compared with the published sequences from C. albicans and Candida tropicalis. Amplimers for gene sequences highly conserved throughout the fungal kingdom were first used; positive PCR results were obtained for C. albicans, T. glabrata, C. krusei, Candida parapsilosis, C. tropicalis, Cryptococcus neoformans, and Trichosporon beigelii DNA extracts. Primers were then selected for a highly variable region of the gene, allowing the species-specific detection from purified DNA of C. albicans, T. glabrata, C. krusei, and C. tropicalis. The assay sensitivity as tested for C. albicans in seeded clinical specimens such as blood, peritoneal fluid, or urine was 10 to 20 cells per 0.1 ml. Compared with results obtained by culture, the sensitivity, specificity, and efficiency of the species-specific nested PCR tested with 80 clinical specimens were 71, 95, and 83% for C. albicans and 100, 97, and 98% for T. glabrata, respectively.

  20. Development of SYBR green-based real-time PCR and duplex nested PCR assays for quantitation and differential detection of species- or type-specific porcine Torque teno viruses.

    PubMed

    Huang, Y W; Dryman, B A; Harrall, K K; Vaughn, E M; Roof, M B; Meng, X J

    2010-12-01

    Porcine Torque teno virus (TTV), a single-stranded circular DNA virus, has been incriminated in swine diseases recently. Multiple infection with porcine TTV species 1 (PTTV1) and species 2 (PTTV2), each consisting of two types (PTTV1a and 1b) or subtypes (PTTV2b and 2c), in a single pig had been reported by our group previously. The present study described three novel assays for quantitation and differential detection of porcine TTV. First, we developed two SYBR green-based real-time PCR assays to quantify viral loads of two porcine TTV species, respectively. The PTTV1- and PTTV2-specific real-time PCR primer sequences were selected to target conserved regions identified by multiple alignments of ten available porcine TTV full-length genomes. Furthermore, by coupling the two singleplex PCR assays, a duplex real-time PCR assay followed by melting curve analysis was established for simultaneous detection and differentiation of PTTV1 and PTTV2. In addition, a type-specific duplex nested PCR was also developed to simultaneously detect and distinguish between the two types, PTTV1a and 1b, in PTTV1 species. These assays provide rapid and practical tools for molecular diagnosis of species- or type-specific porcine TTV.

  1. Phototyping: comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes utilizing sequence-specific primers (PCR-SSP).

    PubMed

    Bunce, M; O'Neill, C M; Barnardo, M C; Krausa, P; Browning, M J; Morris, P J; Welsh, K I

    1995-11-01

    We have developed a single DNA typing method which uses 144 sequence-specific primer (SSP) reactions to simultaneously detect all known HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 and DQB1 specificities in an allele specific or group specific manner using the same method, reagents, PCR parameters and protocols for all loci. The results from this integrated class I & II method can be visualized on a single photographic or electronic image and hence is described as "Phototyping". Phototyping has an overall resolution greater than or equivalent to good serology and results can be obtained in under 3 hours making the method suitable for genotyping potential cadaver donor peripheral blood without serological backup. This in turn produces the potential for reducing cold ischaemia times in renal transplantation as well as the application of prospective matching to cardiac and liver transplantation. The method has capacity to detect new alleles, for example, novel amplification patterns suggestive of 4 new HLA-B alleles have been detected. The Phototyping set has been used as the sole method of HLA typing for over 1010 individuals. Phototyping is not problem-free; deviations from the standard protocol, poor quality DNA and unsuitable PCR machines can result in individual PCR failures or in incorrect assignment of antigens. Approximately 5% of genotypes were repeated (either partially or fully) because of incomplete or equivocal results.

  2. A multiplex nested PCR assay for simultaneous detection of Corchorus golden mosaic virus and a phytoplasma in white jute (Corchorus capsularis L.).

    PubMed

    Biswas, C; Dey, P; Satpathy, S

    2013-05-01

    A multiplex nested PCR assay was developed by optimizing reaction components and reaction cycling parameters for simultaneous detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma (Group 16Sr V-C) causing little leaf and bunchy top in white jute (Corchorus capsularis). Three sets of specific primers viz. a CoGMV specific (DNA-A region) primer, a 16S rDNA universal primer pair P1/P7 and nested primer pair R16F2n/R2 for phytoplasmas were used. The concentrations of the PCR components such as primers, MgCl2 , Taq DNA polymerase, dNTPs and PCR conditions including annealing temperature and amplification cycles were examined and optimized. Expected fragments of 1 kb (CoGMV), 674 bp (phytoplasma) and 370 bp (nested R16F2n/R2) were successfully amplified by this multiplex nested PCR system ensuring simultaneous, sensitive and specific detection of the phytoplasma and the virus. The multiplex nested PCR provides a sensitive, rapid and low-cost method for simultaneous detection of jute little leaf phytoplasma and CoGMV. Based on BLASTn analyses, the phytoplasma was found to belong to the Group 16Sr V-C.

  3. A multiplex nested PCR assay for simultaneous detection of Corchorus golden mosaic virus and a phytoplasma in white jute (Corchorus capsularis L.).

    PubMed

    Biswas, C; Dey, P; Satpathy, S

    2013-05-01

    A multiplex nested PCR assay was developed by optimizing reaction components and reaction cycling parameters for simultaneous detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma (Group 16Sr V-C) causing little leaf and bunchy top in white jute (Corchorus capsularis). Three sets of specific primers viz. a CoGMV specific (DNA-A region) primer, a 16S rDNA universal primer pair P1/P7 and nested primer pair R16F2n/R2 for phytoplasmas were used. The concentrations of the PCR components such as primers, MgCl2 , Taq DNA polymerase, dNTPs and PCR conditions including annealing temperature and amplification cycles were examined and optimized. Expected fragments of 1 kb (CoGMV), 674 bp (phytoplasma) and 370 bp (nested R16F2n/R2) were successfully amplified by this multiplex nested PCR system ensuring simultaneous, sensitive and specific detection of the phytoplasma and the virus. The multiplex nested PCR provides a sensitive, rapid and low-cost method for simultaneous detection of jute little leaf phytoplasma and CoGMV. Based on BLASTn analyses, the phytoplasma was found to belong to the Group 16Sr V-C. PMID:23413927

  4. Phonics Primer

    ERIC Educational Resources Information Center

    Elam, Sandra

    2007-01-01

    This primer lists the 44 sounds in the English language and then gives steps for teaching those 44 sounds and their most common spelling patterns. In addition to learning sounds and spellings, each day the student must read lists of phonetically related words and spell these words from dictation. Phonics instruction must be reinforced by having…

  5. Emaravirus-specific degenerate PCR primers allowed the identification of partial RNA-dependent RNA polymerase sequences of Maize red stripe virus and Pigeonpea sterility mosaic virus.

    PubMed

    Elbeaino, Toufic; Whitfield, Anna; Sharma, Mamta; Digiaro, Michele

    2013-03-01

    Emaravirus is a recently established viral genus that includes two approved virus species: European mountain ash ringspot-associated virus (EMARaV) and Fig mosaic virus (FMV). Other described but unclassified viruses appear to share biological characteristics similar to emaraviruses, including segmented, negative-single stranded RNA genomes with enveloped virions approximately 80-200nm in diameter. Sequence analysis of emaravirus genomes revealed the presence of conserved amino acid sequences in the RNA-dependent RNA polymerase gene (RdRp) denoted as pre-motif A, motifs A and C. Degenerate oligonucleotide primers were developed to these conserved sequences and were shown to amplify in reverse transcription-polymerase chain reaction assay (RT-PCR) DNA fragments of 276bp and 360bp in size. These primers efficiently detected emaraviruses with known sequences available in the database (FMV and EMARaV); they also detected viruses with limited sequence information such as Pigeonpea sterility mosaic virus (PPSMV) and Maize red stripe virus (MRSV). The degenerate primers designed on pre-motif A and motif A sequences successfully amplified the four species used as positive controls (276bp), whereas those of motifs A and C failed to detect only MRSV. The amino acid sequences obtained from PPSMV and MRSV shared the highest identity with those of two other tentative species of the Emaravirus genus, Rose rosette virus (RRV) (69%) and Redbud yellow ringspot virus (RYRV) (60%), respectively. The phylogenetic tree constructed with 92 amino acid-long portions of polypeptide putatively encoded by RNA1 of definitive and tentative emaravirus species clustered PPSMV and MRSV in two separate clades close to RRV and Raspberry leaf blotch virus (RLBV), respectively. The newly developed degenerate primers have proved their efficacy in amplifying new emaravirus-specific sequences; accordingly, they could be useful in identifying new emaravirus-like species in nature.

  6. Detection of minimal residual disease in patients with multiple myeloma using clonotype-specific PCR primers designed from DNA extracted from archival bone marrow slides.

    PubMed

    Takamatsu, Hiroyuki; Ogawa, Yoshiyasu; Kobayashi, Noriko; Obata, Kazue; Narisawa, Tadashi; Nakayama, Kouji; Munemoto, Saori; Aoki, Go; Ohata, Kinya; Kumano, Yoshihisa; Ozaki, Jun; Murata, Ryoichi; Kondo, Yukio; Terasaki, Yasushi; Kurokawa, Toshiro; Miyamoto, Toshihiro; Shimizu, Naomi; Fukushima, Toshihiro; Yoshida, Akira; Ueda, Takanori; Yoshida, Takashi; Nakao, Shinji

    2013-10-01

    Polymerase chain reaction (PCR)-negative molecular complete remission (mCR) can be induced by stem cell transplantation in some patients with multiple myeloma (MM) and is associated with long-term progression-free survival (PFS). The detection of molecular minimal residual disease (MRD), however, requires fresh or frozen materials for designing clone-specific primers, which are not always readily available. In this study, we used DNA extracted from archival bone marrow (BM) slides for PCR to detect MRD in 50 patients with MM who received various induction therapies and autologous peripheral blood stem cell transplantation (ASCT). Clonotype-specific immunoglobulin (Ig) H PCR primers were prepared for 32 of 50 cases (64%) using BM slides, and for 9 of 14 cases (64%) using fresh BM cells. DNA in peripheral blood stem cell autografts of the 22 patients who achieved at least a partial response after ASCT was subjected to PCR to amplify clonotype-specific rearranged IgH gene sequences. The median PFS of the eight patients with MRD-positive autografts was 18 months, whereas that of 14 patients with MRD-negative autografts was not reached at a median follow-up of 27 months (p = 0.012). Post-ASCT PFS of the four patients who achieved mCR was 100% at a median follow-up of 47 months. These results indicate that archival BM slides can serve as a source of DNA for preparing clonotype-specific primers for MRD monitoring in patients with MM whose cryopreserved myeloma cells are not available for DNA preparation. Our results also suggest that patients with MM who received MRD-negative autografts and achieved mCR have a long PFS.

  7. Primers and a specific DNA probe for detecting lactic acid bacteria producing 3-hydroxypropionaldehyde from glycerol in spoiled ciders.

    PubMed

    Claisse, O; Lonvaud-Funel, A

    2001-06-01

    Of the 40 strains isolated from several spoiled ciders where glycerol was degraded, 36 were identified as Lactobacillus collinoides, three were Lactobacillus hilgardii, and one was Lactobacillus mali. However, only 30 L. collinoides and two L. hilgardii could degrade glycerol. The glycerol dehydratase activity was shown. The main product of the transformation was 1.3 propanediol. Two DNA primers GD1 and GD2 were chosen in the region encoding one of the subunits of glycerol dehydratase of Citrobacter freundii, Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella Typhimurium, and Clostridium pasteurianum. A 279-bp amplicon in polymerase chain reaction amplification was obtained with the genomic L. collinoides IOEB 9527 DNA as template. The amino acid sequence deduced from the amplicon DNA sequence showed a very high similarity and identity with the gene of gram-negative and C. pasteurianum species. After labeling, the amplicon was used as DNA probe in dot-blot hybridization with the genomic DNA of all the tested strains. Only strains that could degrade glycerol hybridized. Moreover, polymerase chain reactions using GDI and GD2 revealed only glycerol dehydratase genes of positive L. collinoides and L. hilgardii strains. The primers and the amplicon proved to be suitable and reliable tools to detect the lactic acid bacteria involved in the deterioration of cider. PMID:11403134

  8. Quantitative polymerase chain reaction analysis with allele-specific oligonucleotide primers for individual IgH VDJ regions to evaluate tumor burden in myeloma patients.

    PubMed

    Sata, Hiroshi; Shibayama, Hirohiko; Maeda, Ikuhiro; Habuchi, Yoko; Nakatani, Eiji; Fukushima, Kentaro; Fujita, Jiro; Ezoe, Sachiko; Tadokoro, Seiji; Maeda, Tetsuo; Mizuki, Masao; Kosugi, Satoru; Nakagawa, Masashi; Ueda, Shuji; Iida, Masato; Tokumine, Yukihiro; Azenishi, Yasuhiko; Mitsui, Hideki; Oritani, Kenji; Kanakura, Yuzuru

    2015-05-01

    Quantitative polymerase chain reaction (PCR) with patient-specific, allele-specific oligonucleotide (ASO) primers for individual immunoglobulin H VDJ region (ASO-PCR) amplification was performed using several sources of clinical material, including mRNA from peripheral blood cells (PBMNCs), whole bone marrow cells (BMMNCs), and the CD20+ CD38- B-cell population in bone marrow, as well as cell-free DNA from the sera of patients with multiple myeloma (MM). We designed the ASO primers and produced sufficient PCR fragments to evaluate tumor burden in 20 of 30 bone marrow samples at diagnosis. Polymerase chain reaction amplification efficiency depended on primer sequences because the production of ASO-PCR fragments did not correlate with serum M-protein levels. However, the ASO-PCR levels in BMMNCs showed statistically significant correlations with those in PBMNCs and CD20+ CD38- B-cells. The good association between the BMMNC and PBMNC data indicated that PBMNCs could be a suitable source for monitoring minimal residual disease (MRD). In the case of cell-free DNA, ASO-PCR levels showed a unique pattern and remained high even after treatment. Because the sequence information for each ASO-PCR product was identical to the original, the cell-free DNA might also be useful for evaluating MRD. Moreover, the ASO-PCR products were clearly detected in 17 of 22 mRNA samples from CD20+ CD38- populations, suggesting that MM clones might exist in relatively earlier stages of B cells than in plasma cells. Thus, ASO-PCR analysis using various clinical materials is useful for detecting MRD in MM patients as well as for clarifying MM pathogenesis. PMID:25591497

  9. Quantitative polymerase chain reaction analysis with allele-specific oligonucleotide primers for individual IgH VDJ regions to evaluate tumor burden in myeloma patients.

    PubMed

    Sata, Hiroshi; Shibayama, Hirohiko; Maeda, Ikuhiro; Habuchi, Yoko; Nakatani, Eiji; Fukushima, Kentaro; Fujita, Jiro; Ezoe, Sachiko; Tadokoro, Seiji; Maeda, Tetsuo; Mizuki, Masao; Kosugi, Satoru; Nakagawa, Masashi; Ueda, Shuji; Iida, Masato; Tokumine, Yukihiro; Azenishi, Yasuhiko; Mitsui, Hideki; Oritani, Kenji; Kanakura, Yuzuru

    2015-05-01

    Quantitative polymerase chain reaction (PCR) with patient-specific, allele-specific oligonucleotide (ASO) primers for individual immunoglobulin H VDJ region (ASO-PCR) amplification was performed using several sources of clinical material, including mRNA from peripheral blood cells (PBMNCs), whole bone marrow cells (BMMNCs), and the CD20+ CD38- B-cell population in bone marrow, as well as cell-free DNA from the sera of patients with multiple myeloma (MM). We designed the ASO primers and produced sufficient PCR fragments to evaluate tumor burden in 20 of 30 bone marrow samples at diagnosis. Polymerase chain reaction amplification efficiency depended on primer sequences because the production of ASO-PCR fragments did not correlate with serum M-protein levels. However, the ASO-PCR levels in BMMNCs showed statistically significant correlations with those in PBMNCs and CD20+ CD38- B-cells. The good association between the BMMNC and PBMNC data indicated that PBMNCs could be a suitable source for monitoring minimal residual disease (MRD). In the case of cell-free DNA, ASO-PCR levels showed a unique pattern and remained high even after treatment. Because the sequence information for each ASO-PCR product was identical to the original, the cell-free DNA might also be useful for evaluating MRD. Moreover, the ASO-PCR products were clearly detected in 17 of 22 mRNA samples from CD20+ CD38- populations, suggesting that MM clones might exist in relatively earlier stages of B cells than in plasma cells. Thus, ASO-PCR analysis using various clinical materials is useful for detecting MRD in MM patients as well as for clarifying MM pathogenesis.

  10. The allele-specific probe and primer amplification assay, a new real-time PCR method for fine quantification of single-nucleotide polymorphisms in pooled DNA.

    PubMed

    Billard, A; Laval, V; Fillinger, S; Leroux, P; Lachaise, H; Beffa, R; Debieu, D

    2012-02-01

    The evolution of fungicide resistance within populations of plant pathogens must be monitored to develop management strategies. Such monitoring often is based on microbiological tests, such as microtiter plate assays. Molecular monitoring methods can be considered if the mutations responsible for resistance have been identified. Allele-specific real-time PCR approaches, such as amplification refractory mutation system (ARMS) PCR and mismatch amplification mutation assay (MAMA) PCR, are, despite their moderate efficacy, among the most precise methods for refining SNP quantification. We describe here a new real-time PCR method, the allele-specific probe and primer amplification assay (ASPPAA PCR). This method makes use of mixtures of allele-specific minor groove binder (MGB) TaqMan probes and allele-specific primers for the fine quantification of SNPs from a pool of DNA extracted from a mixture of conidia. It was developed for a single-nucleotide polymorphism (SNP) that is responsible for resistance to the sterol biosynthesis inhibitor fungicide fenhexamid, resulting in the replacement of the phenylalanine residue (encoded by the TTC codon) in position 412 of the enzymatic target (3-ketoreductase) by a serine (TCC), valine (GTC), or isoleucine (ATC) residue. The levels of nonspecific amplification with the ASPPAA PCR were reduced at least four times below the level of currently available allele-specific real-time PCR approaches due to strong allele specificity in amplification cycles, including two allele selectors. This new method can be used to quantify a complex quadriallelic SNP in a DNA pool with a false discovery rate of less than 1%.

  11. Triangular Nests!

    ERIC Educational Resources Information Center

    Powell, R. I.

    2002-01-01

    Shows how integer-sided triangles can be nested, each nest having a single enclosing isosceles triangle. Brings to light what can be seen as a relatively simple generalization of Pythagoras' theorem, a result that should be readily accessible to many secondary school pupils. (Author/KHR)

  12. Explanatory chapter: PCR primer design.

    PubMed

    Álvarez-Fernández, Rubén

    2013-01-01

    This chapter is intended as a guide on polymerase chain reaction (PCR) primer design (for information on PCR, see General PCR and Explanatory Chapter: Troubleshooting PCR). In the next section, general guidelines will be provided, followed by a discussion on primer design for specific applications. A list of recommended software tools is shown at the end.

  13. Identification of lipase encoding genes from Antarctic seawater bacteria using degenerate primers: expression of a cold-active lipase with high specific activity.

    PubMed

    Parra, Loreto P; Espina, Giannina; Devia, Javier; Salazar, Oriana; Andrews, Barbara; Asenjo, Juan A

    2015-01-01

    Cold-active enzymes are valuable catalysts showing high activity at low and moderate temperatures and low thermostability. Among cold-active enzymes, lipases offer a great potential in detergent, cosmetic, biofuel and food or feed industries. In this paper we describe the identification of novel lipase coding genes and the expression of a lipase with high activity at low temperatures. The genomic DNA from Antarctic seawater bacteria showing lipolytic activity at 4°C was used to amplify five DNA fragments that partially encode novel lipases using specifically designed COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOP). All the fragments were found to have a high identity with an α/β-hydrolase domain-containing protein identified by the sequencing of the complete genome of Shewanella frigidimarina NCIMB 400. The complete sequence of one of the lipase-coding gene fragments, lipE13, was obtained by genome walking. Considering that the other fragments had a high identity to the putative lipase from S. frigidimarina NCIMB 400, the complete lipase genes were amplified using oligonucleotide primers designed based on the 5' and 3' regions of the coding sequence of the related protein. This strategy allowed the amplification of 3 lipase-encoding genes of which one was expressed in the periplasm using the Escherichia coli BL21(DE3)/pET-22b(+) expression system. The recombinant protein was obtained with activity toward p-nitrophenyl caproate showing a high specific activity between 15 and 25°C.

  14. RATMAC PRIMER

    SciTech Connect

    Munn, R. J.; Stewart, J. M.; Norden, A. P.; Pagoaga, M. Katherine

    1980-10-01

    The language RATMAC is a direct descendant of one of the most successful structured FORTRAN languages, rational FORTRAN, RATFOR. RATMAC has all of the characteristics of RATFOR, but is augmented by a powerful recursive macro processor which is extremely useful in generating transportable FORTRAN programs. A macro is a collection of programming steps which are associated with a keyword. This keyword uniquely identifies the macro, and whenever it appears in a RATMAC program it is replaced by the collection of steps. This primer covers the language's control and decision structures, macros, file inclusion, symbolic constants, and error messages.

  15. Testing the Specificity of Primers to Environmental Ammonia Monooxygenase (amoA) Genes in Groundwater Treated with Urea to Promote Calcite Precipitation

    SciTech Connect

    Freeman, S.; Reed, D.W.; Fujita, Y.

    2006-01-01

    Bacterial ammonia monooxygenase (amoA) genes in DNA isolated from microorganisms in groundwater were characterized by amplification of amoA DNA using polymerase chain reaction (PCR), Restriction Fragment Length Polymorphism (RFLP) analysis, and sequencing. The amoA gene is characteristic of ammonia oxidizing bacteria (AOB). The DNA extracts were acquired from an experiment where dilute molasses and urea were sequentially introduced into a well in the Eastern Snake River Plain Aquifer (ESRPA) in Idaho to examine whether such amendments could stimulate enhanced ureolytic activity. The hydrolysis of urea into ammonium and carbonate serves as the basis for a potential remediation technique for trace metals and radionuclide contaminants that can co-precipitate in calcite. The ammonium ion resulting from ureolysis can promote the growth of AOB. The goal of this work was to investigate the effectiveness of primers designed for quantitative PCR of environmental amoA genes and to evaluate the effect of the molasses and urea amendments upon the population diversity of groundwater AOB. PCR primers designed to target a portion of the amoA gene were used to amplify amoA gene sequences in the groundwater DNA extracts. Following PCR, amplified gene products were cloned and the clones were characterized by RFLP, a DNA restriction technique that can distinguish different DNA sequences, to gauge the initial diversity. Clones exhibiting unique RFLP patterns were subjected to DNA sequencing. Initial sequencing results suggest that the primers were successful at specific detection of amoA sequences and the RFLP analyses indicated that the diversity of detected amoA sequences in the ESRPA decreased with the additions of molasses and urea.

  16. Identification of new primer binding site mutations at TH01 and D13S317 loci and determination of their corresponding STR alleles by allele-specific PCR.

    PubMed

    Li, Fengrui; Xuan, Jinfeng; Xing, Jiaxin; Ding, Mei; Wang, Baojie; Pang, Hao

    2014-01-01

    Several commercial multiplex PCR kits for the amplification of short tandem repeat (STR) loci have been extensively applied in forensic genetics. Consequently, large numbers of samples have been genotyped, and the number of discordant genotypes observed has also increased. We observed allele dropout with two novel alleles at the STR loci TH01 and D13S317 during paternity testing using the AmpFℓSTR Identifiler PCR Amplification Kit. The lost alleles reappeared when alternative PCR primer pairs were used. A sequence analysis revealed a G-to-A substitution 82 bases downstream of the last TCAT motif of the repeat region at the TH01 locus (GenBank accession: D00269) and a G-to-T substitution 90 bases upstream of the first TATC motif of the repeat region at the D13S317 locus (GenBank accession: G09017). The frequencies of these two point mutations were subsequently investigated in the Chinese population using sequence-specific primer PCR (SSP-PCR), but neither of these mutations was detected in any of the samples tested. In addition, the DNA samples in which the mutations were identified were amplified to type the point mutations by SSP-PCR to determine the corresponding STR alleles at the two loci. Subsequently, the amplified PCR products with different point mutations and STR repeat numbers were directly sequenced because this strategy overcomes the appearance overlapping peaks generated by different STR alleles and accurately characterizes genotypes. Thus, our findings not only provide useful information for DNA databases and forensic identification but also establish an effective strategy for typing STR alleles with primer binding site mutations.

  17. A spot multiplex nested RT-PCR for the simultaneous and generic detection of viruses involved in the aetiology of grapevine leafroll and rugose wood of grapevine.

    PubMed

    Dovas, C I; Katis, N I

    2003-05-01

    A spot nested RT-PCR assay using degenerate deoxyinosine-containing primers was developed, allowing rapid and simultaneous detection of Closterovirus sequences. Nested PCR amplification increased the specificity and sensitivity of detection. The sensitivity was also increased by a factor of 10 by using in addition to the deoxyinosine (dI)-containing primers, respective homologous primers in which dI was substituted by dG in the region of sequence homology. These homologous primers are shorter, having lower degeneracy and higher amplification efficiency than the dI-containing primers. This method was coupled to a similar nested RT-PCR detection method for Vitivirus and Foveavirus sequences. This permitted multiplex RT-PCR amplification of sequences belonging to the three genera in the same reaction tube and the two subsequent nested PCR amplifications (one for closteroviruses and one for viti- and foveaviruses) to run in parallel. Different primers and amplification parameters (additives and thermocycling conditions) were evaluated and optimised, respectively, in order to amplify efficiently all different templates. These improvements permitted the multiplex detection of fovea- and closteroviruses in petiole and cortical scraping preparations from 23 infected field-grown grapevines throughout the year, with the exception of GLRaV-1 in petioles that was only possible from June onwards. Preliminary results show that this method can detect reliably virus species from three genera in grapevine allowing simple, fast and cost-effective testing of a large number of samples in certification schemes.

  18. Salinas primer.

    SciTech Connect

    Walsh, Timothy Francis; Reese, Garth M.; Bhardwaj, Manoj Kumar

    2004-08-01

    Salinas provides a massively parallel implementation of structural dynamics finite element analysis. This capability is required for high fidelity, validated models used in modal, vibration, static and shock analysis of weapons systems. General capabilities for modal, statics and transient dynamics are provided. Salinas is similar to commercial codes like Nastran or Abaqus. It has some nonlinear capability, but excels in linear computation. It is different than the above commercial codes in that it is designed to operate efficiently in a massively parallel environment. Even for an experienced analyst, running a new finite element package can be a challenge. This little primer is intended to make part of this task easier by presenting the basic steps in a simple way. The analyst is referred to the theory manual for details of the mathematics behind the work. The User's Notes should be used for more complex inputs, and will have more details about the process (as well as many more examples). More information can be found on our web pages, 3 or 4. Finite element analysis can be deceptive. Any software can give the wrong answers if used improperly, and occasionally even when used properly. Certainly a solid background in structural mechanics is necessary to build an adequate finite element model and interpret the results. This primer should provide a quick start in answering some of the more common questions that come up in using Salinas.

  19. Optimization of nested polymerase chain reaction assays for identification of Aeromonas salmonicida, Yersinia ruckeri and Flavobacterium psychrophilum

    USGS Publications Warehouse

    Taylor, P.W.; Winton, J.R.

    2002-01-01

    Nested polymerase chain reaction (PCR) assays were developed using first-round primers complementary to highly conserved regions within the bacterial 16S ribosomal RNA (rRNA) gene (universal eubacterial primers) and second-round primers specific for sequences within the 16S rRNA genes of Aeromonas salmonicida, Yersinia ruckeri, andFlavobacterium psychrophilum. Following optimization of the MgCl2 concentration and primer annealing temperature, PCR employing the universal eubacterial primers was used to amplify a 1,500-base-pair (bp) product visible in agarose gels stained with ethidium bromide. The calculated detection limit of this single-round assay was less than 1.4 × 104 colony-forming units (CFU) per reaction for all bacterial species tested. Single-round PCR using primer sets specific for A. salmonicida, Y. ruckeri, and F. psychrophilumamplified bands of 271, 575, and 1,100 bp, respectively, with detection limits of less than 1.4 × 104, 1.4 × 105, and 1.4 × 105 CFU per reaction. Using the universal eubacterial primers in the first round and the species-specific primer sets in the second round of nested PCR assays improved the detection ability by approximately four orders of magnitude to fewer than 14 CFU per sample for each of the three bacterial species. Such nested assays could be adapted to a wide variety of bacterial fish pathogens for which 16S sequences are available.

  20. Nested Cohort

    Cancer.gov

    NestedCohort is an R software package for fitting Kaplan-Meier and Cox Models to estimate standardized survival and attributable risks for studies where covariates of interest are observed on only a sample of the cohort.

  1. Detection of Citrus leprosis virus C using specific primers and TaqMan probe in one-step real-time reverse-transcription polymerase chain reaction assays.

    PubMed

    Choudhary, Nandlal; Wei, G; Govindarajulu, A; Roy, Avijit; Li, Wenbin; Picton, Deric D; Nakhla, M K; Levy, L; Brlansky, R H

    2015-11-01

    Citrus leprosis virus C (CiLV-C), a causal agent of the leprosis disease in citrus, is mostly present in the South and Central America and spreading toward the North America. To enable better diagnosis and inhibit the further spread of this re-emerging virus a quantitative (q) real-time reverse transcription polymerase chain reaction (qRT-PCR) assay is needed for early detection of CiLV-C when the virus is present in low titer in citrus leprosis samples. Using the genomic sequence of CiLV-C, specific primers and probe were designed and synthesized to amplify a 73 nt amplicon from the movement protein (MP) gene. A standard curve of the 73 nt amplicon MP gene was developed using known 10(10)-10(1) copies of in vitro synthesized RNA transcript to estimate the copy number of RNA transcript in the citrus leprosis samples. The one-step qRT-PCR detection assays for CiLV-C were determined to be 1000 times more sensitive when compared to the one-step conventional reverse transcription polymerase chain reaction (RT-PCR) CiLV-C detection method. To evaluate the quality of the total RNA extracts, NADH dehydrogenase gene specific primers (nad5) and probe were included in reactions as an internal control. The one-step qRT-PCR specificity was successfully validated by testing for the presence of CiLV-C in the total RNA extracts of the citrus leprosis samples collected from Belize, Costa Rica, Mexico and Panama. Implementation of the one-step qRT-PCR assays for CiLV-C diagnosis should assist regulatory agencies in surveillance activities to monitor the distribution pattern of CiLV-C in countries where it is present and to prevent further dissemination into citrus growing countries where there is no report of CiLV-C presence. PMID:26341059

  2. Small subunit ribosomal RNA gene sequence of Minchinia teredinis (Haplosporidia: Haplosporidiidae) and a specific DNA probe and PCR primers for its detection.

    PubMed

    Stokes, N A; Siddall, M E; Burreson, E M

    1995-05-01

    Minchinia teredinis is a pathogen of wood-boring molluscs (shipworms), Teredo spp., along the middle Atlantic coast of the U.S. Genomic DNA was extracted from M. teredinis spores and small subunit (SSU) rDNA was amplified by PCR, cloned, and sequenced. The sequence of M. teredinis SSU rDNA was aligned with that of Haplosporidium nelsoni and various protists in GenBank. A 22-base oligonucleotide probe unique to M. teredinis, designated MIN702, was commercially synthesized and tested for sensitivity and specificity. In dot-blot hybridizations the probe detected 500 pg of cloned M. teredinis rDNA. The probe did not hybridize with cloned SSU rDNA of Teredo spp. or H. nelsoni. The probe was further tested for specificity with in situ hybridizations on AFA-fixed, paraffin-embedded tissue sections. The probe hybridized well with M. teredinis plasmodia and immature spores, but poorly with mature spores. The probe did not hybridize with shipworm tissue or with the haplosporidians Haplosporidium louisiana from mud crabs (Panopeus spp.) or H. nelsoni and H. costale from Crassostrea virginica. The probe and a second 18-base oligonucleotide, when used as PCR primers, amplified a 536-bp fragment of the M. teredinis SSU rRNA gene. The PCR assay was able to detect 10 fg of the cloned gene and also detected the presence of M. teredinis DNA in shipworms in which infections were observed microscopically. The 536-bp amplification product was not obtained in one Teredo sp. or in one Bankia gouldi, both categorized as uninfected after microscopic inspection. The DNA probe and PCR primers appear to be specific for M. teredinis and should be useful as diagnostic tools and for life cycle investigations.

  3. Detection of Citrus leprosis virus C using specific primers and TaqMan probe in one-step real-time reverse-transcription polymerase chain reaction assays.

    PubMed

    Choudhary, Nandlal; Wei, G; Govindarajulu, A; Roy, Avijit; Li, Wenbin; Picton, Deric D; Nakhla, M K; Levy, L; Brlansky, R H

    2015-11-01

    Citrus leprosis virus C (CiLV-C), a causal agent of the leprosis disease in citrus, is mostly present in the South and Central America and spreading toward the North America. To enable better diagnosis and inhibit the further spread of this re-emerging virus a quantitative (q) real-time reverse transcription polymerase chain reaction (qRT-PCR) assay is needed for early detection of CiLV-C when the virus is present in low titer in citrus leprosis samples. Using the genomic sequence of CiLV-C, specific primers and probe were designed and synthesized to amplify a 73 nt amplicon from the movement protein (MP) gene. A standard curve of the 73 nt amplicon MP gene was developed using known 10(10)-10(1) copies of in vitro synthesized RNA transcript to estimate the copy number of RNA transcript in the citrus leprosis samples. The one-step qRT-PCR detection assays for CiLV-C were determined to be 1000 times more sensitive when compared to the one-step conventional reverse transcription polymerase chain reaction (RT-PCR) CiLV-C detection method. To evaluate the quality of the total RNA extracts, NADH dehydrogenase gene specific primers (nad5) and probe were included in reactions as an internal control. The one-step qRT-PCR specificity was successfully validated by testing for the presence of CiLV-C in the total RNA extracts of the citrus leprosis samples collected from Belize, Costa Rica, Mexico and Panama. Implementation of the one-step qRT-PCR assays for CiLV-C diagnosis should assist regulatory agencies in surveillance activities to monitor the distribution pattern of CiLV-C in countries where it is present and to prevent further dissemination into citrus growing countries where there is no report of CiLV-C presence.

  4. An Efficient Approach for the Development of Locus Specific Primers in Bread Wheat (Triticum aestivum L.) and Its Application to Re-Sequencing of Genes Involved in Frost Tolerance.

    PubMed

    Babben, Steve; Perovic, Dragan; Koch, Michael; Ordon, Frank

    2015-01-01

    Recent declines in costs accelerated sequencing of many species with large genomes, including hexaploid wheat (Triticum aestivum L.). Although the draft sequence of bread wheat is known, it is still one of the major challenges to developlocus specific primers suitable to be used in marker assisted selection procedures, due to the high homology of the three genomes. In this study we describe an efficient approach for the development of locus specific primers comprising four steps, i.e. (i) identification of genomic and coding sequences (CDS) of candidate genes, (ii) intron- and exon-structure reconstruction, (iii) identification of wheat A, B and D sub-genome sequences and primer development based on sequence differences between the three sub-genomes, and (iv); testing of primers for functionality, correct size and localisation. This approach was applied to single, low and high copy genes involved in frost tolerance in wheat. In summary for 27 of these genes for which sequences were derived from Triticum aestivum, Triticum monococcum and Hordeum vulgare, a set of 119 primer pairs was developed and after testing on Nulli-tetrasomic (NT) lines, a set of 65 primer pairs (54.6%), corresponding to 19 candidate genes, turned out to be specific. Out of these a set of 35 fragments was selected for validation via Sanger's amplicon re-sequencing. All fragments, with the exception of one, could be assigned to the original reference sequence. The approach presented here showed a much higher specificity in primer development in comparison to techniques used so far in bread wheat and can be applied to other polyploid species with a known draft sequence.

  5. An Efficient Approach for the Development of Locus Specific Primers in Bread Wheat (Triticum aestivum L.) and Its Application to Re-Sequencing of Genes Involved in Frost Tolerance

    PubMed Central

    Babben, Steve; Perovic, Dragan; Koch, Michael; Ordon, Frank

    2015-01-01

    Recent declines in costs accelerated sequencing of many species with large genomes, including hexaploid wheat (Triticum aestivum L.). Although the draft sequence of bread wheat is known, it is still one of the major challenges to developlocus specific primers suitable to be used in marker assisted selection procedures, due to the high homology of the three genomes. In this study we describe an efficient approach for the development of locus specific primers comprising four steps, i.e. (i) identification of genomic and coding sequences (CDS) of candidate genes, (ii) intron- and exon-structure reconstruction, (iii) identification of wheat A, B and D sub-genome sequences and primer development based on sequence differences between the three sub-genomes, and (iv); testing of primers for functionality, correct size and localisation. This approach was applied to single, low and high copy genes involved in frost tolerance in wheat. In summary for 27 of these genes for which sequences were derived from Triticum aestivum, Triticum monococcum and Hordeum vulgare, a set of 119 primer pairs was developed and after testing on Nulli-tetrasomic (NT) lines, a set of 65 primer pairs (54.6%), corresponding to 19 candidate genes, turned out to be specific. Out of these a set of 35 fragments was selected for validation via Sanger's amplicon re-sequencing. All fragments, with the exception of one, could be assigned to the original reference sequence. The approach presented here showed a much higher specificity in primer development in comparison to techniques used so far in bread wheat and can be applied to other polyploid species with a known draft sequence. PMID:26565976

  6. Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori

    PubMed Central

    Luo, Xiao-Feng; Jiao, Jian-Hua; Zhang, Wen-Yue; Pu, Han-Ming; Qu, Bao-Jin; Yang, Bing-Ya; Hou, Min; Ji, Min-Jun

    2016-01-01

    AIM: To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR). METHODS: The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion. RESULTS: The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. CONCLUSION: The nested-ASP-PCR assay showed higher

  7. New primers for detecting and quantifying denitrifying anaerobic methane oxidation archaea in different ecological niches.

    PubMed

    Ding, Jing; Ding, Zhao-Wei; Fu, Liang; Lu, Yong-Ze; Cheng, Shuk H; Zeng, Raymond J

    2015-11-01

    The significance of ANME-2d in methane sink in the environment has been overlooked, and there was no any study evaluating the distribution of ANME-2d in the environment. New primers were thus needed to be designed for following research. In this paper, a pair of primers (DP397F and DP569R) was designed to quantify ANME-2d. The specificity and amplification efficiency of this primer pair were acceptable. PCR amplification of another pair of primers (DP142F and DP779R) generated a single, bright targeted band from the enrichment sample, but yielded faint, multiple bands from the environmental samples. Nested PCR was conducted using the primers DP142F/DP779R in the first round and DP142F/DP569R in the second round, which generated a bright targeted band. Further phylogenetic analysis showed that these targeted bands were ANME-2d-related sequences. Real-time PCR showed that the copies of the 16s ribosomal RNA gene of ANME-2d in these samples ranged from 3.72 × 10(4) to 2.30 × 10(5) copies μg(-1) DNA, indicating that the percentage of ANME-2d was greatest in a polluted river sample and least in a rice paddy sample. These results demonstrate that the newly developed real-time PCR primers could sufficiently quantify ANME-2d and that nested PCR with an appropriate combination of the new primers could successfully detect ANME-2d in environmental samples; the latter finding suggests that ANME-2d may spread in environments.

  8. Can selection on nest size from nest predation explain the latitudinal gradient in clutch size?

    USGS Publications Warehouse

    Biancucci, L.; Martin, T.E.

    2010-01-01

    1. Latitudinal variation in clutch sizes of birds is a well described, but poorly understood pattern. Many hypotheses have been proposed, but few have been experimentally tested, and none have been universally accepted by researchers. 2. The nest size hypothesis posits that higher nest predation in the tropics favours selection for smaller nests and thereby constrains clutch size by shrinking available space for eggs and/or nestlings in the nest. We tested this hypothesis with an experiment in a tropical forest and a comparative study between temperate and tropical field sites. 3. Specifically, we tested if: (i) predation increased with nest size; (ii) tropical birds had smaller nests controlled for body size; and (iii) clutch size was explained by nest size controlled for body size. 4. Experimental swapping of nests of different sizes showed that nest predation increased with nest size in the tropical site. Moreover, nest predation rates were higher in species with larger nests in both sites. However, nest size, corrected for body mass and phylogeny, did not differ between sites and was not related to clutch size between sites. 5. Hence, nest predation can exert selection on nest size as predicted by the hypothesis. Nest size increased with adult body mass, such that adult size might indirectly influence reproductive success through effects on nest size and nest predation risk. Ultimately, however, selection from nest predation on nest size does not explain the smaller clutch sizes typical of the tropics.

  9. Branched modular primers in DNA sequencing

    SciTech Connect

    Mugasimangalam, R.C.; Shmulevitz, M. |; Ramanathan, V.

    1997-08-01

    The need to synthesize new sequencing primers, such as in primer walking, can be eliminated by assembling modular primers from oligonucleotide modules selected from presynthesized libraries. Our earlier modular primers consisted of 5-mers, 6-mers or 7-mers, annealing to the template contiguously with each other. Here we introduce a novel {open_quotes}branched{close_quotes} type of modular primer with a distinctly different specificity mechanism. The concept of a {open_quotes}branched{close_quotes} primer involves modules that are physically linked by annealing to each other as well as to the target, forming a branched structure of the 3-way junction type. While contiguous modular primers are made specific by the preference of the polymerase for longer primer, branched primers, in contrast, owe their specificity to cooperative annealing of their modules to the intended site on the template. This cooperativity of annealing to the template is provided by mutually complementary segments in the two modules that bind each other. Thus the primer-template complex is no longer limited to linear sequences, but acquires another, second dimension giving the modular primer new functionality.

  10. Nested PCR protocol for the rapid detection of Escherichia coli in potable water.

    PubMed

    Juck, D; Ingram, J; Prévost, M; Coallier, J; Greer, C

    1996-08-01

    A rapid and sensitive method for the detection of low levels of bacteria in potable water was developed. The fecal indicator bacterium Escherichia coli was used as the test organism in a filtration concentration - nested polymerase chain reaction (PCR) protocol, combined with ethidium bromide visualization of PCR products. Two sets of primers were designed from the E. coli specific beta-glucuronidase gene (uidA), the primary pair producing a 486-bp fragment that was used as template for the nested primer pair delineating a 186-bp fragment. This protocol can detect 1-10 bacterial cells/50 mL water sample within 6-8 h, in contrast to traditional culturing or Southern hybridization methods which require 2-3 days for results.

  11. Negotiations Primer.

    ERIC Educational Resources Information Center

    New Jersey Principals and Supervisors Association, Trenton.

    This booklet provides step-by-step advice for New Jersey school administrators on the organization of bargaining units and on negotiation. The section covering organizing reviews briefly the rights of school employee associations to negotiate, the values of collective negotiation, and the specific requirements and procedures involved in…

  12. Diskette Primer.

    ERIC Educational Resources Information Center

    Chien, Philip

    1986-01-01

    This detailed look into proper diskette maintenance presents general precautions on temperature, humidity, lifespan, labeling, storage, and disk drive maintenance. The Apple UniDisk 3.5 disk drive is described, tips on purchasing diskettes are presented, and information on specific data retrieval and backup software and diskettes is included. (MBR)

  13. Specific primer sets used to amplify by PCR the hepatitis B virus overlapping S/Pol region select different viral variants.

    PubMed

    Cuestas, M L; Mathet, V L; Oubiña, J R

    2012-10-01

    PCR detection of viral genomes has provided new insights into viral diagnosis. Nowadays, it is the most frequently used nucleic acid testing (qualitative and quantitative) technique. The aim of this study was to analyse the major circulating hepatitis B virus (HBV) variants PCR-amplified by three sets of primers in a patient infected with genotype E. The HBV S/Pol overlapping genomic region was amplified from the serum of an infected child using three primer sets previously described. Sequence analysis corresponding to the HBV S/Pol region revealed the presence of different viral populations depending on the set of primers used. D144A S-escape mutant was detected with two of the primer sets, while the rtL217R mutant within the Pol - conferring resistance to Adefovir - could be picked up with a different pair of primer sets. This study undoubtedly implies that the description of viral polymorphisms should be stated together with the sequence of the primers used for PCR amplification when studies of escape and/or antiviral-resistant HBV mutants are carried out.

  14. Specific primer sets used to amplify by PCR the hepatitis B virus overlapping S/Pol region select different viral variants.

    PubMed

    Cuestas, M L; Mathet, V L; Oubiña, J R

    2012-10-01

    PCR detection of viral genomes has provided new insights into viral diagnosis. Nowadays, it is the most frequently used nucleic acid testing (qualitative and quantitative) technique. The aim of this study was to analyse the major circulating hepatitis B virus (HBV) variants PCR-amplified by three sets of primers in a patient infected with genotype E. The HBV S/Pol overlapping genomic region was amplified from the serum of an infected child using three primer sets previously described. Sequence analysis corresponding to the HBV S/Pol region revealed the presence of different viral populations depending on the set of primers used. D144A S-escape mutant was detected with two of the primer sets, while the rtL217R mutant within the Pol - conferring resistance to Adefovir - could be picked up with a different pair of primer sets. This study undoubtedly implies that the description of viral polymorphisms should be stated together with the sequence of the primers used for PCR amplification when studies of escape and/or antiviral-resistant HBV mutants are carried out. PMID:22967107

  15. Swine Leukocyte Antigen (SLA) class I allele typing of Danish swine herds and identification of commonly occurring haplotypes using sequence specific low and high resolution primers.

    PubMed

    Pedersen, Lasse Eggers; Jungersen, Gregers; Sorensen, Maria Rathmann; Ho, Chak-Sum; Vadekær, Dorte Fink

    2014-12-15

    The swine major histocompatibility complex (MHC) genomic region (SLA) is extremely polymorphic comprising high numbers of different alleles, many encoding a distinct MHC class I molecule, which binds and presents endogenous peptides to circulating T cells of the immune system. Upon recognition of such peptide-MHC complexes (pMHC) naïve T cells can become activated and respond to a given pathogen leading to its elimination and the generation of memory cells. Hence SLA plays a crucial role in maintaining overall adaptive immunologic resistance to pathogens. Knowing which SLA alleles that are commonly occurring can be of great importance in regard to future vaccine development and the establishment of immune protection in swine through broad coverage, highly specific, subunit based vaccination against viruses such as swine influenza, porcine reproductive and respiratory syndrome virus, vesicular stomatitis virus, foot-and-mouth-disease virus and others. Here we present the use of low- and high-resolution PCR-based typing methods to identify individual and commonly occurring SLA class I alleles in Danish swine. A total of 101 animals from seven different herds were tested, and by low resolution typing the top four most frequent SLA class I alleles were those of the allele groups SLA-3*04XX, SLA-1*08XX, SLA-2*02XX, and SLA-1*07XX, respectively. Customised high resolution primers were used to identify specific alleles within the above mentioned allele groups as well as within the SLA-2*05XX allele group. Our studies also suggest the most common haplotype in Danish pigs to be Lr-4.0 expressing the SLA-1*04XX, SLA-2*04XX, and SLA-3*04XX allele combination.

  16. Laser Doppler velocimetry primer

    NASA Technical Reports Server (NTRS)

    Bachalo, William D.

    1985-01-01

    Advanced research in experimental fluid dynamics required a familiarity with sophisticated measurement techniques. In some cases, the development and application of new techniques is required for difficult measurements. Optical methods and in particular, the laser Doppler velocimeter (LDV) are now recognized as the most reliable means for performing measurements in complex turbulent flows. And such, the experimental fluid dynamicist should be familiar with the principles of operation of the method and the details associated with its application. Thus, the goals of this primer are to efficiently transmit the basic concepts of the LDV method to potential users and to provide references that describe the specific areas in greater detail.

  17. [DNA amplification using PCR with abutting primers].

    PubMed

    Garafutdinov, R R; Galimova, A A; Sakhabutdinova, A R; Vakhitov, V A; Chemeris, A V

    2015-01-01

    DNA analysis of ñîmplex biological objects (wastewater, soil, archaeological and forensic samples, etc.) is currently of great interest. DNA of these objects is characterized by low suitability for research due to the violation of its integrity and chemical structure; thus, the detection of specific nucleic acid fragments can be achieved by PCR with contiguous primers. In this paper, we present the results that clarify the specific characteristics of PCR with abutting primers. The 3'-ends of these primers are annealed at adjacent nucleotides of complementary chains of DNA target. It has been shown that the proximity of primers enables the formation of specific reaction products with a higher sensitivity and less reaction time. Using artificially damaged DNA and DNA from the soil we demonstrated that the abutting primers provide assured detection of specific DNA fragments. The results of this work may be taken into account in PCR with degraded (fragmented) DNA.

  18. Evaluating Primers for Profiling Anaerobic Ammonia Oxidizing Bacteria within Freshwater Environments

    PubMed Central

    Sonthiphand, Puntipar; Neufeld, Josh D.

    2013-01-01

    Anaerobic ammonia oxidizing (anammox) bacteria play an important role in transforming ammonium to nitrogen gas and contribute to fixed nitrogen losses in freshwater environments. Understanding the diversity and abundance of anammox bacteria requires reliable molecular tools, and these are not yet well established for these important Planctomycetes. To help validate PCR primers for the detection of anammox bacteria within freshwater ecosystems, we analyzed representative positive controls and selected samples from Grand River and groundwater sites, both from Ontario, Canada. The objectives of this study were to identify a suitable anammox denaturing gradient gel electrophoresis (DGGE) fingerprint method by using GC-clamp modifications to existing primers, and to verify the specificity of anammox-specific primers used for DGGE, cloning and qPCR methods. Six primer combinations were tested from four published primer sets (i.e. A438f/A684r, Amx368f/Amx820r, An7f/An1388r, and Pla46/1392r) for both direct and nested PCR amplifications. All PCR products were run subsequently on DGGE gels to compare the resulting patterns. Two anammox-specific primer combinations were also used to generate clone libraries and quantify anammox bacterial 16S rRNA genes with qPCR. The primer set A438f/A684r was highly specific to anammox bacteria, provided reliable DGGE fingerprints and generated a high proportion of anammox-related clones. A second primer set (Amx368f/Amx820r) was anammox specific, based on clone library analysis, but PCR products from different candidate species of anammox bacteria resolved poorly using DGGE analysis. Both DGGE and cloning results revealed that Ca. Brocadia and an uncharacterized anammox bacterial cluster represented the majority of anammox bacteria found in Grand River sediment and groundwater samples, respectively. Together, our results demonstrate that although Amx368f/Amx820r was useful for anammox-specific qPCR and clone library analysis, A438f/A684r

  19. Evaluating primers for profiling anaerobic ammonia oxidizing bacteria within freshwater environments.

    PubMed

    Sonthiphand, Puntipar; Neufeld, Josh D

    2013-01-01

    Anaerobic ammonia oxidizing (anammox) bacteria play an important role in transforming ammonium to nitrogen gas and contribute to fixed nitrogen losses in freshwater environments. Understanding the diversity and abundance of anammox bacteria requires reliable molecular tools, and these are not yet well established for these important Planctomycetes. To help validate PCR primers for the detection of anammox bacteria within freshwater ecosystems, we analyzed representative positive controls and selected samples from Grand River and groundwater sites, both from Ontario, Canada. The objectives of this study were to identify a suitable anammox denaturing gradient gel electrophoresis (DGGE) fingerprint method by using GC-clamp modifications to existing primers, and to verify the specificity of anammox-specific primers used for DGGE, cloning and qPCR methods. Six primer combinations were tested from four published primer sets (i.e. A438f/A684r, Amx368f/Amx820r, An7f/An1388r, and Pla46/1392r) for both direct and nested PCR amplifications. All PCR products were run subsequently on DGGE gels to compare the resulting patterns. Two anammox-specific primer combinations were also used to generate clone libraries and quantify anammox bacterial 16S rRNA genes with qPCR. The primer set A438f/A684r was highly specific to anammox bacteria, provided reliable DGGE fingerprints and generated a high proportion of anammox-related clones. A second primer set (Amx368f/Amx820r) was anammox specific, based on clone library analysis, but PCR products from different candidate species of anammox bacteria resolved poorly using DGGE analysis. Both DGGE and cloning results revealed that Ca. Brocadia and an uncharacterized anammox bacterial cluster represented the majority of anammox bacteria found in Grand River sediment and groundwater samples, respectively. Together, our results demonstrate that although Amx368f/Amx820r was useful for anammox-specific qPCR and clone library analysis, A438f/A684r

  20. Single-tube nested PCR in the diagnosis of tuberculosis.

    PubMed Central

    Chan, C M; Yuen, K Y; Chan, K S; Yam, W C; Yim, K H; Ng, W F; Ng, M H

    1996-01-01

    AIMS: To evaluate the usefulness of a single-tube nested polymerase chain reaction (PCR) assay in the diagnosis of tuberculosis in 1497 pulmonary and 536 extrapulmonary specimens. METHODS: A single-tube nested PCR, utilising two sets of primers with different melting temperatures (88 degrees C for external primers; 70 degrees C for internal primers) to augment sensitivity and specificity without increasing the risk of amplicon contamination, was evaluated. Specimens were initially tested for the repetitive IS6110 sequences and if negative, retested for the universal 38 kilodalton sequence and for inhibitors. dUTP/Uracil-N-glycosylase and Instagene treatment were used to minimise contamination and the effect of inhibitors, respectively. RESULTS: Using culture as the gold standard, the overall sensitivity of the assay was 89% for pulmonary and 42% for extrapulmonary specimens. Sensitivity varied greatly with respect to sample type (92% for follow up specimens from a chest hospital and 70% for non-follow up specimens from a general hospital). The smear positivity rates were 15% for extrapulmonary specimens, and 69% and 45%, respectively, for follow up and non-follow up specimens from pulmonary sites. Specificity was 99.7%. Inhibitors were present more frequently in extrapulmonary than in pulmonary specimens (13.4% v 2.7%). CONCLUSION: Despite the high sensitivity of the PCR assay for the diagnosis of tuberculosis in pulmonary specimens, it was less effective in the extrapulmonary samples. This is probably because of the lower bacterial load in extrapulmonary specimens, the presence of more inhibitors adversely affecting the PCR assay and the higher volume of specimens used for culture. Images PMID:8655703

  1. Myelin basic protein-specific T lymphocyte repertoire in multiple sclerosis. Complexity of the response and dominance of nested epitopes due to recruitment of multiple T cell clones.

    PubMed Central

    Meinl, E; Weber, F; Drexler, K; Morelle, C; Ott, M; Saruhan-Direskeneli, G; Goebels, N; Ertl, B; Jechart, G; Giegerich, G

    1993-01-01

    The human T cell response to the myelin basic protein (MBP) has been studied with respect to T cell receptor (TCR) usage, HLA class II restriction elements, and epitope specificity using a total of 215 long-term MBP-specific T cell lines (TCL) isolated from the peripheral blood of 13 patients with multiple sclerosis (MS) and 10 healthy donors. In most donors, the anti-MBP response was exceedingly heterogeneous. Using a panel of overlapping synthetic peptides spanning the entire length of human MBP, at least 26 epitopes recognized by human TCL could be distinguished. The MBP domain most commonly recognized was sequence 80-105 (31% of MS TCL, and 24% of control TCL). Sequence 29-48 was recognized more frequently by control-derived TCL (24%) than by TCL from MS patients (5%). The MBP epitopes were recognized in the context of DRB1 *0101, DRB5*0101, DRB1*1501, DRB1*0301, DRB1*0401, DRB1*1402, and DRB3*0102, as demonstrated using a panel of DR gene-transfected L cells. The TCR gene usage was also heterogeneous. V beta 5.2, a peptide of which is currently being used in a clinical trial for treatment of MS patients, was expressed by only one of our TCL. However, within this complex pattern of MBP-specific T cell responses, a minority of MS patients were found to exhibit a more restricted response with respect to their TCL epitope specificity. In these patients 75-87% of the TCL responded to a single, patient-specific cluster of immunodominant T cell epitopes located within a small (20-amino acid) domain of MBP. These nested clusters of immunodominant epitopes were noted within the amino acids 80-105, 108-131, and 131-153. The T cell response to the immunodominant epitopes was not monoclonal, but heterogeneous, with respect to fine specificity, TCR usage, and even HLA restriction. In one patient (H.K.), this restricted epitope profile remained stable for > 2 yr. The TCR beta chain sequences of TCL specific for the immunodominant region of HK are consistent with an

  2. Identification of Pythium insidiosum by nested PCR in cutaneous lesions of Brazilian horses and rabbits.

    PubMed

    Botton, Sonia A; Pereira, Daniela I B; Costa, Mateus M; Azevedo, Maria Isabel; Argenta, Juliana S; Jesus, Francielli P K; Alves, Sydney Hartz; Santurio, Janio Morais

    2011-04-01

    Pythium insidiosum is a fungus-like organism present in subtropical and tropical areas, such as Brazil, known to infect humans and various animal species. P. insidiosum is the etiological agent of pythiosis, an emerging and granulomatous disease characterized mainly by cutaneous and subcutaneous lesions in horses, the principal species affected. Accurate diagnosis of pythiosis and identification of its causal agent by microbiological and serological tests can be often difficult and inconclusive principally for horses and humans. The aim of this study was to evaluate the application of the previously described P. insidiosum-specific nested polymerase chain reaction (PCR) assay to directly detect P. insidiosum DNA in clinical and experimental lesions. Universal fungal primers (ITS1 and ITS4) were used during the first-round of PCR to amplify ITS1, 5.8s, and ITS2. A second-round of PCR was conducted with P. insidiosum-specific primers (PI1 and PI2) to amplify a variable region within this ITS1. In this study, a total of 21 equine clinical samples (kunkers) and 28 specimens from experimentally infected rabbits were analyzed by nested PCR. The first-round of PCR generated 800-base pair products, and the second-round produced 105-base pair amplicons for each P. insidiosum-specific sample; no amplicons were generated in negative control samples. Our results suggest that nested PCR is an important and efficient tool for diagnosis of both endemic (horse samples) and experimental (rabbit samples) pythiosis. PMID:21188592

  3. The feather-degrading bacterial community in two soils as revealed by a specific primer targeting serine-type keratinolytic proteases.

    PubMed

    Gu, Zhenhong; Zhu, Honghui; Xie, Xiaolin; Wang, Yonghong; Liu, Xiaodi; Yao, Qing

    2016-10-01

    Feather waste represents a huge resource of protein, but is underutilized due to its recalcitrant nature. Feather-degrading bacteria can biologically degrade feathers and have great potential for industries. In this study, we first designed a primer set (BC) suitable for exploring the diversity of the keratinolytic bacterial community with denatured gradient gel electrophoresis (DGGE). With the BC primer set, the difference in the keratinolytic bacterial community between a feather-dumping (FD) soil and a non feather-dumping (NFD) soil and the influence of feather addition (enrichment culture) on the keratinolytic bacterial community were investigated. DGGE and sequencing showed that keratinolytic bacteria in these soils belong to 2 phyla (Actinobacteria and Proteobacteria) and 9 genera (Micromonospora, Verrucosispora, Actinopolymorpha, Knoellia, Hyalangium, Stigmatella, Archangium, Cystobacter, and Luteimonas). Feather addition decreased the species richness of the keratinolytic bacteria in FD soil, but greatly increased the diversity, species richness and abundance in NFD soil. Moreover, feather addition to NFD soil induced some keratinolytic bacteria that were absent in all of the other soils. Collectively, these data indicate that keratinolytic bacteria are diverse in both FD and NFD soil, and some novel keratinolytic bacteria taxa might be revealed by using the BC primer set.

  4. The feather-degrading bacterial community in two soils as revealed by a specific primer targeting serine-type keratinolytic proteases.

    PubMed

    Gu, Zhenhong; Zhu, Honghui; Xie, Xiaolin; Wang, Yonghong; Liu, Xiaodi; Yao, Qing

    2016-10-01

    Feather waste represents a huge resource of protein, but is underutilized due to its recalcitrant nature. Feather-degrading bacteria can biologically degrade feathers and have great potential for industries. In this study, we first designed a primer set (BC) suitable for exploring the diversity of the keratinolytic bacterial community with denatured gradient gel electrophoresis (DGGE). With the BC primer set, the difference in the keratinolytic bacterial community between a feather-dumping (FD) soil and a non feather-dumping (NFD) soil and the influence of feather addition (enrichment culture) on the keratinolytic bacterial community were investigated. DGGE and sequencing showed that keratinolytic bacteria in these soils belong to 2 phyla (Actinobacteria and Proteobacteria) and 9 genera (Micromonospora, Verrucosispora, Actinopolymorpha, Knoellia, Hyalangium, Stigmatella, Archangium, Cystobacter, and Luteimonas). Feather addition decreased the species richness of the keratinolytic bacteria in FD soil, but greatly increased the diversity, species richness and abundance in NFD soil. Moreover, feather addition to NFD soil induced some keratinolytic bacteria that were absent in all of the other soils. Collectively, these data indicate that keratinolytic bacteria are diverse in both FD and NFD soil, and some novel keratinolytic bacteria taxa might be revealed by using the BC primer set. PMID:27562599

  5. Polyacid macromolecule primers

    DOEpatents

    Sugama, Toshifumi.

    1989-12-26

    Hydrophilic polyacids are described, such as macromolecules of polyitaconic acid and polyacrylic acid, where such macromolecules have molecular weights >50,000 as primers between a polymeric top coating, such as polyurethane, and an oxidized aluminum or aluminum alloy. A near monolayer of primer is used in polymeric adhesive/oxidized aluminum adhered joint systems in 0.05% primer concentration to give superior results in standard peel tests. 2 figs.

  6. Polyacid macromolecule primers

    DOEpatents

    Sugama, Toshifumi

    1989-01-01

    Hydrophylic polyacids, such as macromolecules of polyitaconic acid and polyacrylic acid, where such macromolecules have molecular weights >50,000 as primers between a polymeric top coating, such as polyurethane, and an oxidized aluminum or aluminum alloy. A near monolayer of primer is used in polymeric adhesive/oxidized aluminum adhered joint systems in 0.05% primer concentration to give superior results in standard peel tests.

  7. Detection of HLA-DRB1 microchimerism using nested polymerase chain reaction and single-strand conformation polymorphism analysis.

    PubMed

    Song, Eun Young; Chung, Hye Yoon; Joo, Shin Young; Roh, Eun Youn; Seong, Moon-Woo; Shin, Yunsu; Park, Myoung Hee

    2012-03-01

    For the detection of microchimerism, molecular methods detecting donor-specific HLA-DRB1 alleles in the recipient are most commonly used. Nested polymerase chain reaction sequence specific primer (nested PCR-SSP) methods widely used to increase the sensitivity of detection have been reported to give frequent false-positive reactions. We have developed a new method combining nested PCR with single-strand conformation polymorphism analysis (nested PCR-SSCP) and tested the 1 to 0.00001% level of microchimerism for 27 different HLA-DRB1 alleles. For most (26/27) of the HLA-DRB1 alleles tested, this method could detect 0.01 to 0.001% of microchimerism and its sensitivity was equal to or better than that of nested PCR-SSP tested in parallel. Its specificity was verified by visualizing particular DRB1-specific SSCP bands under test. Nested PCR-SSP indicated frequent false-positive reactions, mainly caused by nonspecific amplification of DRB3/B4/B5 alleles present in the major (recipient) DNAs. We have compared a real-time quantitative PCR for non-human leukocyte antigen (HLA) target (insertion/deletion marker) using a commercial kit (AlleleSEQR Chimerism assay), and its microchimerism detection sensitivity (around 0.1%) was 1 step (10 times) lower than that of nested PCR-SSP or -SSCP methods for HLA-DRB1 alleles. We validated that the newly designed nested PCR-SSCP affords good sensitivity and specificity and may be useful for studying microchimerism in clinical settings.

  8. Development of a Highly Sensitive Nested-PCR Procedure Using a Single Closed Tube for Detection of Erwinia amylovora in Asymptomatic Plant Material

    PubMed Central

    Llop, Pablo; Bonaterra, Anna; Peñalver, Javier; López, María M.

    2000-01-01

    A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of E. amylovora, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. The specificity and sensitivity were greater than those of standard PCR procedures that used a single primer pair. The presence of inhibitors in plant material, very common in E. amylovora hosts, is overcome with this system in combination with a simple DNA extraction protocol because it eliminates many of the inhibitory compounds. In addition, it needs a very small sample volume (1 μl of DNA extracted). With 83 samples of naturally infected material, this method achieved better results than any other PCR technique: standard PCR detected 55% of positive samples, two-tube nested PCR detected 71% of positive samples, and nested PCR in a single closed tube detected 78% of positive samples. When analyzing asymptomatic plant material, the number of positive samples detected by the developed nested PCR was also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method is proposed for the detection of endophytic and epiphytic populations of E. amylovora in epidemiological studies and for routine use in quarantine surveys, due to its high sensitivity, specificity, speed, and simplicity. PMID:10788384

  9. Education Vouchers. A Primer.

    ERIC Educational Resources Information Center

    Richter, Philip C.; Hollender, Mary Jo

    This document is intended to serve both as a primer and as an annotated bibliography about educational vouchers. As a primer, it introduces the reader to the concept of vouchers and to the variety of issues--including political, economic, legal, and educational issues--associated with vouchers. As an annotated bibliography, it provides a summary…

  10. Detection and resolution of Cryptosporidium species and species mixtures by genus-specific nested PCR-restriction fragment length polymorphism analysis, direct sequencing, and cloning.

    PubMed

    Ruecker, Norma J; Hoffman, Rebecca M; Chalmers, Rachel M; Neumann, Norman F

    2011-06-01

    Molecular methods incorporating nested PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene of Cryptosporidium species were validated to assess performance based on limit of detection (LoD) and for detecting and resolving mixtures of species and genotypes within a single sample. The 95% LoD was determined for seven species (Cryptosporidium hominis, C. parvum, C. felis, C. meleagridis, C. ubiquitum, C. muris, and C. andersoni) and ranged from 7 to 11 plasmid template copies with overlapping 95% confidence limits. The LoD values for genomic DNA from oocysts on microscope slides were 7 and 10 template copies for C. andersoni and C. parvum, respectively. The repetitive nested PCR-RFLP slide protocol had an LoD of 4 oocysts per slide. When templates of two species were mixed in equal ratios in the nested PCR-RFLP reaction mixture, there was no amplification bias toward one species over another. At high ratios of template mixtures (>1:10), there was a reduction or loss of detection of the less abundant species by RFLP analysis, most likely due to heteroduplex formation in the later cycles of the PCR. Replicate nested PCR was successful at resolving many mixtures of Cryptosporidium at template concentrations near or below the LoD. The cloning of nested PCR products resulted in 17% of the cloned sequences being recombinants of the two original templates. Limiting-dilution nested PCR followed by the sequencing of PCR products resulted in no sequence anomalies, suggesting that this method is an effective and accurate way to study the species diversity of Cryptosporidium, particularly for environmental water samples, in which mixtures of parasites are common.

  11. Eggs in the Freezer: Energetic Consequences of Nest Site and Nest Design in Arctic Breeding Shorebirds

    PubMed Central

    Tulp, Ingrid; Schekkerman, Hans; de Leeuw, Joep

    2012-01-01

    Birds construct nests for several reasons. For species that breed in the Arctic, the insulative properties of nests are very important. Incubation is costly there and due to an increasing surface to volume ratio, more so in smaller species. Small species are therefore more likely to place their nests in thermally favourable microhabitats and/or to invest more in nest insulation than large species. To test this hypothesis, we examined characteristics of nests of six Arctic breeding shorebird species. All species chose thermally favourable nesting sites in a higher proportion than expected on the basis of habitat availability. Site choice did not differ between species. Depth to frozen ground, measured near the nests, decreased in the course of the season at similar non-species-specific speeds, but this depth increased with species size. Nest cup depth and nest scrape depth (nest cup without the lining) were unrelated to body mass (we applied an exponent of 0.73, to account for metabolic activity of the differently sized species). Cup depth divided by diameter2 was used as a measure of nest cup shape. Small species had narrow and deep nests, while large species had wide shallow nests. The thickness of nest lining varied between 0.1 cm and 7.6 cm, and decreased significantly with body mass. We reconstruct the combined effect of different nest properties on the egg cooling coefficient using previously published quantitative relationships. The predicted effect of nest cup depth and lining depth on heat loss to the frozen ground did not correlate with body mass, but the sheltering effect of nest cup diameter against wind and the effects of lining material on the cooling coefficient increased with body mass. Our results suggest that small arctic shorebirds invest more in the insulation of their nests than large species. PMID:22701596

  12. Eggs in the freezer: energetic consequences of nest site and nest design in Arctic breeding shorebirds.

    PubMed

    Tulp, Ingrid; Schekkerman, Hans; de Leeuw, Joep

    2012-01-01

    Birds construct nests for several reasons. For species that breed in the Arctic, the insulative properties of nests are very important. Incubation is costly there and due to an increasing surface to volume ratio, more so in smaller species. Small species are therefore more likely to place their nests in thermally favourable microhabitats and/or to invest more in nest insulation than large species. To test this hypothesis, we examined characteristics of nests of six Arctic breeding shorebird species. All species chose thermally favourable nesting sites in a higher proportion than expected on the basis of habitat availability. Site choice did not differ between species. Depth to frozen ground, measured near the nests, decreased in the course of the season at similar non-species-specific speeds, but this depth increased with species size. Nest cup depth and nest scrape depth (nest cup without the lining) were unrelated to body mass (we applied an exponent of 0.73, to account for metabolic activity of the differently sized species). Cup depth divided by diameter(2) was used as a measure of nest cup shape. Small species had narrow and deep nests, while large species had wide shallow nests. The thickness of nest lining varied between 0.1 cm and 7.6 cm, and decreased significantly with body mass. We reconstruct the combined effect of different nest properties on the egg cooling coefficient using previously published quantitative relationships. The predicted effect of nest cup depth and lining depth on heat loss to the frozen ground did not correlate with body mass, but the sheltering effect of nest cup diameter against wind and the effects of lining material on the cooling coefficient increased with body mass. Our results suggest that small arctic shorebirds invest more in the insulation of their nests than large species. PMID:22701596

  13. Testing ecological and behavioral correlates of nest predation

    USGS Publications Warehouse

    Fontaine, J.J.; Martel, M.; Markland, H.M.; Niklison, Alina M.; Decker, Karie L.; Martin, T.E.

    2007-01-01

    Variation in nest predation rates among bird species are assumed to reflect differences in risk that are specific to particular nest sites. Theoretical and empirical studies suggest that parental care behaviors can evolve in response to nest predation risk and thereby differ among ecological conditions that vary in inherent risk. However, parental care also can influence predation risk. Separating the effects of nest predation risk inherent to a nest site from the risk imposed by parental strategies is needed to understand the evolution of parental care. Here we identify correlations between risks inherent to nest sites, and risk associated with parental care behaviors, and use an artificial nest experiment to assess site-specific differences in nest predation risk across nesting guilds and between habitats that differed in nest predator abundance. We found a strong correlation between parental care behaviors and inherent differences in nest predation risk, but despite the absence of parental care at artificial nests, patterns of nest predation risk were similar for real and artificial nests both across nesting guilds and between predator treatments. Thus, we show for the first time that inherent risk of nest predation varies with nesting guild and predator abundance independent of parental care. ?? Oikos.

  14. Helicobacter pylori in dental plaque: a comparison of different PCR primer sets.

    PubMed

    Song, Q; Haller, B; Schmid, R M; Adler, G; Bode, G

    1999-03-01

    This study was designed to compare different primer sets for PCR analysis of H. pylori in the same series of 40 dental plaque samples. Three pairs of primers, HPU1/HPU2, HP1/HP2, and EHC-U/EHC-L, directed to the urease A gene, 16S rRNA gene, or 860-bp DNA of H. pylori, respectively, were used. Our results demonstrate that EHC-L/EHC-U were more specific and sensitive for H. pylori added to saliva or dental plaque than HPU1/HPU2 and HP1/HP2. The detection rates for H. pylori DNA in dental plaque samples from randomly selected adult patients from the Dental Clinic of the University of Ulm were 26.5% (9/34) for HPU1/HPU2, 78.9% (30/38) for HP1/HP2, and 100% (40/40) for EHC-U/EHC-L (P < 0.001). Nested PCR using primers directed to the 860-bp DNA of H. pylori further confirmed the presence of H. pylori DNA (40/40) in all these samples. Our results indicate that primers EHC-U/EHC-L are to be recommended for PCR detection of H. pylori in the oral cavity.

  15. PHUSER (Primer Help for USER): a novel tool for USER fusion primer design.

    PubMed

    Olsen, Lars Rønn; Hansen, Niels Bjørn; Bonde, Mads Tvillinggaard; Genee, Hans Jasper; Holm, Dorte Koefoed; Carlsen, Simon; Hansen, Bjarne Gram; Patil, Kiran Raosaheb; Mortensen, Uffe Hasbro; Wernersson, Rasmus

    2011-07-01

    Uracil-Specific Exision Reagent (USER) fusion is a recently developed technique that allows for assembly of multiple DNA fragments in a few simple steps. However, designing primers for USER fusion is both tedious and time consuming. Here, we present the Primer Help for USER (PHUSER) software, a novel tool for designing primers specifically for USER fusion and USER cloning applications. We also present proof-of-concept experimental validation of its functionality. PHUSER offers quick and easy design of PCR optimized primers ensuring directionally correct fusion of fragments into a plasmid containing a customizable USER cassette. Designing primers using PHUSER ensures that the primers have similar annealing temperature (T(m)), which is essential for efficient PCR. PHUSER also avoids identical overhangs, thereby ensuring correct order of assembly of DNA fragments. All possible primers are individually analysed in terms of GC content, presence of GC clamp at 3'-end, the risk of primer dimer formation, the risk of intra-primer complementarity (secondary structures) and the presence of polyN stretches. Furthermore, PHUSER offers the option to insert linkers between DNA fragments, as well as highly flexible cassette options. PHUSER is publicly available at http://www.cbs.dtu.dk/services/phuser/. PMID:21622660

  16. Detection and Typing of Human Papilloma Viruses by Nested Multiplex Polymerase Chain Reaction Assay in Cervical Cancer

    PubMed Central

    Jalal Kiani, Seyed; Shatizadeh Malekshahi, Somayeh; Yousefi Ghalejoogh, Zohreh; Ghavvami, Nastaran; Shafiei Jandaghi, Nazanin Zahra; Shahsiah, Reza; Jahanzad, Isa; Yavarian, Jila

    2015-01-01

    Background: Cervical cancer is the leading cause of death from cancer in under-developed countries. Human papilloma virus (HPV) 16 and 18 are the most prevalent types associated with carcinogenesis in the cervix. Conventional Polymerase Chain Reaction (PCR), type-specific and consensus primer-based PCR followed by sequencing, Restriction Fragment Length Polymorphism (RFLP) or hybridization by specific probes are common methods for HPV detection and typing. In addition, some researchers have developed a multiplex PCR for simultaneous detection and typing of different HPVs. Objectives: The aim of the present study was to investigate the prevalence of HPV infection and its types in cervical Squamous Cell Carcinoma (SCC) using the Nested Multiplex PCR (NMPCR) assay. Patients and Methods: Sixty-six samples with histologically confirmed SCC were evaluated. Total DNA was isolated by phenol–chloroform extraction and ethanol precipitation. Nested multiplex PCR was performed with first-round PCR by GP-E6/E7 consensus primers for amplification of the genomic DNA of all known mucosal HPV genotypes and second-round PCR by type-specific multiplex PCR primer cocktails. Results: Human papilloma virus infection was detected in 78.8% of samples, with the highest prevalence of HPV 16 (60.6%) while concurrent infections with two types was detected in 10.6%. Conclusions: The NMPCR assay is more convenient and easy for analysis of results, which is important for fast diagnosis and patient management, in a type-specific manner. PMID:26865940

  17. Waterbird nest-site selection is influenced by neighboring nests and island topography

    USGS Publications Warehouse

    Hartman, Christopher; Ackerman, Josh; Takekawa, John Y.; Herzog, Mark

    2016-01-01

    Avian nest-site selection is influenced by factors operating across multiple spatial scales. Identifying preferred physical characteristics (e.g., topography, vegetation structure) can inform managers to improve nesting habitat suitability. However, social factors (e.g., attraction, territoriality, competition) can complicate understanding physical characteristics preferred by nesting birds. We simultaneously evaluated the physical characteristics and social factors influencing selection of island nest sites by colonial-nesting American avocets (Recurvirostra americana) and Forster's terns (Sterna forsteri) at 2 spatial scales in San Francisco Bay, 2011–2012. At the larger island plot (1 m2) scale, we used real-time kinematics to produce detailed topographies of nesting islands and map the distribution of nests. Nesting probability was greatest in island plots between 0.5 m and 1.5 m above the water surface, at distances <10 m from the water's edge, and of moderately steep (avocets) or flat (terns) slopes. Further, avocet and tern nesting probability increased as the number of nests initiated in adjacent plots increased up to a peak of 11–12 tern nests, and then decreased thereafter. Yet, avocets were less likely to nest in plots adjacent to plots with nesting avocets, suggesting an influence of intra-specific territoriality. At the smaller microhabitat scale, or the area immediately surrounding the nest, we compared topography, vegetation, and distance to nearest nest between nest sites and paired random sites. Topography had little influence on selection of the nest microhabitat. Instead, nest sites were more likely to have vegetation present, and greater cover, than random sites. Finally, avocet, and to a lesser extent tern, nest sites were closer to other active conspecific or heterospecific nests than random sites, indicating that social attraction played a role in selection of nest microhabitat. Our results demonstrate key differences in nest

  18. Quick spacecraft charging primer

    SciTech Connect

    Larsen, Brian Arthur

    2014-03-12

    This is a presentation in PDF format which is a quick spacecraft charging primer, meant to be used for program training. It goes into detail about charging physics, RBSP examples, and how to identify charging.

  19. Characterization of polybacterial clinical samples using a set of group-specific broad-range primers targeting the 16S rRNA gene followed by DNA sequencing and RipSeq analysis

    PubMed Central

    Lekang, Katrine; Langeland, Nina; Wiker, Harald G.

    2011-01-01

    The standard use of a single universal broad-range PCR in direct 16S rDNA sequencing from polybacterial samples leaves the minor constituents at risk of remaining undetected because all bacterial DNA will be competing for the same reagents. In this article we introduce a set of three broad-range group-specific 16S rDNA PCRs that together cover the clinically relevant bacteria and apply them in the investigation of 25 polybacterial clinical samples. Mixed DNA chromatograms from samples containing more than one species per primer group were analysed using RipSeq Mixed (iSentio, Norway), a web-based application for the interpretation of chromatograms containing up to three different species. The group-specific PCRs reduced complexity in the resulting DNA chromatograms and made the assay more sensitive in situations with unequal species concentrations. Together this allowed for identification of a significantly higher number of bacterial species than did standard direct sequencing with a single universal primer pair and RipSeq analysis (95 vs 51). The method could improve microbiological diagnostics for important groups of patients and can be established in any laboratory with experience in direct 16S rDNA sequencing. PMID:21436365

  20. Use of primer selection and restriction enzymes to assess bacterial community diversity in an agricultural soil used for potato production via terminal restriction fragment length polymorphism.

    PubMed

    Fortuna, Ann-Marie; Marsh, Terence L; Honeycutt, C Wayne; Halteman, William A

    2011-08-01

    Terminal restriction fragment length polymorphism (T-RFLP) can be used to assess how land use management changes the dominant members of bacterial communities. We compared T-RFLP profiles obtained via amplification with forward primers (27, 63F) each coupled with the fluorescently labeled reverse primer (1392R) and multiple restriction enzymes to determine the best combination for interrogating soil bacterial populations in an agricultural soil used for potato production. Both primer pairs provide nearly universal recognition of a 1,400-bp sequence of the bacterial domain in the V(1)-V(3) region of the 16S ribosomal RNA (rRNA) gene relative to known sequences. Labeling the reverse primer allowed for direct comparison of each forward primer and the terminal restriction fragments' relative migration units obtained with each primer pair and restriction enzyme. Redundancy analysis (RDA) and nested multivariate analysis of variance (MANOVA) were used to assess the effects of primer pair and choice of restriction enzyme on the measured relative migration units. Our research indicates that the 63F-1392R amplimer pair provides a more complete description with respect to the bacterial communities present in this potato (Solanum tuberosum L.)-barley (Hordeum vulgare L.) rotation over seeded to crimson clover (Trifolium praense L.). Domain-specific 16S rRNA gene primers are rigorously tested to determine their ability to amplify across a target region of the gene. Yet, variability within or between T-RFLP profiles can result from factors independent of the primer pair. Therefore, researchers should use RDA and MANOVA analyses to evaluate the effects that additional laboratory and environmental variables have on bacterial diversity.

  1. Development of a highly sensitive one-tube nested real-time PCR for detecting Mycobacterium tuberculosis.

    PubMed

    Choi, Yeonim; Jeon, Bo-Young; Shim, Tae Sun; Jin, Hyunwoo; Cho, Sang-Nae; Lee, Hyeyoung

    2014-12-01

    Rapid, accurate detection of Mycobacterium tuberculosis is crucial in the diagnosis of tuberculosis (TB), but conventional diagnostic methods have limited sensitivity and specificity or are time consuming. A new highly sensitive nucleic acid amplification test, combined nested and real-time polymerase chain reaction (PCR) in a single tube (one-tube nested real-time PCR), was developed for detecting M. tuberculosis, which takes advantage of two PCR techniques, i.e., nested PCR and real-time PCR. One-tube nested real-time PCR was designed to have two sequential reactions with two sets of primers and dual probes for the insertion sequence (IS) 6110 sequence of M. tuberculosis in a single closed tube. The minimum limits of detection of IS6110 real-time PCR and IS6110 one-tube nested real-time PCR were 100 fg/μL and 1 fg/μL of M. tuberculosis DNA, respectively. AdvanSure TB/non-tuberculous mycobacteria (NTM) real-time PCR, IS6110 real-time PCR, and two-tube nested real-time PCR showed 100% sensitivity and 100% specificity for clinical M. tuberculosis isolates and NTM isolates. In comparison, the sensitivities of AdvanSure TB/NTM real-time PCR, single IS6110 real-time PCR, and one-tube nested real-time PCR were 91% (152/167), 94.6% (158/167), and 100% (167/167) for sputum specimens, respectively. In conclusion, IS6110 one-tube nested real-time PCR is useful for detecting M. tuberculosis due to its high sensitivity and simple manipulation.

  2. Detection of Mycoplasma hyopneumoniae by Air Sampling with a Nested PCR Assay

    PubMed Central

    Stärk, Katharina D. C.; Nicolet, Jacques; Frey, Joachim

    1998-01-01

    This article describes the first successful detection of airborne Mycoplasma hyopneumoniae under experimental and field conditions with a new nested PCR assay. Air was sampled with polyethersulfone membranes (pore size, 0.2 μm) mounted in filter holders. Filters were processed by dissolution and direct extraction of DNA for PCR analysis. For the PCR, two nested pairs of oligonucleotide primers were designed by using an M. hyopneumoniae-specific DNA sequence of a repeated gene segment. A nested PCR assay was developed and used to analyze samples collected in eight pig houses where respiratory problems had been common. Air was also sampled from a mycoplasma-free herd. The nested PCR was highly specific and 104 times as sensitive as a one-step PCR. Under field conditions, the sampling system was able to detect airborne M. hyopneumoniae on 80% of farms where acute respiratory disease was present. No airborne M. hyopneumoniae was detected on infected farms without acute cases. The chance of successful detection was increased if air was sampled at several locations within a room and at a lower air humidity. PMID:9464391

  3. Identification of latent neosporosis in sheep in Tehran, Iran by polymerase chain reaction using primers specific for the Nc-5 gene.

    PubMed

    Arbabi, Mohsen; Abdoli, Amir; Dalimi, Abdolhossein; Pirestani, Majid

    2016-01-01

    Little is known about latent infection and molecular characterisation of Neospora caninum in sheep (Ovis aries). In this study, 330 sheep samples (180 hearts and 150 brains) were analysed for N. caninum DNA by nested polymerase chain reaction (PCR) targeting the Nc-5 gene. Neospora caninum DNA was detected in 3.9% (13/330) of sheep samples. The parasite's DNA was detected in 6.7% of heart samples (12/180) and 0.7% (1/150) of brain samples. No clinical signs were recorded from infected or uninfected animals. Sequencing of the genomic DNA revealed 96% - 99% similarity with each other and 95.15% - 100% similarity with N. caninum sequences deposited in GenBank. To our knowledge, this is the first report on the use of PCR to identify latent neosporosis in sheep in Iran. The results of this study have the potential to contribute to our understanding of the role of N. caninum-infected sheep in the epidemiology of neosporosis. PMID:27543149

  4. Identification of latent neosporosis in sheep in Tehran, Iran by polymerase chain reaction using primers specific for the Nc-5 gene.

    PubMed

    Arbabi, Mohsen; Abdoli, Amir; Dalimi, Abdolhossein; Pirestani, Majid

    2016-01-01

    Little is known about latent infection and molecular characterisation of Neospora caninum in sheep (Ovis aries). In this study, 330 sheep samples (180 hearts and 150 brains) were analysed for N. caninum DNA by nested polymerase chain reaction (PCR) targeting the Nc-5 gene. Neospora caninum DNA was detected in 3.9% (13/330) of sheep samples. The parasite's DNA was detected in 6.7% of heart samples (12/180) and 0.7% (1/150) of brain samples. No clinical signs were recorded from infected or uninfected animals. Sequencing of the genomic DNA revealed 96% - 99% similarity with each other and 95.15% - 100% similarity with N. caninum sequences deposited in GenBank. To our knowledge, this is the first report on the use of PCR to identify latent neosporosis in sheep in Iran. The results of this study have the potential to contribute to our understanding of the role of N. caninum-infected sheep in the epidemiology of neosporosis.

  5. PyMultiNest: Python interface for MultiNest

    NASA Astrophysics Data System (ADS)

    Buchner, Johannes

    2016-06-01

    PyMultiNest provides programmatic access to MultiNest (ascl:1109.006) and PyCuba, integration existing Python code (numpy, scipy), and enables writing Prior & LogLikelihood functions in Python. PyMultiNest can plot and visualize MultiNest's progress and allows easy plotting, visualization and summarization of MultiNest results. The plotting can be run on existing MultiNest output, and when not using PyMultiNest for running MultiNest.

  6. PrimerIdent: A web based tool for conserved primer design

    PubMed Central

    Pessoa, Alberto M; Pereira, Susana; Teixeira, Jorge

    2010-01-01

    Conserved primers across multiple species and simultaneously specific for a certain isozyme can be rare and difficult to find. PrimerIdent was developed aiming to automate this primer design and selection process in a given nucleotide sequence alignment, providing an intuitive, easy to interpret graphical result, which offers a list of all possible primers that meet the user criteria, with a colour-code identity to each sequence in the alignment. The software here presented is a simple and intuitive web based tool that is suitable for distinguishing very similar nucleotide sequences, such as isozymes­coding sequences, to enable the conserved primer design across multiple species, necessary for approaches that rely on knowing if a primer is suitable for a certain set of pre-aligned sequences, to design a specific primer to a certain sequence variation, or a combination thereof. This extremely useful software can, therefore, be used as a tool for the specific amplification of individual members of multigenic families across related species and also to evaluate the differential expression of isogenes for a given species. Availability http://primerident.up.pt PMID:21346862

  7. Detection and typing of human papillomavirus by e6 nested multiplex PCR.

    PubMed

    Sotlar, K; Diemer, D; Dethleffs, A; Hack, Y; Stubner, A; Vollmer, N; Menton, S; Menton, M; Dietz, K; Wallwiener, D; Kandolf, R; Bültmann, B

    2004-07-01

    A nested multiplex PCR (NMPCR) assay that combines degenerate E6/E7 consensus primers and type-specific primers was evaluated for the detection and typing of human papillomavirus (HPV) genotypes 6/11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, 59, 66, and 68 using HPV DNA-containing plasmids and cervical scrapes (n = 1,525). The performance of the NMPCR assay relative to that of conventional PCR with MY09-MY11 and GP5+-GP6+ primers, and nested PCR with these two primer sets (MY/GP) was evaluated in 495 cervical scrapes with corresponding histologic and cytologic findings. HPV prevalence rates determined with the NMPCR assay were 34.7% (102 of 294) in the absence of cervical intraepithelial neoplasia (CIN 0), 94.2% (113 of 120) in the presence of mild or moderate dysplasia (CIN I/II), and 97.8% (44 of 45) in the presence of severe dysplasia (CIN III). The combination of all four HPV detection methods applied in the study was taken as "gold standard": in all three morphological subgroups the NMPCR assay had significantly (P < 0.0001) higher sensitivities than the MY09-MY11 and GP5+-GP6+ assays and sensitivities comparable or equal to those of the MY/GP assay. All 18 HPV genotypes investigated were detected among the clinical samples. The ratio of high- to low-risk HPV genotypes increased from 4:1 (80 of 103) in CIN 0 to 19:1 (149 of 157) in CIN I to III. Multiple infections were detected in 47.9% (124 of 259) of the patients. In conclusion, the novel NMPCR method is a sensitive and useful tool for HPV DNA detection, especially when exact HPV genotyping and the identification of multiple HPV infections are required.

  8. China Energy Primer

    SciTech Connect

    Ni, Chun Chun

    2009-11-16

    Based on extensive analysis of the 'China Energy Databook Version 7' (October 2008) this Primer for China's Energy Industry draws a broad picture of China's energy industry with the two goals of helping users read and interpret the data presented in the 'China Energy Databook' and understand the historical evolution of China's energy inustry. Primer provides comprehensive historical reviews of China's energy industry including its supply and demand, exports and imports, investments, environment, and most importantly, its complicated pricing system, a key element in the analysis of China's energy sector.

  9. Application of nested PCR-DGGE (denaturing gradient gel electrophoresis) for the analysis of ciliate communities in soils.

    PubMed

    Shimano, Satoshi; Sambe, Mitsuo; Kasahara, Yasuhiro

    2012-01-01

    Ciliates play important roles as prey and predators in ecosystems. Changes in the ciliate community can affect the composition and population of microfauna and microflora in ecosystems. To investigate the structure of ciliate communities, we developed a nested PCR-DGGE method, which combines a universal eukaryotic-specific primer set in the first PCR step with a ciliate-specific primer set in the second PCR step, to amplify 18S rRNA genes from ciliates. The 300 bp DGGE fragments generated more bands on the gel than the 600 bp DGGE fragments. Prior to bead beating, DNA extraction of ciliates from soil samples was optimized with a combination of freeze-thaw cycles and ultrasonication. We applied this nested PCR-DGGE method to agricultural soils amended with 0, 120, 300, and 600 t ha⁻¹ year⁻¹ of livestock slurry. The results from the DGGE profiles and principal component analysis (PCA) revealed that the supplement of slurry to soils influenced the ciliate communities. From phylogenetic analysis, 108 DGGE bands were assigned to six classes, which included Spirotrichea and Colpodea, of the subphylum Intramacronucleata, and one class of the subphylum Postciliodesmatophora. These results indicated that a wide variety of taxonomic groups were detected by DGGE profiling. Thus, the nested PCR-DGGE method described here could clearly differentiate between ciliate communities within soil samples and allowed for the phylogenetic identification of these ciliates at the class level.

  10. Development of primer sets for loop-mediated isothermal amplification that enables rapid and specific detection of Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three ...

  11. Primer on Social Economics.

    ERIC Educational Resources Information Center

    Darcy, Robert L.

    An elaboration of the author's booklet entitled "First Steps Toward Economic Understanding," this primer is designed to help the reader develop a functional understanding of the economic process so that he can make wiser decisions on issues of social policy and on matters affecting his economic well-being. The document is not "economics in one…

  12. An SAT® Validity Primer

    ERIC Educational Resources Information Center

    Shaw, Emily J.

    2015-01-01

    This primer should provide the reader with a deeper understanding of the concept of test validity and will present the recent available validity evidence on the relationship between SAT® scores and important college outcomes. In addition, the content examined on the SAT will be discussed as well as the fundamental attention paid to the fairness of…

  13. ANL supplement to the UNICOS primer

    SciTech Connect

    Wiley, M.S.; Karlovsky, S.R.

    1991-06-01

    The ANL Supplement to the UNICOS Primer (ANL/TM 460) introduces the Cray X-MP interactive and batch services available at Argonne National Laboratory. It serves as a companion to the UNICOS Primer (Cray publication SG-2010 6.0). Whereas the UNICOS Primer discusses standard Unix issues of Cray computing, this manual discusses those issues specific to Cray computing at ANL. If this is your first experience on a Unix-based system, we assume that you have read at least Chapters 1 through 3 of the UNICOS Primer. The Glossary at the back of the UNICOS Primer will also be useful to you. If you are already familiar with a Unix system, it should suffice to keep the UNICOS Primer handy as you use this document. To learn about Unix programming in greater detail, we recommend A Practical Guide to the Unix System, by Mark G. Sobell. This manual and all other sources referred to in this document are available for purchase at the Document Distribution Counter in Building 221, Room A-134. We assume that you have already read the Guide to Computing at ANL (ANL/TM 336) to get an overview of all the computing facilities and services available at Argonne National Laboratory. You should also refer to Recommended Documentation for Computer Users at ANL (ANL/TM 379) for additional guidance in selecting available documentation that will best fill your particular computing needs.

  14. State-of-the-art monitoring of cytomegalovirus-specific cell-mediated immunity after organ transplant: a primer for the clinician.

    PubMed

    Egli, Adrian; Humar, Atul; Kumar, Deepali

    2012-12-01

    Cytomegalovirus (CMV) is one of the most common infections after solid organ transplantation. Improved assays to predict viral replication and disease would help refine current preventive strategies. Monitoring of CMV-specific T-cell responses may help guide clinical decision making. Several techniques are now available to quantify CMV-specific T-cell responses, including flow cytometry, enzyme-linked immunosorbent spot assay, and enzyme-linked immunosorbent assay. Standardization and validation of these assays have the potential to significantly change the monitoring and treatment of CMV and further personalize CMV prevention strategies. In this review, we discuss the measurement of CMV-specific T-cell responses and their clinical impact on the management of CMV after organ transplantation.

  15. Creating State-Specific Resources: A Technical Assistance Model That Works. Primers on Implementing Special Education in Charter Schools. Special Report

    ERIC Educational Resources Information Center

    Lange, Cheryl M.

    2008-01-01

    A model developed by the Technical Assistance Customizer (TAC) Project offers state education agencies and other organizations an approach to assist them with converting national findings and resources to state-specific materials in a timely and cost-effective manner. Moreover, the approach recognizes the importance of collaboration with and the…

  16. A unified approach to analyzing nest success

    USGS Publications Warehouse

    Shaffer, T.L.

    2004-01-01

    Logistic regression has become increasingly popular for modeling nest success in terms of nest-specific explanatory variables. However, logistic regression models for nest fate are inappropriate when applied to data from nests found at various ages, for the same reason that the apparent estimator of nest success is biased (i.e. older clutches are more likely to be successful than younger clutches). A generalized linear model is presented and illustrated that gives ornithologists access to a flexible, suitable alternative to logistic regression that is appropriate when exposure periods vary, as they usually do. Unlike the Mayfield method and the logistic regression method of Aebischer (1999), the logistic-exposure model requires no assumptions about when nest losses occur. Nest survival models involving continuous and categorical explanatory variables, multi-way classifications, and time-specific (e.g. nest age) and random effects are easily implemented with the logistic-exposure model. Application of the model to a sample of Yellow-breasted Chat (Icteria virens) nests shows that logistic-exposure estimates for individual levels of categorical explanatory variables agree closely with estimates obtained with Johnson's (1979) constant-survival estimator. Use of the logistic-exposure method to model time-specific effects of nest age and date on survival of Blue-winged Teal (Anas discors) and Mallard (A. platyrhynchos) nests gives results comparable to those reported by Klett and Johnson (1982). However, the logistic-exposure approach is less subjective and much easier to implement than Klett and Johnson's method. In addition, logistic-exposure survival rate estimates are constrained to the (0,1) interval, whereas Klett and Johnson estimates are not. When applied to a sample of Mountain Plover (Charadrius montanus) nests, the logistic-exposure method gives results either identical to, or similar to, those obtained with the nest survival model in program MARK. I

  17. Rapid DNA typing for HLA-C using sequence-specific primers (PCR-SSP): identification of serological and non-serologically defined HLA-C alleles including several new alleles.

    PubMed

    Bunce, M; Welsh, K I

    1994-01-01

    Detection of HLA-C antigens by complement mediated cytotoxicity using human alloantisera is often difficult. Between 20 to 40% of individuals in every race have undetectable HLA-C locus antigens and 9 out of the 29 sequenced HLA-C alleles so far published encode serologically undetected antigens. In addition, HLA-C molecules are expressed at the cell surface at about 10% of the levels of HLA-A and HLA-B. Recently, amplification of DNA using sequence-specific primers (PCR-SSP) has proved a reliable and rapid method for typing HLA-DR, HLA-DQA and HLA-DQB genes. PCR-SSP takes two hours to perform and is therefore suitable for the genotyping of cadaveric donors. We have designed a set of primers which will positively identify the HLA-C alleles corresponding to the serologically defined series HLA-Cw1, Cw2, Cw3, Cw4, Cw5, Cw6, Cw7 and Cw8. The serologically undetectable alleles have also been detected in groups according to sequence homology. In addition, three new unsequenced variants have been identified. DNA samples from 56 International Histocompatibility Workshop reference cell lines and 103 control individuals have been typed by the HLA-C PCR-SSP technique. 4/56 cell line types and 11/103 normal control individuals types were discrepant with the reported serological types. All combinations of serologically detectable and most of the serologically blank HLA-C antigens can be readily identified. DNA typing for HLA-Cw by PCR-SSP can take as little as 130 minutes from start to finish, including DNA preparation.

  18. Nested PCR detection of abalone shriveling syndrome-associated virus in China.

    PubMed

    Jiang, Jing-Zhe; Liang, Ya-Yu; Luo, Li-Juan; Guo, Zhi-Xun; Zhuang, Jun; Liu, Guang-Feng; Su, You-Lu; Wang, Jiang-Yong

    2012-09-01

    Haliotis diversicolor (small abalone) is an economic seafood found off the Southern coast of China. Since 1999, the cultured abalone yields in China have been affected severely by continual outbreaks of a fatal epidemic disease caused by abalone shriveling syndrome associated virus (AbSV), a double-stranded DNA virus. Although the pathogenicity and genome of AbSV have been ascertained, the epidemiology of AbSV infection remains to be investigated. In the present study, four pairs of AbSV-specific primers were designed on the basis of open reading frame (ORF)24 and ORF25 sequences in the AbSV genome. Two nested PCR detection methods were established by optimization of the annealing temperatures of primers. The results showed that the specificity of primers for AbSV detection could not be interfered with by the host genome and other aquaculture species or viruses. The detection limits of the two methods were about 10 copies of recombinant plasmid containing AbSV genes in 20μL reaction mixture. The results of detection of the AbSV epidemic showed that AbSV was still present in juvenile abalones in some farms along the Southern coast of China (Fujian and Guangdong).

  19. Subtyping of new Brazilian avian metapneumovirus isolates from chickens and turkeys by reverse transcriptase-nested-polymerase chain reaction.

    PubMed

    D'Arce, Regina C F; Coswig, Lia T; Almeida, Renata S; Trevisol, Iara M; Monteiro, Maria C B; Rossini, Lavínia I; Di Fabio, José; Hafez, Hafez M; Arns, Clarice W

    2005-04-01

    The aim of this study was to improve a reverse transcriptase (RT)-nested-polymerase chain reaction (PCR) able to differentiate avian pneumovirus (APV) subtypes A and B, and to characterize new Brazilian isolates. Representative APV strains and clinical field samples from chickens and turkey flocks were amplified in the chicken embryo-related cell line. Viral RNA was extracted from harvested cells, and submitted to cDNA synthesis. The primers utilized for RT-PCR were compatible with the G gene of both the A and B subtypes of APV, while the nested primers were subtype specific. This approach showed that three new APVs from chickens and one from turkeys were subtype A, confirmed by sequencing. This is the first report of APV isolation from turkeys in Brazil. Four other APVs were detected and classified as subtype A by RT-nested-PCR. These optimized techniques could be useful for differentiation of APV subtypes A and B, proving to be a valuable molecular epidemiological tool.

  20. Nested Neural Networks

    NASA Technical Reports Server (NTRS)

    Baram, Yoram

    1992-01-01

    Report presents analysis of nested neural networks, consisting of interconnected subnetworks. Analysis based on simplified mathematical models more appropriate for artificial electronic neural networks, partly applicable to biological neural networks. Nested structure allows for retrieval of individual subpatterns. Requires fewer wires and connection devices than fully connected networks, and allows for local reconstruction of damaged subnetworks without rewiring entire network.

  1. Marsh nesting by mallards

    USGS Publications Warehouse

    Krapu, G.L.; Talent, L.G.; Dwyer, T.J.

    1979-01-01

    Nest-site selection by mallard (Anas platyrhynchos) hens was studied on a 52-km2, privately owned area in the Missouri Coteau of south-central North Dakota during 1974-77. Sixty-six percent of 53 nests initiated by radio-marked and unmarked hens were in wetlands in dense stands of emergent vegetation and usually within 50 m of the wetland edge. These findings and other sources of information suggest that significant numbers of mallards breeding in the Prairie Pothole Region nest in marsh habitat. Potential factors contributing to mallard use of marsh habitat for nesting purposes are discussed. Management considerations associated with marsh nesting by mallards are described and research needs are identified.

  2. Crystalline Silica Primer

    USGS Publications Warehouse

    ,

    1992-01-01

    substance and will present a nontechnical overview of the techniques used to measure crystalline silica. Because this primer is meant to be a starting point for anyone interested in learning more about crystalline silica, a list of selected readings and other resources is included. The detailed glossary, which defines many terms that are beyond the scope of this publication, is designed to help the reader move from this presentation to a more technical one, the inevitable next step.

  3. Coal Bed Methane Primer

    SciTech Connect

    Dan Arthur; Bruce Langhus; Jon Seekins

    2005-05-25

    During the second half of the 1990's Coal Bed Methane (CBM) production increased dramatically nationwide to represent a significant new source of income and natural gas for many independent and established producers. Matching these soaring production rates during this period was a heightened public awareness of environmental concerns. These concerns left unexplained and under-addressed have created a significant growth in public involvement generating literally thousands of unfocused project comments for various regional NEPA efforts resulting in the delayed development of public and fee lands. The accelerating interest in CBM development coupled to the growth in public involvement has prompted the conceptualization of this project for the development of a CBM Primer. The Primer is designed to serve as a summary document, which introduces and encapsulates information pertinent to the development of Coal Bed Methane (CBM), including focused discussions of coal deposits, methane as a natural formed gas, split mineral estates, development techniques, operational issues, producing methods, applicable regulatory frameworks, land and resource management, mitigation measures, preparation of project plans, data availability, Indian Trust issues and relevant environmental technologies. An important aspect of gaining access to federal, state, tribal, or fee lands involves education of a broad array of stakeholders, including land and mineral owners, regulators, conservationists, tribal governments, special interest groups, and numerous others that could be impacted by the development of coal bed methane. Perhaps the most crucial aspect of successfully developing CBM resources is stakeholder education. Currently, an inconsistent picture of CBM exists. There is a significant lack of understanding on the parts of nearly all stakeholders, including industry, government, special interest groups, and land owners. It is envisioned the Primer would being used by a variety of

  4. Primer on molecular genetics

    SciTech Connect

    Not Available

    1992-04-01

    This report is taken from the April 1992 draft of the DOE Human Genome 1991--1992 Program Report, which is expected to be published in May 1992. The primer is intended to be an introduction to basic principles of molecular genetics pertaining to the genome project. The material contained herein is not final and may be incomplete. Techniques of genetic mapping and DNA sequencing are described.

  5. eDNA and specific primers for early detection of invasive species--A case study on the bivalve Rangia cuneata, currently spreading in Europe.

    PubMed

    Ardura, Alba; Zaiko, Anastasija; Martinez, Jose L; Samulioviene, Aurelija; Semenova, Anna; Garcia-Vazquez, Eva

    2015-12-01

    Intense human activities facilitate the successful spread and establishment of non-indigenous aquatic organisms in marine and freshwater ecosystems. In some cases such intrusions result in noticeable and adverse changes in the recipient environments. In the Baltic Sea, the discovery and rapid initial spread of the North American wedge clam Rangia cuneata represents a new wave of invasion which may trigger unpredictable changes of the local benthic communities. In this study we present a species-specific DNA-based marker developed in silico and experimentally tested on environmental samples. Marker specificity and sensitivity were assessed in vitro from water samples containing different mixtures of the target species and other five bivalves currently present in the region: the native Cerastoderma glaucum, Macoma balthica and Mytilus trossulus, the invasive Dreissena polymorpha and the cryptogenic Mya arenaria. Cross-species amplification was not found in any case. The method allows to detecting at least 0.4 ng of R. cuneata DNA per μl, and 0.1 g of tissue per liter of water. Finally, the marker performance was assessed in water samples from the Baltic Sea and Vistula Lagoon. The coincidence between independent visual observations of R. cuneata and positive PCR amplification of the marker from the water samples confirmed the efficiency of this highly reproducible, fast, and technically easy method. R. cuneata traces can be detected from environmental DNA even when the population is sparse and small, enabling rapid management responses and allowing to track the invasion dynamics.

  6. eDNA and specific primers for early detection of invasive species--A case study on the bivalve Rangia cuneata, currently spreading in Europe.

    PubMed

    Ardura, Alba; Zaiko, Anastasija; Martinez, Jose L; Samulioviene, Aurelija; Semenova, Anna; Garcia-Vazquez, Eva

    2015-12-01

    Intense human activities facilitate the successful spread and establishment of non-indigenous aquatic organisms in marine and freshwater ecosystems. In some cases such intrusions result in noticeable and adverse changes in the recipient environments. In the Baltic Sea, the discovery and rapid initial spread of the North American wedge clam Rangia cuneata represents a new wave of invasion which may trigger unpredictable changes of the local benthic communities. In this study we present a species-specific DNA-based marker developed in silico and experimentally tested on environmental samples. Marker specificity and sensitivity were assessed in vitro from water samples containing different mixtures of the target species and other five bivalves currently present in the region: the native Cerastoderma glaucum, Macoma balthica and Mytilus trossulus, the invasive Dreissena polymorpha and the cryptogenic Mya arenaria. Cross-species amplification was not found in any case. The method allows to detecting at least 0.4 ng of R. cuneata DNA per μl, and 0.1 g of tissue per liter of water. Finally, the marker performance was assessed in water samples from the Baltic Sea and Vistula Lagoon. The coincidence between independent visual observations of R. cuneata and positive PCR amplification of the marker from the water samples confirmed the efficiency of this highly reproducible, fast, and technically easy method. R. cuneata traces can be detected from environmental DNA even when the population is sparse and small, enabling rapid management responses and allowing to track the invasion dynamics. PMID:26453004

  7. Molecular analysis of single oocyst of Eimeria by whole genome amplification (WGA) based nested PCR.

    PubMed

    Wang, Yunzhou; Tao, Geru; Cui, Yujuan; Lv, Qiyao; Xie, Li; Li, Yuan; Suo, Xun; Qin, Yinghe; Xiao, Lihua; Liu, Xianyong

    2014-09-01

    PCR-based molecular tools are widely used for the identification and characterization of protozoa. Here we report the molecular analysis of Eimeria species using combined methods of whole genome amplification (WGA) and nested PCR. Single oocyst of Eimeria stiedai or Eimeriamedia was directly used for random amplification of the genomic DNA with either primer extension preamplification (PEP) or multiple displacement amplification (MDA), and then the WGA product was used as template in nested PCR with species-specific primers for ITS-1, 18S rDNA and 23S rDNA of E. stiedai and E. media. WGA-based PCR was successful for the amplification of these genes from single oocyst. For the species identification of single oocyst isolated from mixed E. stiedai or E. media, the results from WGA-based PCR were exactly in accordance with those from morphological identification, suggesting the availability of this method in molecular analysis of eimerian parasites at the single oocyst level. WGA-based PCR method can also be applied for the identification and genetic characterization of other protists.

  8. Evaluation for the clinical diagnosis of Pythium insidiosum using a single-tube nested PCR.

    PubMed

    Thongsri, Yordhathai; Wonglakorn, Lumyai; Chaiprasert, Angkana; Svobodova, Lucie; Hamal, Petr; Pakarasang, Maitree; Prariyachatigul, Chularut

    2013-12-01

    Pythiosis is a rare infectious disease caused by Pythium insidiosum, which typically occurs in tropical and subtropical regions. The high mortality rate may be in consequence of the lack of diagnosis. The objective of this study was to evaluate reliability of a new single-tube nested PCR for detection of P. insidiosum DNA. A total of 78 clinical isolates of various fungi and bacteria, 106 clinical specimens and 80 simulated positive blood samples were tested. The developed primer pairs CPL6-CPR8 and YTL1-YTR1 are located on 18S subunit of the rRNA gene of P. insidiosum. The specificity, negative and positive predictive values were 100, 100 and 87.5 %, respectively, as compared with direct microscopy and cultivation. The detection limit of the single-tube nested PCR was 21 zoospores corresponding to 2.7 pg of the DNA. The results demonstrate that the new single-tube nested PCR offers a highly sensitive, specific and rapid genetic method for detecting P. insidiosum. PMID:23948967

  9. Forest Interpreter's Primer on Fire Management.

    ERIC Educational Resources Information Center

    Zelker, Thomas M.

    Specifically prepared for the use of Forest Service field-based interpreters of the management, protection, and use of forest and range resources and the associated human, cultural, and natural history found on these lands, this book is the second in a series of six primers on the multiple use of forest and range resources. Following an…

  10. Nested neural networks

    NASA Technical Reports Server (NTRS)

    Baram, Yoram

    1988-01-01

    Nested neural networks, consisting of small interconnected subnetworks, allow for the storage and retrieval of neural state patterns of different sizes. The subnetworks are naturally categorized by layers of corresponding to spatial frequencies in the pattern field. The storage capacity and the error correction capability of the subnetworks generally increase with the degree of connectivity between layers (the nesting degree). Storage of only few subpatterns in each subnetworks results in a vast storage capacity of patterns and subpatterns in the nested network, maintaining high stability and error correction capability.

  11. Nested sampling with demons

    NASA Astrophysics Data System (ADS)

    Habeck, Michael

    2015-01-01

    This article looks at Skilling's nested sampling from a physical perspective and interprets it as a microcanonical demon algorithm. Using key quantities of statistical physics we investigate the performance of nested sampling on complex systems such as Ising, Potts and protein models. We show that releasing multiple demons helps to smooth the truncated prior and eases sampling from it because the demons keep the particle off the constraint boundary. For continuous systems it is straightforward to extend this approach and formulate a phase space version of nested sampling that benefits from correlated explorations guided by Hamiltonian dynamics.

  12. The use of singleplex and nested PCR to detect Batrachochytrium dendrobatidis in free-living frogs

    PubMed Central

    Coutinho, Selene Dall'Acqua; Burke, Julieta Catarina; de Paula, Catia Dejuste; Rodrigues, Miguel Trefaut; Catão-Dias, José Luiz

    2015-01-01

    Many microorganisms are able to cause diseases in amphibians, and in the past few years one of the most reported has been Batrachochytrium dendrobatidis. This fungus was first reported in Brazil in 2005; following this, other reports were made in specimens deposited in museum collections, captive and free-living frogs. The aim of this study was to compare singleplex and nested-PCR techniques to detect B. dendrobatidis in free-living and apparently healthy adult frogs from the Brazilian Atlantic Forest. The sample collection area was a protected government park, with no general entrance permitted and no management of the animals there. Swabs were taken from the skin of 107 animals without macroscopic lesions and they were maintained in ethanol p.a. Fungal DNA was extracted and identification of B. dendrobatidis was performed using singleplex and nested-PCR techniques, employing specific primers sequences. B. dendrobatidis was detected in 61/107 (57%) and 18/107 (17%) animals, respectively by nested and singleplex-PCR. Nested-PCR was statistically more sensible than the conventional for the detection of B. dendrobatidis (Chi-square = 37.1; α = 1%) and the agreement between both techniques was considered just fair (Kappa = 0.27). The high prevalence obtained confirms that these fungi occur in free-living frogs from the Brazilian Atlantic Forest with no macroscopic lesions, characterizing the state of asymptomatic carrier. We concluded that the nested-PCR technique, due to its ease of execution and reproducibility, can be recommended as one of the alternatives in epidemiological surveys to detect B. dendrobatidis in healthy free-living frog populations. PMID:26273273

  13. Absolute quantification of the alleles in somatic point mutations by bioluminometric methods based on competitive polymerase chain reaction in the presence of a locked nucleic acid blocker or an allele-specific primer.

    PubMed

    Iliadi, Alexandra; Petropoulou, Margarita; Ioannou, Penelope C; Christopoulos, Theodore K; Anagnostopoulos, Nikolaos I; Kanavakis, Emmanuel; Traeger-Synodinos, Jan

    2011-09-01

    In somatic (acquired) point mutations, the challenge is to quantify minute amounts of the mutant allele in the presence of a large excess of the normal allele that differs only in a single base pair. We report two bioluminometric methods that enable absolute quantification of the alleles. The first method exploits the ability of a locked nucleic acid (LNA) oligonucleotide to bind to and inhibit effectively the polymerase chain reaction (PCR) amplification of the normal allele while the amplification of the mutant allele remains unaffected. The second method employs allele-specific PCR primers, thereby allowing the amplification of the corresponding allele only. DNA internal standards (competitors) are added to the PCR mixture to compensate for any sample-to-sample variation in the amplification efficiency. The amplification products from the two alleles and the internal standards are quantified by a microtiter well-based bioluminometric hybridization assay using the photoprotein aequorin as a reporter. The methods allow absolute quantification of less than 300 copies of the mutant allele even in samples containing less than 1% of the mutant allele.

  14. Multiplexed Primer Prediction for PCR

    SciTech Connect

    2007-07-23

    MPP predicts sets of multiplex-compatible primers for Polymerase Chain Reaction (PCR), finding a near minimal set of primers such that at least one amplicon will be generated from every target sequence in the input file. The code finds highly conserved oligos that are suitable as primers, according to user-specified desired primer characteristics such as length, melting temperature, and amplicon length. The primers are predicted not to form unwanted dimer or hairpin structures. The target sequences used as input can be diverse, since no multiple sequence alighment is required. The code is scalable, taking up to tens of thousands of sequences as input, and works, for example, to find a "universal primer set" for all viral genomes provided as a single input file. The code generates a periodic check-point file, thus in the event of premature execution termination, the application can be restarted from the last check-point file.

  15. Limited-life cartridge primers

    DOEpatents

    Makowiecki, Daniel M.; Rosen, Robert S.

    1998-01-01

    A cartridge primer which utilizes an explosive that can be designed to become inactive in a predetermined period of time: a limited-life primer. The explosive or combustible material of the primer is an inorganic reactive multilayer (RML). The reaction products of the RML are sub-micron grains of non-corrosive inorganic compounds that would have no harmful effects on firearms or cartridge cases. Unlike use of primers containing lead components, primers utilizing RML's would not present a hazard to the environment. The sensitivity of an RML is determined by the physical structure and the stored interfacial energy. The sensitivity lowers with time due to a decrease in interfacial energy resulting from interdiffusion of the elemental layers. Time-dependent interdiffusion is predictable, thereby enabling the functional lifetime of an RML primer to be predetermined by the initial thickness and materials selection of the reacting layers.

  16. Limited-life cartridge primers

    DOEpatents

    Makowiecki, Daniel M.; Rosen, Robert S.

    2005-04-19

    A cartridge primer which utilizes an explosive that can be designed to become inactive in a predetermined period of time: a limited-life primer. The explosive or combustible material of the primer is an inorganic reactive multilayer (RML). The reaction products of the RML are sub-micron grains of non-corrosive inorganic compounds that would have no harmful effects on firearms or cartridge cases. Unlike use of primers containing lead components, primers utilizing RML's would not present a hazard to the environment. The sensitivity of an RML is determined by the physical structure and the stored interfacial energy. The sensitivity lowers with time due to a decrease in interfacial energy resulting from interdiffusion of the elemental layers. Time-dependent interdiffusion is predictable, thereby enabling the functional lifetime of an RML primer to be predetermined by the initial thickness and materials selection of the reacting layers.

  17. Limited-life cartridge primers

    DOEpatents

    Makowiecki, D.M.; Rosen, R.S.

    1998-06-30

    A cartridge primer is described which utilizes an explosive that can be designed to become inactive in a predetermined period of time: a limited-life primer. The explosive or combustible material of the primer is an inorganic reactive multilayer (RML). The reaction products of the RML are sub-micron grains of non-corrosive inorganic compounds that would have no harmful effects on firearms or cartridge cases. Unlike use of primers containing lead components, primers utilizing RML`s would not present a hazard to the environment. The sensitivity of an RML is determined by the physical structure and the stored interfacial energy. The sensitivity lowers with time due to a decrease in interfacial energy resulting from interdiffusion of the elemental layers. Time-dependent interdiffusion is predictable, thereby enabling the functional lifetime of an RML primer to be predetermined by the initial thickness and materials selection of the reacting layers. 10 figs.

  18. Multiplexed Primer Prediction for PCR

    2007-07-23

    MPP predicts sets of multiplex-compatible primers for Polymerase Chain Reaction (PCR), finding a near minimal set of primers such that at least one amplicon will be generated from every target sequence in the input file. The code finds highly conserved oligos that are suitable as primers, according to user-specified desired primer characteristics such as length, melting temperature, and amplicon length. The primers are predicted not to form unwanted dimer or hairpin structures. The target sequencesmore » used as input can be diverse, since no multiple sequence alighment is required. The code is scalable, taking up to tens of thousands of sequences as input, and works, for example, to find a "universal primer set" for all viral genomes provided as a single input file. The code generates a periodic check-point file, thus in the event of premature execution termination, the application can be restarted from the last check-point file.« less

  19. Do ducks and songbirds initiate more nests when the probability of survival is greater?

    USGS Publications Warehouse

    Grant, Todd A.; Shaffer, Terry L.

    2015-01-01

    Nesting chronology in grassland birds can vary by species, locality, and year. The date a nest is initiated can influence the subsequent probability of its survival in some grassland bird species. Because predation is the most significant cause of nest loss in grassland birds, we examined the relation between timing of nesting and nest survival. Periods of high nest survival that correspond with the peak of nesting activity might reflect long-term adaptations to specific predation pressures commonly recurring during certain periods of the nesting cycle. We evaluated this theory by comparing timing of nesting with date-specific nest survival rates for several duck and passerine species breeding in north-central North Dakota during 1998–2003. Nest survival decreased seasonally with date for five of the seven species we studied. We found little evidence to support consistent relations between timing of nesting, the number of nest initiations, and nest survival for any species we studied, suggesting that factors other than nest predation may better explain nesting chronology for these species. The apparent mismatch between date-specific patterns of nest survival and nest initiation underscores uncertainty about the process of avian nest site selection driven mainly by predation. Although timing of nesting differed among species, the general nesting period was fairly predictable across all years of study, suggesting the potential for research activities or management actions to be timed to take advantage of known periods when nests are active (or inactive). However, our results do not support the notion that biologists can take advantage of periods when many nests are active and survival is also high.

  20. Size matters: nest colonization patterns for twig-nesting ants.

    PubMed

    Jiménez-Soto, Estelí; Philpott, Stacy M

    2015-08-01

    Understanding the drivers of ant diversity and co-occurrence in agroecosystems is fundamental because ants participate in interactions that influence agroecosystem processes. Multiple local and regional factors influence ant community assembly.We examined local factors that influence the structure of a twig-nesting ant community in a coffee system in Mexico using an experimental approach. We investigated whether twig characteristics (nest entrance size and diversity of nest entrance sizes) and nest strata (canopy shade tree or coffee shrub) affected occupation, species richness, and community composition of twig-nesting ants and whether frequency of occupation of ant species varied with particular nest entrance sizes or strata.We conducted our study in a shaded coffee farm in Chiapas, Mexico, between March and June 2012. We studied ant nest colonization by placing artificial nests (bamboo twigs) on coffee shrubs and shade trees either in diverse or uniform treatments. We also examined whether differences in vegetation (no. of trees, canopy cover and coffee density) influenced nest colonization.We found 33 ant species occupying 73% of nests placed. Nest colonization did not differ with nest strata or size. Mean species richness of colonizing ants was significantly higher in the diverse nest size entrance treatment, but did not differ with nest strata. Community composition differed between strata and also between the diverse and uniform size treatments on coffee shrubs, but not on shade trees. Some individual ant species were more frequently found in certain nest strata and in nests with certain entrance sizes.Our results indicate that twig-nesting ants are nest-site limited, quickly occupy artificial nests of many sizes, and that trees or shrubs with twigs of a diversity of entrance sizes likely support higher ant species richness. Further, individual ant species more frequently occupy nests with different sized entrances promoting ant richness on individual coffee

  1. Size matters: nest colonization patterns for twig-nesting ants

    PubMed Central

    Jiménez-Soto, Estelí; Philpott, Stacy M

    2015-01-01

    Understanding the drivers of ant diversity and co-occurrence in agroecosystems is fundamental because ants participate in interactions that influence agroecosystem processes. Multiple local and regional factors influence ant community assembly. We examined local factors that influence the structure of a twig-nesting ant community in a coffee system in Mexico using an experimental approach. We investigated whether twig characteristics (nest entrance size and diversity of nest entrance sizes) and nest strata (canopy shade tree or coffee shrub) affected occupation, species richness, and community composition of twig-nesting ants and whether frequency of occupation of ant species varied with particular nest entrance sizes or strata. We conducted our study in a shaded coffee farm in Chiapas, Mexico, between March and June 2012. We studied ant nest colonization by placing artificial nests (bamboo twigs) on coffee shrubs and shade trees either in diverse or uniform treatments. We also examined whether differences in vegetation (no. of trees, canopy cover and coffee density) influenced nest colonization. We found 33 ant species occupying 73% of nests placed. Nest colonization did not differ with nest strata or size. Mean species richness of colonizing ants was significantly higher in the diverse nest size entrance treatment, but did not differ with nest strata. Community composition differed between strata and also between the diverse and uniform size treatments on coffee shrubs, but not on shade trees. Some individual ant species were more frequently found in certain nest strata and in nests with certain entrance sizes. Our results indicate that twig-nesting ants are nest-site limited, quickly occupy artificial nests of many sizes, and that trees or shrubs with twigs of a diversity of entrance sizes likely support higher ant species richness. Further, individual ant species more frequently occupy nests with different sized entrances promoting ant richness on individual

  2. Strategies for nest-site selection by king eiders

    USGS Publications Warehouse

    Bentzen, R.L.; Powell, A.N.; Suydam, R.S.

    2009-01-01

    Nest site selection is a critical component of reproduction and has presumably evolved in relation to predation, local resources, and microclimate. We investigated nest-site choice by king eiders (Somateria spectabilis) on the coastal plain of northern Alaska, USA, 2003-2005. We hypothesized that nest-site selection is driven by predator avoidance and that a variety of strategies including concealment, seclusion, and conspecific or inter-specific nest defense might lead to improved nesting success. We systematically searched wetland basins for king eider nests and measured habitat and social variables at nests (n = 212) and random locations (n = 493). King eiders made use of both secluded and concealed breeding strategies; logistic regression models revealed that females selected nests close to water, on islands, and in areas with high willow (Salix spp.) cover but did not select sites near conspecific or glaucous gull (Larus hyperboreus) nests. The most effective nest-placement strategy may vary depending on density and types of nest predators; seclusion is likely a mammalian-predator avoidance tactic whereas concealment may provide protection from avian predators. We recommend that managers in northern Alaska attempt to maintain wetland basins with islands and complex shorelines to provide potential nest sites in the vicinity of water. ?? The Wildlife Society.

  3. STR primer concordance study.

    PubMed

    Budowle, B; Masibay, A; Anderson, S J; Barna, C; Biega, L; Brenneke, S; Brown, B L; Cramer, J; DeGroot, G A; Douglas, D; Duceman, B; Eastman, A; Giles, R; Hamill, J; Haase, D J; Janssen, D W; Kupferschmid, T D; Lawton, T; Lemire, C; Llewellyn, B; Moretti, T; Neves, J; Palaski, C; Schueler, S; Sgueglia, J; Sprecher, C; Tomsey, C; Yet, D

    2001-12-15

    Over 1500 population database samples comprising African Americans, Caucasians, Hispanics, Native Americans, Chamorros and Filipinos were typed using the PowerPlex 16 and the Profiler Plus/COfiler kits. Except for the D8S1179 locus in Chamorros and Filipinos from Guam, there were eight examples in which a typing difference due to allele dropout was observed. At the D8S1179 locus in the population samples from Guam, there were 13 examples of allele dropout observed when using the Profiler Plus kit. The data support that the primers used in the PowerPlex 16, Profiler Plus, and COfiler kits are reliable for typing reference samples that are for use in CODIS. In addition, allele frequency databases have been established for the STR loci Penta D and Penta E. Both loci are highly polymorphic. PMID:11741760

  4. CRISPR Primer Designer: Design primers for knockout and chromosome imaging CRISPR-Cas system.

    PubMed

    Yan, Meng; Zhou, Shi-Rong; Xue, Hong-Wei

    2015-07-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-associated system enables biologists to edit genomes precisely and provides a powerful tool for perturbing endogenous gene regulation, modulation of epigenetic markers, and genome architecture. However, there are concerns about the specificity of the system, especially the usages of knocking out a gene. Previous designing tools either were mostly built-in websites or ran as command-line programs, and none of them ran locally and acquired a user-friendly interface. In addition, with the development of CRISPR-derived systems, such as chromosome imaging, there were still no tools helping users to generate specific end-user spacers. We herein present CRISPR Primer Designer for researchers to design primers for CRISPR applications. The program has a user-friendly interface, can analyze the BLAST results by using multiple parameters, score for each candidate spacer, and generate the primers when using a certain plasmid. In addition, CRISPR Primer Designer runs locally and can be used to search spacer clusters, and exports primers for the CRISPR-Cas system-based chromosome imaging system.

  5. Nest site characteristics, nesting movements, and lack of long-term nest site fidelity in Agassiz's desert tortoises at a wind energy facility in southern California

    USGS Publications Warehouse

    Lovich, Jeffrey E.; Agha, Mickey; Yackulic, Charles B.; Meyer-Wilkins, Kathie; Bjurlin, Curtis; Ennen, Joshua R.; Arundel, Terry R.; Austin, Meaghan

    2014-01-01

    Nest site selection has important consequences for maternal and offspring survival and fitness. Females of some species return to the same nesting areas year after year. We studied nest site characteristics, fidelity, and daily pre-nesting movements in a population of Agassiz’s desert tortoises (Gopherus agassizii) at a wind energy facility in southern California during two field seasons separated by over a decade. No females returned to the same exact nest site within or between years but several nested in the same general area. However, distances between first and second clutches within a year (2000) were not significantly different from distances between nests among years (2000 and 2011) for a small sample of females, suggesting some degree of fidelity within their normal activity areas. Environmental attributes of nest sites did not differ significantly among females but did among years due largely to changes in perennial plant structure as a result of multiple fires. Daily pre-nesting distances moved by females decreased consistently from the time shelled eggs were first visible in X-radiographs until oviposition, again suggesting some degree of nest site selection. Tortoises appear to select nest sites that are within their long-term activity areas, inside the climate-moderated confines of one of their self-constructed burrows, and specifically, at a depth in the burrow that minimizes exposure of eggs and embryos to lethal incubation temperatures. Nesting in “climate-controlled” burrows and nest guarding by females relaxes some of the constraints that drive nest site selection in other oviparous species.

  6. Superposition Enhanced Nested Sampling

    NASA Astrophysics Data System (ADS)

    Martiniani, Stefano; Stevenson, Jacob D.; Wales, David J.; Frenkel, Daan

    2014-07-01

    The theoretical analysis of many problems in physics, astronomy, and applied mathematics requires an efficient numerical exploration of multimodal parameter spaces that exhibit broken ergodicity. Monte Carlo methods are widely used to deal with these classes of problems, but such simulations suffer from a ubiquitous sampling problem: The probability of sampling a particular state is proportional to its entropic weight. Devising an algorithm capable of sampling efficiently the full phase space is a long-standing problem. Here, we report a new hybrid method for the exploration of multimodal parameter spaces exhibiting broken ergodicity. Superposition enhanced nested sampling combines the strengths of global optimization with the unbiased or athermal sampling of nested sampling, greatly enhancing its efficiency with no additional parameters. We report extensive tests of this new approach for atomic clusters that are known to have energy landscapes for which conventional sampling schemes suffer from broken ergodicity. We also introduce a novel parallelization algorithm for nested sampling.

  7. Serenbe Nest Cottages

    SciTech Connect

    Butler, T.; Curtis, O.; Kim, E.; Roberts, S.; Stephenson, R.

    2012-12-01

    As part of the NAHB Research Center Industry Partnership, Southface partnered with Martin Dodson Builders and the Serenbe community on the construction of a new test home in the suburbs of Atlanta, GA in the mixed humid climate zone. The most recent subdivision within the Serenbe community, the Nest, will contain 15 small footprint cottage style homes, and Southface has selected Lot Nine, as the test home for this study. This Nest subdivision serves as a project showcase for both the builder partner and the Serenbe community as a whole. The planning and design incorporated into the Nest cottages will be implemented in each home within the subdivision. These homes addresses Building America Savings targets and serve as a basis of design for other homes Martin Dodson plans to build within the Serenbe community.

  8. Serenbe Nest Cottages

    SciTech Connect

    Butler, T.; Curtis, O.; Kim, E.; Roberts, S.; Stephenson, R.

    2012-12-01

    As part of the NAHB Research Center Industry Partnership, Southface partnered with Martin Dodson Builders and the Serenbe community on the construction of a new test home in the suburbs of Atlanta, GA, in the mixed humid climate zone. The most recent subdivision within the Serenbe community, the Nest, will contain 15 small footprint cottage-style homes, and Southface has selected Lot Nine, as the test home for this study. This Nest subdivision serves as a project showcase for both the builder partner and the Serenbe community as a whole. The planning and design incorporated into the Nest cottages will be implemented in each home within the subdivision. These homes addresses Building America savings targets and serve as a basis of design for other homes Martin Dodson plans to build within the Serenbe community.

  9. Rust transformation/rust compatible primers

    NASA Technical Reports Server (NTRS)

    Emeric, Dario A.; Miller, Christopher E.

    1993-01-01

    Proper surface preparation has been the key to obtain good performance by a surface coating. The major obstacle in preparing a corroded or rusted surface is the complete removal of the contaminants and the corrosion products. Sandblasting has been traditionally used to remove the corrosion products before painting. However, sandblasting can be expensive, may be prohibited by local health regulations and is not applicable in every situation. To get around these obstacles, Industry developed rust converters/rust transformers and rust compatible primers (high solids epoxies). The potential use of these products for military equipment led personnel of the Belvoir Research, Development and Engineering Center (BRDEC) to evaluate the commercially available rust transformers and rust compatible primers. Prior laboratory experience with commercially available rust converters, as well as field studies in Hawaii and Puerto Rico, revealed poor performance, several inherent limitations, and lack of reliability. It was obvious from our studies that the performance of rust converting products was more dependent on the amount and type of rust present, as well as the degree of permeability of the coating, than on the product's ability to form an organometallic complex with the rust. Based on these results, it was decided that the Military should develop their own rust converter formulation and specification. The compound described in the specification is for use on a rusted surface before the application of an organic coating (bituminous compounds, primer or topcoat). These coatings should end the need for sandblasting or the removing of the adherent corrosion products. They also will prepare the surface for the application of the organic coating. Several commercially available rust compatible primers (RCP) were also tested using corroded surfaces. All of the evaluated RCP failed our laboratory tests for primers.

  10. [Detection of JAK2V617F mutation rate by real-time fluorescent quantitative PCR using allele specific primer and TaqMan-MGB probe for dual inhibiting amplification of wild type alleles].

    PubMed

    Liang, Guo-Wei; Shao, Dong-Hua; He, Mei-Ling; Cao, Qing-Yun

    2012-12-01

    This study was purposed to develop a real-time PCR assay for sensitive quantification of JAK2V617F allele burden in peripheral blood and to evaluate the clinical value of this method. Both allele-specific mutant reverse primer and wild-type TaqMan-MGB probe were used for dual-inhibiting amplification of wild-type alleles in a real-time PCR, and then the JAK2V617F mutant alleles were amplified specially. The standard curve for quantification of JAK2V617F was established by percentages of JAK2V617F alleles with threshold cycle (Ct) values in a real-time PCR. Furthermore, 89 apparent healthy donors were tested by this method. The results showed that the quantitative lower limit of this method for JAK2V617F was 0.1%, and the intra- and inter-assay average variability for quantifying percentage of JAK2V617F in total DNA was 4.1% and 6.1%, respectively. Two JAK2V617F-positive individuals were identified (the percentage of JAK2V617F alleles were 0.64% and 0.98%, respectively) using this method in blood from 89 apparently healthy donors. It is concluded that the developed method with highly sensitive and reproducible quantification of JAK2V617F mutant burden can be used clinically for diagnosis and evaluation of disease prognosis and efficacy of therapy in patients with myeloproliferative neoplasms. Moreover, this technique can be also used for quantitative detection of variety of single nucleotide mutation.

  11. Density-dependent nest predation in waterfowl: the relative importance of nest density versus nest dispersion

    USGS Publications Warehouse

    Ackerman, Joshua T.; Ringelman, KM; Eadie, J.M.

    2012-01-01

    When nest predation levels are very high or very low, the absolute range of observable nest success is constrained (a floor/ceiling effect), and it may be more difficult to detect density-dependent nest predation. Density-dependent nest predation may be more detectable in years with moderate predation rates, simply because there can be a greater absolute difference in nest success between sites. To test this, we replicated a predation experiment 10 years after the original study, using both natural and artificial nests, comparing a year when overall rates of nest predation were high (2000) to a year with moderate nest predation (2010). We found no evidence for density-dependent predation on artificial nests in either year, indicating that nest predation is not density-dependent at the spatial scale of our experimental replicates (1-ha patches). Using nearest-neighbor distances as a measure of nest dispersion, we also found little evidence for “dispersion-dependent” predation on artificial nests. However, when we tested for dispersion-dependent predation using natural nests, we found that nest survival increased with shorter nearest-neighbor distances, and that neighboring nests were more likely to share the same nest fate than non-adjacent nests. Thus, at small spatial scales, density-dependence appears to operate in the opposite direction as predicted: closer nearest neighbors are more likely to be successful. We suggest that local nest dispersion, rather than larger-scale measures of nest density per se, may play a more important role in density-dependent nest predation.

  12. Combining nested PCR and restriction digest of the internal transcribed spacer region to characterize arbuscular mycorrhizal fungi on roots from the field.

    PubMed

    Renker, Carsten; Heinrichs, Jochen; Kaldorf, Michael; Buscot, François

    2003-08-01

    Identification of arbuscular mycorrhizal fungi (AMF) on roots is almost impossible with morphological methods and, due to the presence of contaminating fungi, it is also difficult with molecular biological techniques. To allow broad investigation of the population structure of AMF in the field, we have established a new method to selectively amplify the internal transcribed spacer (ITS) region of most AMF with a unique primer set. Based on available sequences of the rDNA, one primer pair specific for AMF and a few other fungal groups was designed and combined in a nested PCR with the already established primer pair ITS5/ITS4. Amplification from contaminating organisms was reduced by an AluI restriction after the first reaction of the nested PCR. The method was assessed at five different field sites representing different types of habitats. Members of all major groups within the Glomeromycota (except Archaeosporaceae) were detected at the different sites. Gigasporaceae also proved detectable with the method based on cultivated strains. PMID:12938031

  13. DNA sequencing technology, walking with modular primers. Final report

    SciTech Connect

    Ulanovsky, L.

    1996-12-31

    The success of the Human Genome Project depends on the development of adequate technology for rapid and inexpensive DNA sequencing, which will also benefit biomedical research in general. The authors are working on DNA technologies that eliminate primer synthesis, the main bottleneck in sequencing by primer walking. They have developed modular primers that are assembled from three 5-mer, 6-mer or 7-mer modules selected from a presynthesized library of as few as 1,000 oligonucleotides ({double_bond}4, {double_bond}5, {double_bond}7). The three modules anneal contiguously at the selected template site and prime there uniquely, even though each is not unique for the most part when used alone. This technique is expected to speed up primer walking 30 to 50 fold, and reduce the sequencing cost by a factor of 5 to 15. Time and expensive will be saved on primer synthesis itself and even more so due to closed-loop automation of primer walking, made possible by the instant availability of primers. Apart from saving time and cost, closed-loop automation would also minimize the errors and complications associated with human intervention between the walks. The author has also developed two additional approaches to primer-library based sequencing. One involves a branched structure of modular primers which has a distinctly different mechanism of achieving priming specificity. The other introduces the concept of ``Differential Extension with Nucleotide Subsets`` as an approach increasing priming specificity, priming strength and allowing cycle sequencing. These approaches are expected to be more robust than the original version of the modular primer technique.

  14. MRI Biosensors: A Short Primer

    PubMed Central

    Louie, Angelique

    2013-01-01

    Interest in Magnetic Resonance Imaging (MRI) contrast agents for molecular imaging of biological function experienced a surge of excitement approximately 20 years ago with the development of the first activatable contrast agents that could act as biosensors and turn “on” in response to a specific biological activity. This brief tutorial, based on a short course lecture from the 2011 ISMRM meeting, provides an overview of underlying principles governing the design of biosensing contrast agents. We describe mechanisms by which a magnetic resonance imaging (MRI) contrast agent can be made into a sensor for both T1 and T2 types contrast agents. Examples of biological activities that can interact with a contrast agent are discussed using specific examples from the recent literature to illustrate the primary mechanisms of action that have been utilized to achieve activation. MRI sensors for pH, ion binding, enzyme cleavage, and oxidation-reduction are presented. This article is not meant to be an exhaustive review, but an illustrative primer to explain how activation can be achieved for an MRI contrast agent. Chemical exchange saturation transfer (CEST) is not covered as these agents were covered in a separate lecture. PMID:23996662

  15. Variability in nest survival rates and implications to nesting studies

    USGS Publications Warehouse

    Klett, A.T.; Johnson, D.H.

    1982-01-01

    We used four reasonably large samples (83-213) of Mallard (Anas platyrhynchos) and Blue-winged Teal (A. discors) nests on an interstate highway right-of-way in southcentral North Dakota to evaluate potential biases in hatch-rate estimates. Twelve consecutive, weekly searches for nests were conducted with a cable-chain drag in 1976 and 1977. Nests were revisited at weekly intervals. Four methods were used to estimate hatch rates for the four data sets: the Traditional Method, the Mayfield Method, and two modifications of the Mayfield Method that are sometimes appropriate when daily mortality rates of nests are not constant. Hatch rates and the average age of nests at discovery declined as the interval between searches decreased, suggesting that mortality rates were not constant in our samples. An analysis of variance indicated that daily mortality rates varied with the age of nests in all four samples. Mortality was generally highest during the early laying period, moderately high during the late laying period, and lowest during incubation. We speculate that this relationship of mortality to nest age might be due to the presence of hens at nests or to differences in the vulnerability of nest sites to predation. A modification of the Mayfield Method that accounts for age-related variation in nest mortality was most appropriate for our samples. We suggest methods for conducting nesting studies and estimating nest success for species possessing similar nesting habits.

  16. Feathering Your Nest

    ERIC Educational Resources Information Center

    Nabors, Martha L.; Edwards, Linda Carol; Decker, Suzanne

    2010-01-01

    The first-grade classroom was like a natural history museum. Bird nests of every shape and size lay on top of bookshelves that lined two walls. Methods students, who were visiting the classroom in preparation for the science lessons they would teach there, were immediately inspired by the collection. They used the collection as a springboard for…

  17. Nested multiplex PCR--a feasible technique to study partial community of arbuscular mycorrhizal fungi in field-growing plant root.

    PubMed

    Dong, Xiuli; Zhao, Bin

    2006-08-01

    Plant can be infected by different arbuscular mycorrhizal fungi, but little is known about the interaction between them within root tissues mainly because different species cannot be distinguished on the basis of fungal structure. Accurate species identification of Arbuscular mycorrhizal fungi (AMF) colonized in plant roots is the cornerstone of mycorrhizal study, yet this fundamental step is impossible through its morphological character alone. For accurate, rapid and inexpensive detection of partial mycorrhizal fungal community in plant roots, a nested multiplex polymerase chain reaction (PCR) was developed in this study. Five discriminating primers designed based on the variable region of the 5' end of the large ribosomal subunit were used in the experiment for testing their specificity and the sensitivity in nested PCR by using spores from Glomus mosseae (BEG12), Glomus intraradices (BEG141), Scutellospora castaneae (BEG1) and two unidentified Glomus sp. HAUO3 and HAUO4. The feasibility assay of nested multiplex PCR was conducted by use of spore mixture, Astragalus sinicum roots co-inoculated with 4 species of arbuscular mycorrhizal fungi from pot cultures and 15 different field-growing plant roots respectively after analyses of the compatibility of primers. The result indicated that the sensitivity was in the same range as that of the corresponding single PCR reaction. Overall accuracy was 95%. The efficiency and sensitivity of this multiplex PCR procedure provided a rapid and easy way to simultaneously detect several of arbuscular mycorrhiza fungal species in a same plant root system. PMID:16989281

  18. Novel method for detection of small amounts of RNA based on the semi-nested polymerase chain reaction.

    PubMed

    Sanders, S; Thomas, G A

    1997-04-01

    A technique for the amplification of very small quantities of cDNA based on a semi-nested polymerase chain reaction system has been developed. The technique uses an additional blocking oligonucleotide "primer" in the second round nest which is complementary to one of the first round primers. This prevents amplification of first round product or of any other unwanted products dependent on that primer, but allows amplification of second round product, using first round product as the template. The second round primers are added through the oil layer, eliminating the possibility of introducing first round product aerosol into the atmosphere, as would be the case in a fully nested system. Contamination is therefore minimised, while amplification of the desired product is maximised. PMID:9231160

  19. CO2 efflux from subterranean nests of ant communities in a seasonal tropical forest, Thailand.

    PubMed

    Hasin, Sasitorn; Ohashi, Mizue; Yamada, Akinori; Hashimoto, Yoshiaki; Tasen, Wattanachai; Kume, Tomonori; Yamane, Seiki

    2014-10-01

    Many ant species construct subterranean nests. The presence of their nests may explain soil respiration "hot spots", an important factor in the high CO2 efflux from tropical forests. However, no studies have directly measured CO2 efflux from ant nests. We established 61 experimental plots containing 13 subterranean ant species to evaluate the CO2 efflux from subterranean ant nests in a tropical seasonal forest, Thailand. We examined differences in nest CO2 efflux among ant species. We determined the effects of environmental factors on nest CO2 efflux and calculated an index of nest structure. The mean CO2 efflux from nests was significantly higher than those from the surrounding soil in the wet and dry seasons. The CO2 efflux was species-specific, showing significant differences among the 13 ant species. The soil moisture content significantly affected nest CO2 efflux, but there was no clear relationship between nest CO2 efflux and nest soil temperature. The diameter of the nest entrance hole affected CO2 efflux. However, there was no significant difference in CO2 efflux rates between single-hole and multiple-hole nests. Our results suggest that in a tropical forest ecosystem the increase in CO2 efflux from subterranean ant nests is caused by species-specific activity of ants, the nest soil environment, and nest structure.

  20. Hermetic G-16 percussion primer

    SciTech Connect

    Durand, N.A.; Weinmaster, R.R.; Massis, T.M.

    1988-01-01

    Studies were conducted to optimize a Hermetic percussion primer capable of surviving temperatures of 200/degree/C for up to 48 hours. These studies included work with typical brass percussion primers and the pyrotechnic composition designed G-16. The G-16 mixture is composed of antimony sulfide, calcium silicide, and potassium. The hermetically sealed assembly consists of a brass cup and anvil loaded with G-16 pyrotechnic mixture and assembly includes a steel disc which is laser welded over the sealing mechanisms cause negligible changes. This assembly can be used with other primers and is capable of enhanced output for specialized applications. 4 refs., 8 figs., 4 tabs.

  1. PCR Amplicon Prediction from Multiplex Degenerate Primer and Probe Sets

    2013-08-08

    Assessing primer specificity and predicting both desired and off-target amplification products is an essential step for robust PCR assay design. Code is described to predict potential polymerase chain reaction (PCR) amplicons in a large sequence database such as NCBI nt from either singleplex or a large multiplexed set of primers, allowing degenerate primer and probe bases, with target mismatch annotates amplicons with gene information automatically downloaded from NCBI, and optionally it can predict whether theremore » are also TaqMan/Luminex probe matches within predicted amplicons.« less

  2. PCR Amplicon Prediction from Multiplex Degenerate Primer and Probe Sets

    SciTech Connect

    Gardner, S. N.

    2013-08-08

    Assessing primer specificity and predicting both desired and off-target amplification products is an essential step for robust PCR assay design. Code is described to predict potential polymerase chain reaction (PCR) amplicons in a large sequence database such as NCBI nt from either singleplex or a large multiplexed set of primers, allowing degenerate primer and probe bases, with target mismatch annotates amplicons with gene information automatically downloaded from NCBI, and optionally it can predict whether there are also TaqMan/Luminex probe matches within predicted amplicons.

  3. Selection of primers for polymerase chain reaction.

    PubMed

    Rychlik, W

    1995-04-01

    One of the most important factors affecting the quality of PCR is the choice of primers. In general, the longer the PCR product the more difficult it is to select efficient primers and set appropriate designing primers, and in general, the more DNA sequence information is available, the better the chance of finding an optimal primer pair. Efficient primers can be designed by avoiding the following flaws: primer-dimer formation, self-complementarity, too low Tm of the primers, and/or their incorrect internal stability profile. Tips on subcloning PCR products, calculating duplex stability (predicting dimer formation strength), and designing degenerate primers are given.

  4. Preliminary evaluation of a nest usage sensor to detect double nest occupations of laying hens.

    PubMed

    Zaninelli, Mauro; Costa, Annamaria; Tangorra, Francesco Maria; Rossi, Luciana; Agazzi, Alessandro; Savoini, Giovanni

    2015-01-01

    Conventional cage systems will be replaced by housing systems that allow hens to move freely. These systems may improve hens' welfare, but they lead to some disadvantages: disease, bone fractures, cannibalism, piling and lower egg production. New selection criteria for existing commercial strains should be identified considering individual data about laying performance and the behavior of hens. Many recording systems have been developed to collect these data. However, the management of double nest occupations remains critical for the correct egg-to-hen assignment. To limit such events, most systems adopt specific trap devices and additional mechanical components. Others, instead, only prevent these occurrences by narrowing the nest, without any detection and management. The aim of this study was to develop and test a nest usage "sensor", based on imaging analysis, that is able to automatically detect a double nest occupation. Results showed that the developed sensor correctly identified the double nest occupation occurrences. Therefore, the imaging analysis resulted in being a useful solution that could simplify the nest construction for this type of recording system, allowing the collection of more precise and accurate data, since double nest occupations would be managed and the normal laying behavior of hens would not be discouraged by the presence of the trap devices. PMID:25629704

  5. Rapid and sensitive detection of Sclerotium rolfsii associated with collar rot disease of Amorphophallus paeoniifolius by species-specific polymerase chain reaction assay.

    PubMed

    Pravi, V; Jeeva, M L; Archana, P V

    2014-09-01

    Collar rot disease caused by Sclerotium rolfsii is an economically important disease prevailing in all Amorphophallus growing areas. The pathogen propagules surviving in soil and planting material are the major sources of inoculum. A nested PCR assay has been developed for specific detection of S. rolfsii in soil and planting material. The PCR detection limit was 10 pg in conventional assay whereas 0.1 pg in nested assay. The primers designed were found to be highly specific and could be used for accurate identification of pathogen up to species level. The protocol was standardized for detection of the pathogen in artificially and naturally infected field samples. PMID:24788585

  6. Nested Hierarchical Dirichlet Processes.

    PubMed

    Paisley, John; Wang, Chong; Blei, David M; Jordan, Michael I

    2015-02-01

    We develop a nested hierarchical Dirichlet process (nHDP) for hierarchical topic modeling. The nHDP generalizes the nested Chinese restaurant process (nCRP) to allow each word to follow its own path to a topic node according to a per-document distribution over the paths on a shared tree. This alleviates the rigid, single-path formulation assumed by the nCRP, allowing documents to easily express complex thematic borrowings. We derive a stochastic variational inference algorithm for the model, which enables efficient inference for massive collections of text documents. We demonstrate our algorithm on 1.8 million documents from The New York Times and 2.7 million documents from Wikipedia. PMID:26353240

  7. Characteristic distribution pattern of Helicobacter pylori in dental plaque and saliva detected with nested PCR.

    PubMed

    Song, Q; Lange, T; Spahr, A; Adler, G; Bode, G

    2000-04-01

    The precise mode of transmission and the natural reservoir for Helicobacter pylori are unknown. PCR assays have proved to be highly sensitive and specific and are regarded as the method of choice for detecting H. pylori DNA in the oral cavity. The aim of this study was to investigate the prevalence and distribution of H. pylori in the oral cavity. Forty-two patients undergoing gastroscopy were investigated for the presence of H. pylori in dental plaque and saliva by nested PCR, and in the stomach by the 13C-urea breath test. Samples tested comprised dental plaque from molars, premolars and incisors and saliva. Two sets of primers homologous to the 860-bp fragment of H. pylori DNA, which have been shown previously to be highly sensitive and specific, were used for nested PCR. Eleven patients (26.2%) were infected with H. pylori in the stomach. H. pylori DNA was identified in dental plaque samples from 41 patients (97%) and in 23 saliva samples (55%). The prevalence in dental plaque from molars, premolars and incisors was 82%, 64% and 59%, with an odds ratio of 3.18, 1.24 and 1 (reference), respectively. In conclusion, H. pylori was present in the oral cavity of 97% of tested patients, with a characteristic distribution that was independent of the infection status of the stomach. Thus H. pylori may belong to the normal oral microflora.

  8. Testing Nested Distributions

    NASA Astrophysics Data System (ADS)

    Economou, P.

    2010-09-01

    A number of criteria, test statistics and diagnostic plots have been developed in order to test the adapted distribution assumption. Usually, a simpler distribution is tested against a more complicated one (by adding an extra parameter), which include the first distribution as a special case (nested distributions). A characteristic example of such cases is the Burr XII distribution which can be obtained under the proportional hazards frailty model by assuming a Weibull baseline function and a Gamma frailty distribution with mean frailty equal to 1 and variance equal to θ. In this work, two new easy to construct and to interpret tests, a diagnostic plot and an asymptotic test, are presented in order to test nested distributions. The asymptotic test is based on the approximation of the difference of the two estimated nested distribution functions using the first two terms of the Taylor's expansion while the diagnostic plot is constructed using the exact difference of the two fitted distribution functions. Simulation results, using data sets with and without censored observations, demonstrate that the proposed tests perform, in most of the cases, better than other test statistics such as the LR and the Wald.

  9. PyNEST: A Convenient Interface to the NEST Simulator.

    PubMed

    Eppler, Jochen Martin; Helias, Moritz; Muller, Eilif; Diesmann, Markus; Gewaltig, Marc-Oliver

    2008-01-01

    The neural simulation tool NEST (http://www.nest-initiative.org) is a simulator for heterogeneous networks of point neurons or neurons with a small number of compartments. It aims at simulations of large neural systems with more than 10(4) neurons and 10(7) to 10(9) synapses. NEST is implemented in C++ and can be used on a large range of architectures from single-core laptops over multi-core desktop computers to super-computers with thousands of processor cores. Python (http://www.python.org) is a modern programming language that has recently received considerable attention in Computational Neuroscience. Python is easy to learn and has many extension modules for scientific computing (e.g. http://www.scipy.org). In this contribution we describe PyNEST, the new user interface to NEST. PyNEST combines NEST's efficient simulation kernel with the simplicity and flexibility of Python. Compared to NEST's native simulation language SLI, PyNEST makes it easier to set up simulations, generate stimuli, and analyze simulation results. We describe how PyNEST connects NEST and Python and how it is implemented. With a number of examples, we illustrate how it is used. PMID:19198667

  10. Why wasp foundresses change nests: relatedness, dominance, and nest quality.

    PubMed

    Seppä, Perttu; Queller, David C; Strassmann, Joan E

    2012-01-01

    The costs and benefits of different social options are best understood when individuals can be followed as they make different choices, something that can be difficult in social insects. In this detailed study, we follow overwintered females of the social wasp Polistes carolina through different nesting strategies in a stratified habitat where nest site quality varies with proximity to a foraging area, and genetic relatedness among females is known. Females may initiate nests, join nests temporarily or permanently, or abandon nests. Females can become helpers or egglayers, effectively workers or queens. What they actually do can be predicted by a combination of ecological and relatedness factors. Advantages through increased lifetime success of individuals and nests drives foundresses of the social wasp Polistes from solitary to social nest founding. We studied reproductive options of spring foundresses of P. carolina by monitoring individually-marked wasps and assessing reproductive success of each foundress by using DNA microsatellites. We examined what behavioral decisions foundresses make after relaxing a strong ecological constraint, shortage of nesting sites. We also look at the reproductive consequences of different behaviors. As in other Polistes, the most successful strategy for a foundress was to initiate a nest as early as possible and then accept others as subordinates. A common feature for many P. carolina foundresses was, however, that they reassessed their reproductive options by actively monitoring other nests at the field site and sometimes moving permanently to new nests should that offer better (inclusive) fitness prospects compared to their original nests. A clear motivation for moving to new nests was high genetic relatedness; by the end of the foundress period all females were on nests with full sisters.

  11. Formalizing narratives using nested circumscription

    SciTech Connect

    Baral, C.; Gabaldon, A.; Provetti, A.

    1996-12-31

    The representation of narratives of actions and observations is a current issue in Knowledge Representation, where traditional plan-oriented treatments of action seem to fall short. To address narratives, Pinto and Reiter have extended Situation Calculus axioms, Kowalski and Sergot have introduced the Event Calculus in Logic Programming, and Baral et al. have defined the specification language L which allows to express actual and hypothetical situations in a uniform setting. The L entailment relation can formalize several forms of reasoning about actions and change. In this paper we illustrate a translation of L theories into Nested Abnormality Theories, a novel form of circumscription. The proof of soundness and completeness of the translation is the main technical result of the paper, but attention is also devoted to the features of Nested Abnormality Theories to capture commonsense reasoning in general and to clarify which assumptions a logical formalization forces upon a domain. These results also help clarifying the relationship between L and other recent circumscriptive formalization for narratives, such as Miller and Shanahan`s.

  12. PD5: A General Purpose Library for Primer Design Software

    PubMed Central

    Riley, Michael C.; Aubrey, Wayne; Young, Michael; Clare, Amanda

    2013-01-01

    Background Complex PCR applications for large genome-scale projects require fast, reliable and often highly sophisticated primer design software applications. Presently, such applications use pipelining methods to utilise many third party applications and this involves file parsing, interfacing and data conversion, which is slow and prone to error. A fully integrated suite of software tools for primer design would considerably improve the development time, the processing speed, and the reliability of bespoke primer design software applications. Results The PD5 software library is an open-source collection of classes and utilities, providing a complete collection of software building blocks for primer design and analysis. It is written in object-oriented C++ with an emphasis on classes suitable for efficient and rapid development of bespoke primer design programs. The modular design of the software library simplifies the development of specific applications and also integration with existing third party software where necessary. We demonstrate several applications created using this software library that have already proved to be effective, but we view the project as a dynamic environment for building primer design software and it is open for future development by the bioinformatics community. Therefore, the PD5 software library is published under the terms of the GNU General Public License, which guarantee access to source-code and allow redistribution and modification. Conclusions The PD5 software library is downloadable from Google Code and the accompanying Wiki includes instructions and examples: http://code.google.com/p/primer-design PMID:24278254

  13. On the mechanism of the modular primer effect.

    PubMed Central

    Beskin, A D; Zevin-Sonkin, D; Sobolev, I A; Ulanovsky, L E

    1995-01-01

    Modular primers are strings of three contiguously annealed unligated oligonucleotides (modules) as short as 5- or 6-mers, selected from a presynthesized library. It was previously found that such strings can prime DNA sequencing reactions specifically, thus eliminating the need for the primer synthesis step in DNA sequencing by primer walking. It has remained largely a mystery why modular primers prime uniquely, while a single module, used alone in the same conditions, often shows alternative priming of comparable strength. In a puzzling way, the single module, even in a large excess over the template, no longer primes at the alternative sites, when modules with which it can form a contiguous string are also present. Here we describe experiments indicating that this phenomenon cannot be explained by cooperative annealing of the modules to the template. Instead, the mechanism seems to involve competition between different primers for the available polymerase. In this competition, the polymerase is preferentially engaged by longer primers, whether modular or conventional, at the expense of shorter primers, even though the latter can otherwise prime with similar or occasionally higher efficiency. Images PMID:7659510

  14. Collecting in collections: a PCR strategy and primer set for DNA barcoding of decades-old dried museum specimens.

    PubMed

    Mitchell, Andrew

    2015-09-01

    Natural history museums are vastly underutilized as a source of material for DNA analysis because of perceptions about the limitations of DNA degradation in older specimens. Despite very few exceptions, most DNA barcoding projects, which aim to obtain sequence data from all species, generally use specimens collected specifically for that purpose, instead of the wealth of identified material in museums, constrained by the lack of suitable PCR methods. Any techniques that extend the utility of museum specimens for DNA analysis therefore are highly valuable. This study first tested the effects of specimen age and PCR amplicon size on PCR success rates in pinned insect specimens, then developed a PCR primer set and amplification strategy allowing greatly increased utilization of older museum specimens for DNA barcoding. PCR success rates compare favourably with the few published studies utilizing similar aged specimens, and this new strategy has the advantage of being easily automated for high-throughput laboratory workflows. The strategy uses hemi-nested, degenerate, M13-tailed PCR primers to amplify two overlapping amplicons, using two PCRs per amplicon (i.e. four PCRs per DNA sample). Initial PCR products are reamplified using an internal primer and a M13 primer. Together the two PCR amplicons yield 559 bp of the COI gene from Coleoptera, Lepidoptera, Diptera, Hemiptera, Odonata and presumably also other insects. BARCODE standard-compliant data were recovered from 67% (56 of 84) of specimens up to 25 years old, and 51% (102 of 197) of specimens up to 55 years old. Given the time, cost and specialist expertise required for fieldwork and identification, 'collecting in collections' is a viable alternative allowing researchers to capitalize on the knowledge captured by curation work in decades past.

  15. Colour preferences in nest-building zebra finches.

    PubMed

    Muth, Felicity; Steele, Matthew; Healy, Susan D

    2013-10-01

    Some bird species are selective in the materials they choose for nest building, preferring, for example, materials of one colour to others. However, in many cases the cause of these preferences is not clear. One of those species is the zebra finch, which exhibits strong preferences for particular colours of nest material. In an attempt to determine why these birds strongly prefer one colour of material over another, we compared the preferences of paired male zebra finches for nest material colour with their preferences for food of the same colours. We found that birds did indeed prefer particular colours of nest material (in most cases blue) but that they did not generally prefer food of one colour over the other colours. It appears, then, that a preference for one colour or another of nest material is specific to the nest-building context. This article is part of a Special Issue entitled: insert SI title.

  16. On piecewise smooth vector fields tangent to nested tori

    NASA Astrophysics Data System (ADS)

    Carvalho, Tiago; Teixeira, Marco A.

    2016-10-01

    In this paper we present a number of results involving 3D nonsmooth dynamical systems tangent to a foliation. We study one-parameter families of systems Zε passing through a specific model Z0 whose phase portrait is foliated by invariant nested tori. For each positive integer k we, explicitly, construct a family Zεk possessing exactly k nested tori.

  17. EAGLES NEST WILDERNESS, COLORADO.

    USGS Publications Warehouse

    Tweto, Ogden; Williams, Frank E.

    1984-01-01

    On the basis of a geologic and mineral survey, a primitive area that constitutes the nucleus of the Eagles Nest Wilderness, Colorado was appraised to offer little promise for the occurrence of mineral or energy resources. Among the additional areas later incorporated in the wilderness, only a strip near a major fault west and northwest of Frisco and Dillon is classed as having probable mineral-resource potential. If mineral deposits exist, they probably are of the silver-lead-zinc or fluorspar types.

  18. Exciton Primer-mediated SNP detection in SmartAmp2 reactions.

    PubMed

    Lezhava, Alexander; Ishidao, Takefumi; Ishizu, Yuri; Naito, Kana; Hanami, Takeshi; Katayama, Atsuko; Kogo, Yasushi; Soma, Takahiro; Ikeda, Shuji; Murakami, Kayoko; Nogawa, Chihiro; Itoh, Masayoshi; Mitani, Yasumasa; Harbers, Matthias; Okamoto, Akimitsu; Hayashizaki, Yoshihide

    2010-02-01

    Most commonly used intercalating fluorescent dyes in DNA detection are lacking any sequence specificity, whereas so-called Exciton Primers can overcome this limitation by functioning as "sequence-specific dyes." After hybridization to complementary sequences, the fluorescence of Exciton Primers provides sequence-specific signals for real-time monitoring of amplification reactions. Applied to the SmartAmp2 mutation detection process, Exciton Primers show high signal strength with low background leading to a superior specificity and sensitivity compared to SYBR Green I. Signal strength can be further enhanced using multiple dyes within one Exciton Primer or use of multiple Exciton Primers in the same amplification reaction. Here we demonstrate the use of Exciton Primers for genotyping a single nucleotide polymorphism (SNP) in the VKORC1 locus (-1639G>A) relevant for Warfarin dosing as an example for Exciton Primers mediated genotyping by SmartAmp2. The genotyping assay can use only one labeled Exciton Primer for endpoint detection, or simultaneously by real-time monitoring detect wild-type and mutant alleles in a one-tube reaction using two Exciton Primers having different dyes. Working directly from blood samples, Exciton Primer mediated genotyping by SmartAmp2 offers superior solutions for rapid point-of-care testing.

  19. New primers for DNA barcoding of digeneans and cestodes (Platyhelminthes).

    PubMed

    Van Steenkiste, Niels; Locke, Sean A; Castelin, Magalie; Marcogliese, David J; Abbott, Cathryn L

    2015-07-01

    Digeneans and cestodes are species-rich taxa and can seriously impact human health, fisheries, aqua- and agriculture, and wildlife conservation and management. DNA barcoding using the COI Folmer region could be applied for species detection and identification, but both 'universal' and taxon-specific COI primers fail to amplify in many flatworm taxa. We found that high levels of nucleotide variation at priming sites made it unrealistic to design primers targeting all flatworms. We developed new degenerate primers that enabled acquisition of the COI barcode region from 100% of specimens tested (n = 46), representing 23 families of digeneans and 6 orders of cestodes. This high success rate represents an improvement over existing methods. Primers and methods provided here are critical pieces towards redressing the current paucity of COI barcodes for these taxa in public databases.

  20. A Home Computer Primer.

    ERIC Educational Resources Information Center

    Stone, Antonia

    1982-01-01

    Provides general information on currently available microcomputers, computer programs (software), hardware requirements, software sources, costs, computer games, and programing. Includes a list of popular microcomputers, providing price category, model, list price, software (cassette, tape, disk), monitor specifications, amount of random access…

  1. Universal primers that amplify RNA from all three flavivirus subgroups

    PubMed Central

    Maher-Sturgess, Sheryl L; Forrester, Naomi L; Wayper, Paul J; Gould, Ernest A; Hall, Roy A; Barnard, Ross T; Gibbs, Mark J

    2008-01-01

    Background Species within the Flavivirus genus pose public health problems around the world. Increasing cases of Dengue and Japanese encephalitis virus in Asia, frequent outbreaks of Yellow fever virus in Africa and South America, and the ongoing spread of West Nile virus throughout the Americas, show the geographical burden of flavivirus diseases. Flavivirus infections are often indistinct from and confused with other febrile illnesses. Here we review the specificity of published primers, and describe a new universal primer pair that can detect a wide range of flaviviruses, including viruses from each of the recognised subgroups. Results Bioinformatic analysis of 257 published full-length Flavivirus genomes revealed conserved regions not previously targeted by primers. Two degenerate primers, Flav100F and Flav200R were designed from these regions and used to generate an 800 base pair cDNA product. The region amplified encoded part of the methyltransferase and most of the RNA-dependent-RNA-polymerase (NS5) coding sequence. One-step RT-PCR testing was successful using standard conditions with RNA from over 60 different flavivirus strains representing about 50 species. The cDNA from each virus isolate was sequenced then used in phylogenetic analyses and database searches to confirm the identity of the template RNA. Conclusion Comprehensive testing has revealed the broad specificity of these primers. We briefly discuss the advantages and uses of these universal primers. PMID:18218114

  2. Neste plans three projects

    SciTech Connect

    Not Available

    1993-02-03

    Neste Chemicals (Helsinki) is discussing three joint ventures with local authorities in China, says Mikko Haapavaara, v.p./Asia. The projects should help the Finnish producer to increase sales in Asia by a considerable amount by 2000, he says. The plan involves production of polyethylene (PE), unsaturated polyester resins and PE compounding-all core operations. Sites have not been selected, but Shanghai is the favored location for the PE operations. The company is also looking at a site in the south, near Hong Kong, and at locations near Beijing. The PE plant would need to be near an ethylene unit, says Haapavaara. The PE resin plant would be designed to produce about 150,000 m.t./year and would cost about No. 150 million. A part of the output would need to be exported to take care of the financing, the company says. A feasibility study now under way with the potential Chinese partners should be completed by the end of March. The plant would use Neste's linear low-density PE process, proved in a world-scale plant at Beringen, Belgium. The compounding units would produce specialty PE material for the wire and cable and pipe industry. The company is a joint venture partner in a propane dehydrogenation/polypropylene (PP) plant and a minority partner in a Qualipoly, the 20,000 m.t./year unsaturated polyester resin producer.

  3. Lifespan Analyses of Forest Raptor Nests: Patterns of Creation, Persistence and Reuse

    PubMed Central

    Jiménez-Franco, María V.; Martínez, José E.; Calvo, José F.

    2014-01-01

    Structural elements for breeding such as nests are key resources for the conservation of bird populations. This is especially true when structural elements require a specific and restricted habitat, or if the construction of nests is costly in time and energy. The availability of nesting-platforms is influenced by nest creation and persistence. In a Mediterranean forest in southeastern Spain, nesting-platforms are the only structural element for three forest-dwelling raptor species: booted eagle Aquila pennata, common buzzard Buteo buteo and northern goshawk Accipiter gentilis. From 1998 to 2013, we tracked the fate of 157 nesting-platforms built and reused by these species with the aim of determining the rates of creation and destruction of nesting-platforms, estimating nest persistence by applying two survival analyses, describing the pattern of nest reuse and testing the effects of nest use on breeding success. Nest creation and destruction rates were low (0.14 and 0.05, respectively). Using Kaplan Meier survival estimates and Cox proportional-hazards regression models we found that median nest longevity was 12 years and that this was not significantly affected by nest characteristics, nest-tree dimensions, nest-builder species, or frequency of use of the platform. We also estimated a transition matrix, considering the different stages of nest occupation (vacant or occupied by one of the focal species), to obtain the fundamental matrix and the average life expectancies of nests, which varied from 17.9 to 19.7 years. Eighty six percent of nests were used in at least one breeding attempt, 67.5% were reused and 17.8% were successively occupied by at least two of the study species. The frequency of nest use had no significant effects on the breeding success of any species. We conclude that nesting-platforms constitute an important resource for forest raptors and that their longevity is sufficiently high to allow their reuse in multiple breeding attempts. PMID

  4. Lifespan analyses of forest raptor nests: patterns of creation, persistence and reuse.

    PubMed

    Jiménez-Franco, María V; Martínez, José E; Calvo, José F

    2014-01-01

    Structural elements for breeding such as nests are key resources for the conservation of bird populations. This is especially true when structural elements require a specific and restricted habitat, or if the construction of nests is costly in time and energy. The availability of nesting-platforms is influenced by nest creation and persistence. In a Mediterranean forest in southeastern Spain, nesting-platforms are the only structural element for three forest-dwelling raptor species: booted eagle Aquila pennata, common buzzard Buteo buteo and northern goshawk Accipiter gentilis. From 1998 to 2013, we tracked the fate of 157 nesting-platforms built and reused by these species with the aim of determining the rates of creation and destruction of nesting-platforms, estimating nest persistence by applying two survival analyses, describing the pattern of nest reuse and testing the effects of nest use on breeding success. Nest creation and destruction rates were low (0.14 and 0.05, respectively). Using Kaplan Meier survival estimates and Cox proportional-hazards regression models we found that median nest longevity was 12 years and that this was not significantly affected by nest characteristics, nest-tree dimensions, nest-builder species, or frequency of use of the platform. We also estimated a transition matrix, considering the different stages of nest occupation (vacant or occupied by one of the focal species), to obtain the fundamental matrix and the average life expectancies of nests, which varied from 17.9 to 19.7 years. Eighty six percent of nests were used in at least one breeding attempt, 67.5% were reused and 17.8% were successively occupied by at least two of the study species. The frequency of nest use had no significant effects on the breeding success of any species. We conclude that nesting-platforms constitute an important resource for forest raptors and that their longevity is sufficiently high to allow their reuse in multiple breeding attempts.

  5. Nest survival estimation: a review of alternatives to the Mayfield estimator

    USGS Publications Warehouse

    Jehle, G.; Yackel Adams, A.A.; Savidge, J.A.; Skagen, S.K.

    2004-01-01

    Reliable estimates of nest survival are essential for assessing strategies for avian conservation. We review the history of modifications and alternatives for estimating nest survival, with a focus on four techniques: apparent nest success, the Mayfield estimator, the Stanley method, and program MARK. The widely used Mayfield method avoids the known positive bias inherent in apparent nest success by estimating daily survival rates using the number of exposure days, eliminating the need to monitor nests from initiation. Concerns that some of Mayfield's assumptions were restrictive stimulated the development of new techniques. Stanley's method allows for calculation of stage-specific daily survival rates when transition and failure dates are unknown, and eliminates Mayfield's assumption that failure occurred midway through the nest-check interval. Program MARK obviates Mayfield's assumption of constant daily survival within nesting stages and evaluates variation in nest survival as a function of biologically relevant factors. These innovative methods facilitate the evaluation of nest survival using an information-theoretic approach. We illustrate use of these methods with Lark Bunting (Calamospiza melanocorys) nest data from the Pawnee National Grassland, Colorado. Nest survival estimates calculated using Mayfield, Stanley, and MARK methods were similar, but apparent nest success estimates ranged 1-24% greater than the other estimates. MARK analysis revealed that survival of Lark Bunting nests differed between site-year groups, declined with both nest age and time in season, but did not vary with weather parameters. We encourage researchers to use these approaches to gain reliable and meaningful nest survival estimates.

  6. A Primer on Prototyping.

    PubMed

    Lynch, Dylan; Biron, David

    2015-01-01

    Standard mechanical components, such as adapters or mounts, are ubiquitous in research laboratories, C. elegans labs included. Recently, in-house prototyping and fabricating both standard and custom mechanical parts has become simple and cost effective. Here we describe the basic steps, equipment, and considerations required for rapid prototyping of a handful of simple yet useful designs. These examples were chosen for their simplicity, as well as for demonstrating specific practicalities. They are thus appropriate as training exercises. PMID:26423979

  7. A Primer on Prototyping.

    PubMed

    Lynch, Dylan; Biron, David

    2015-01-01

    Standard mechanical components, such as adapters or mounts, are ubiquitous in research laboratories, C. elegans labs included. Recently, in-house prototyping and fabricating both standard and custom mechanical parts has become simple and cost effective. Here we describe the basic steps, equipment, and considerations required for rapid prototyping of a handful of simple yet useful designs. These examples were chosen for their simplicity, as well as for demonstrating specific practicalities. They are thus appropriate as training exercises.

  8. Detection of hepatopancreatic parvovirus (HPV) in wild shrimp from India by nested polymerase chain reaction (PCR).

    PubMed

    Manjanaik, B; Umesha, K R; Karunasagar, Indrani; Karunasagar, Iddya

    2005-02-28

    The prevalence of hepatopancreatic parvovirus (HPV) in wild penaeid shrimp samples from India was studied by nested polymerase chain reaction (PCR) using primers designed in our laboratory. The virus could be detected in 9 out of 119 samples by non-nested PCR. However, by nested PCR 69 out of 119 samples were positive. The PCR results were confirmed by hybridization with digoxigenin-labelled DNA probe. Shrimp species positive by non-nested PCR included Penaeus monodon, Penaeus indicus and Penaeus semisulcatus and by nested PCR Parapenaeopsis stylifera, Penaeus japonicus, Metapenaeus monoceros, M. affinis, M. elegans, M. dobsoni, M. ensis and Solenocera choprai. This is the first report on the prevalence of HPV in captured wild shrimp from India. PMID:15819441

  9. Charter School Primer. Peter Lang Primer. Volume 34

    ERIC Educational Resources Information Center

    Tryjankowski, Anne Marie

    2012-01-01

    The "Charter School Primer" presents an overview of public charter schools in the United States. The book discusses what charter schools are; the history of public charter school choice in the United States; the role of teachers, parents, boards, and unions in the charter school movement; and gives examples of innovations in education made…

  10. Vygotsky on Education Primer. Peter Lang Primer. Volume 30

    ERIC Educational Resources Information Center

    Lake, Robert

    2012-01-01

    The "Vygotsky on Education Primer" serves as an introduction to the life and work of the Russian psychologist Lev Vygotsky. Even though he died almost eighty years ago, his life's work remains both relevant and significant to the field of education today. This book examines Vygotsky's emphasis on the role of cultural and historical context in…

  11. Influence of primer sequences and DNA extraction method on detection of non-O157 Shiga toxin-producing Escherichia coli in ground beef by real-time PCR targeting the eae, stx, and serogroup-specific genes.

    PubMed

    Wasilenko, Jamie L; Fratamico, Pina M; Narang, Neelam; Tillman, Glenn E; Ladely, Scott; Simmons, Mustafa; Cray, William C

    2012-11-01

    Non-O157 Shiga toxin-producing Escherichia coli (STEC) infections, particularly those caused by the "big six" or "top six" non-O157 serogroups (O26, O45, O103, O111, O121, and O145) can result in severe illness and complications. Because of their significant public health impact and the notable prevalence of STEC in cattle, methods for detection of the big six non-O157 STEC in ground beef have been established. Currently, the U.S. Department of Agriculture, Food Safety and Inspection Service detection methods for screening beef samples for non-O157 STEC target the stx(1), stx(2), and eae virulence genes, with the 16S rRNA gene as an internal control, in a real-time PCR multiplex assay. Further, the serogroup is determined by PCR targeting genes in the E. coli O-antigen gene clusters of the big six non-O157 serogroups. The method that we previously reported was improved so that additional stx variants, stx(1d), stx(2e), and stx(2g), are detected. Additionally, alignments of the primers targeting the eae gene were used to improve the detection assay so that eae subtypes that could potentially be of clinical significance would also be detected. Therefore, evaluation of alternative real-time PCR assay primers and probes for the stx and eae reactions was carried out in order to increase the stx and eae subtypes detected. Furthermore, a Tris-EDTA DNA extraction method was compared with a previously used procedure that was based on a commercially available reagent. The Tris-EDTA DNA extraction method significantly decreased the cycle threshold values for the stx assay (P < 0.0001) and eae assay (P < 0.0001), thereby increasing the ability to detect the targets. The use of different stx primers and probes increased the subtypes detected to include stx(1d), stx(2e), and stx(2g), and sequence data showed that modification of the eae primer should allow the known eae subtypes to be detected.

  12. Hubble Space Telescope Primer for Cycle 21

    NASA Astrophysics Data System (ADS)

    Gonzaga, S.; et al.

    2012-12-01

    The Hubble Space Telescope Primer for Cycle 21 is a companion document to the HST Call for Proposals1. It provides an overview of the Hubble Space Telescope (HST), with basic information about telescope operations, instrument capabilities, and technical aspects of the proposal preparation process. A thorough understanding of the material in this document is essential for the preparation of a competitive proposal. This document is available as an online HTML document and a PDF file. The HTML version, optimized for online browsing, contains many links to additional information. The PDF version is optimized for printing, but online PDF readers have search capabilities for quick retrieval of specific information.

  13. Protein sulfation analysis--A primer.

    PubMed

    Monigatti, Flavio; Hekking, Brian; Steen, Hanno

    2006-12-01

    The aim of this review is to present an overview of protein sulfation in the context of 'modificomics', i.e. post-translational modification-specific proteome research. In addition to a short introduction to the biology of protein sulfation (part 1), we will provide detailed discussion regarding (i) methods and tools for prediction of protein tyrosine sulfation sites (part 2), (ii) biochemical techniques used for protein sulfation analysis (part 3.1), and (iii) mass spectrometric strategies and methods applied to protein sulfation analysis (part 3.2). We will highlight strengths and limitations of different strategies and approaches (including references), providing a primer for newcomers to protein sulfation analysis.

  14. Nest morphology and body size of Ross' Geese and Lesser Snow Geese

    USGS Publications Warehouse

    McCracken, K.G.; Afton, A.D.; Alisauskas, R.T.

    1997-01-01

    Arctic-nesting geese build large, insulated nests to protect developing embryos from cold ambient temperatures. Ross' Geese (Chen rossii) are about two-thirds the mass of Lesser Snow Geese (C. caerulescens caerulescens), have higher mass-specific metabolic rate, and maintain lower nest attentiveness, yet they hatch goslings with more functionally mature gizzards and more protein for their size than do Lesser Snow Geese. We compared nest size (a reflection of nest insulation) in four distinct habitats in a mixed breeding colony of Ross' Geese and Lesser Snow Geese at Karrak Lake, Northwest Territories, Canada. After adjusting measurements for nest-specific egg size and clutch size, we found that overall nest morphology differed between species and among habitats. Nest size increased progressively among heath, rock, mixed, and moss habitats. When nesting materials were not limiting, nests were smaller in habitats that provided cover from wind and precipitation than in habitats that did not provide cover. Ross' Geese constructed relatively larger, more insulated nests than did Lesser Snow Geese, which may hasten embryonic development, minimize energy expenditure during incubation, and minimize embryonic cooling during recesses. We suggest that relative differences in nest morphology reflect greater selection for Ross' Geese to improve nest insulation because of their smaller size (adults and embryos), higher mass-specific metabolic rate, and lower incubation constancy.

  15. Transferability of retrotransposon primers derived from Persimmon (Diospyros kaki Thunb.) across other plant species.

    PubMed

    Du, X Y; Hu, Q N; Zhang, Q L; Wang, Y B; Luo, Z R

    2013-06-06

    Retrotransposon-based molecular markers are powerful molecular tools. However, these markers are not readily available due to the difficulty in obtaining species-specific retrotransposon primers. Although recent techniques enabling the rapid isolation of retrotransposon sequences have facilitated primer development, this process nonetheless remains time-consuming and costly. Therefore, research into the transferability of retrotransposon primers developed from one plant species onto others would be of great value. The present study investigated the transferability of retrotransposon primers derived from 'Luotian-tianshi' persimmon (Diospyros kaki Thunb.) across other fruit crops, as well as within the genus using inter-retrotransposon amplified polymorphism molecular marker. Fourteen of the 26 retrotransposon primers tested (53.85%) produced robust and reproducible amplification products across all fruit crops tested, indicating their applicability across plant species. Four of the 13 fruit crops showed the best transferability performances: persimmon, grape, citrus, and peach. Furthermore, similarity coefficients and UPGMA clustering indicated that these primers could further offer a potential tool for germplasm differentiation, parentage identification, genetic diversity assessment, classification, and phylogenetic studies across a variety of plant species. Transferability was further confirmed by examining published primers derived from Rosaceae, Gramineae, and Solanaceae. This study is one of the few currently available studies concerning the transferability of retrotransposon primers across plant species in general, and is the first successful study of the transferability of retrotransposon primers derived from persimmon. The primers presented here will help reduce costs for future retrotransposon primer development and therefore contribute to the popularization of retrotransposon molecular markers.

  16. Inflatable nested toroid structure

    NASA Technical Reports Server (NTRS)

    Johnson, Christopher J. (Inventor); Raboin, Jasen L. (Inventor); Spexarth, Gary R. (Inventor)

    2011-01-01

    An inflatable structure comprises at least two generally toroidal, inflatable modules. When in a deployed mode, the first, inner module has a major diameter less than that of a second, outer module and is positioned within the inner circumference of the outer module such that the first module is nested circumferentially alongside the second module. The inflatable structure, in a non-deployed, non-inflated mode, is of compact configuration and adapted to be transported to a site of deployment. When deployed, the inflatable structure is of substantially increased interior volume. In one embodiment, access between the interior of the first module and the second module is provided by at least one port or structural pass-through. In another embodiment, the inflatable structure includes at least one additional generally toroidal module external of and circumferentially surrounding the second module.

  17. Using Artificial Nests to Study Nest Predation in Birds

    ERIC Educational Resources Information Center

    Belthoff, James R.

    2005-01-01

    A simple and effective field exercise that demonstrates factors affecting predation on bird nests is described. With instructor guidance, students in high school biology or college-level biology, ecology, animal behavior, wildlife management or ornithology laboratory courses can collaborate to design field experiments related to nest depredation.

  18. Primer vector theory and applications

    NASA Technical Reports Server (NTRS)

    Jezewski, D. J.

    1975-01-01

    A method developed to compute two-body, optimal, N-impulse trajectories was presented. The necessary conditions established define the gradient structure of the primer vector and its derivative for any set of boundary conditions and any number of impulses. Inequality constraints, a conjugate gradient iterator technique, and the use of a penalty function were also discussed.

  19. A Hearing Aid Primer 1

    ERIC Educational Resources Information Center

    Yetter, Carol J.

    2009-01-01

    This hearing aid primer is designed to define the differences among the three levels of hearing instrument technology: conventional analog circuit technology (most basic), digitally programmable/analog circuit technology (moderately advanced), and fully digital technology (most advanced). Both moderate and advanced technologies mean that hearing…

  20. Freshwater Wetlands: A Citizen's Primer.

    ERIC Educational Resources Information Center

    Catskill Center for Conservation and Development, Inc., Hobart, NY.

    The purpose of this "primer" for the general public is to describe the general characteristics of wetlands and how wetland alteration adversely affects the well-being of humans. Particular emphasis is placed on wetlands in New York State and the northeast. Topics discussed include wetland values, destruction of wetlands, the costs of wetland…

  1. Coal mining: A petex primer

    SciTech Connect

    Not Available

    1985-01-01

    This book is an introduction to the coal industry - from planning a mine to delivering coal to a power plant. The primer covers what coal is and how it is used, modern underground and surface mining practices, coal preparation and transport, and the relation between coal and the environment.

  2. Postsecondary Data Connections: A Primer

    ERIC Educational Resources Information Center

    Data Quality Campaign, 2011

    2011-01-01

    There is an increasing focus at the state and federal levels on linking data across the P-20/Workforce spectrum to help inform policies and practices. This primer is intended to provide policymakers with: (1) An overview of the status of states vis-a-vis the linking of postsecondary data to K-12 and workforce data; (2) A subset of questions…

  3. Computational intelligence-based polymerase chain reaction primer selection based on a novel teaching-learning-based optimisation.

    PubMed

    Cheng, Yu-Huei

    2014-12-01

    Specific primers play an important role in polymerase chain reaction (PCR) experiments, and therefore it is essential to find specific primers of outstanding quality. Unfortunately, many PCR constraints must be simultaneously inspected which makes specific primer selection difficult and time-consuming. This paper introduces a novel computational intelligence-based method, Teaching-Learning-Based Optimisation, to select the specific and feasible primers. The specified PCR product lengths of 150-300 bp and 500-800 bp with three melting temperature formulae of Wallace's formula, Bolton and McCarthy's formula and SantaLucia's formula were performed. The authors calculate optimal frequency to estimate the quality of primer selection based on a total of 500 runs for 50 random nucleotide sequences of 'Homo species' retrieved from the National Center for Biotechnology Information. The method was then fairly compared with the genetic algorithm (GA) and memetic algorithm (MA) for primer selection in the literature. The results show that the method easily found suitable primers corresponding with the setting primer constraints and had preferable performance than the GA and the MA. Furthermore, the method was also compared with the common method Primer3 according to their method type, primers presentation, parameters setting, speed and memory usage. In conclusion, it is an interesting primer selection method and a valuable tool for automatic high-throughput analysis. In the future, the usage of the primers in the wet lab needs to be validated carefully to increase the reliability of the method.

  4. Computational intelligence-based polymerase chain reaction primer selection based on a novel teaching-learning-based optimisation.

    PubMed

    Cheng, Yu-Huei

    2014-12-01

    Specific primers play an important role in polymerase chain reaction (PCR) experiments, and therefore it is essential to find specific primers of outstanding quality. Unfortunately, many PCR constraints must be simultaneously inspected which makes specific primer selection difficult and time-consuming. This paper introduces a novel computational intelligence-based method, Teaching-Learning-Based Optimisation, to select the specific and feasible primers. The specified PCR product lengths of 150-300 bp and 500-800 bp with three melting temperature formulae of Wallace's formula, Bolton and McCarthy's formula and SantaLucia's formula were performed. The authors calculate optimal frequency to estimate the quality of primer selection based on a total of 500 runs for 50 random nucleotide sequences of 'Homo species' retrieved from the National Center for Biotechnology Information. The method was then fairly compared with the genetic algorithm (GA) and memetic algorithm (MA) for primer selection in the literature. The results show that the method easily found suitable primers corresponding with the setting primer constraints and had preferable performance than the GA and the MA. Furthermore, the method was also compared with the common method Primer3 according to their method type, primers presentation, parameters setting, speed and memory usage. In conclusion, it is an interesting primer selection method and a valuable tool for automatic high-throughput analysis. In the future, the usage of the primers in the wet lab needs to be validated carefully to increase the reliability of the method. PMID:25429503

  5. Does nonrandom nest placement imply nonrandom nest predation? - A reply

    USGS Publications Warehouse

    Cooper, R.J.; Wilson, R.R.; Zenitsky, G.D.; Mullin, S.J.; Dececco, J.D.; Marshall, M.R.; Wolf, D.J.; Pomara, L.Y.

    1999-01-01

    In response to the critique by Schmidt and Whelan (Condor 101(4):916-920, 1999), we find that the relationship between nest success and tree selectivity is dependent upon inclusion or exclusion of particular tree species, whether or not years are pooled, and the selectivity index used. We question their use of point estimates of nest success with extremely high variances, defend our index, question the application of the Chesson (1983) index to our data, and explain the need to analyze years separately. Bottomland hardwood forest systems are extremely variable; hydroperiods alter the suitability of nesting substrates, availability of alternative food, and behavior of predators and their prey. Given these features, actively searching for Acadian Flycatcher (Empidonax virescens) nests is seldom an efficient predator foraging strategy. Therefore, these predation events are best described as random; nests are principally encountered opportunistically by generalist predators while searching for other prey.

  6. Olfactory response of megachilid bees, Osmia lignaria, Megachile rotundata, and M. pugnata, to individual cues from old nest cavities.

    PubMed

    Pitts-Singer, Theresa L

    2007-04-01

    The megachilid bees Osmia lignaria Say, Megachile rotundata (F.), and M. pugnata Say were tested for attraction to various components associated with their old nest cavities, or chemical extracts of these components, using a Y-tube olfactory response bioassay. Female bees of these species are known to nest in or near old nest cavities, implying that remnant nest components are important cues for bees looking for nest cavities. Significant results show that female bees were attracted to components that may provide species-specific cues or indicate conspecific nesting activity. Specifically, O. lignaria females showed attraction only to the female cocoon. M. rotundata females were attracted to intact nest cells, the fecal material on the outside of a cocoon, leaf pieces used as nest cell lining, and the extract of leaf pieces. M. pugnata females were attracted to the whole nest cell, the paper straw nesting material with attached cocoon, and feces. PMID:17445375

  7. Impact of special early harvest seasons on subarctic-nesting and temperate-nesting Canada geese

    USGS Publications Warehouse

    Sheaffer, S.E.; Kendall, W.L.; Bowers, E. Frank

    2005-01-01

    Dramatic changes in wintering distributions of Canada geese (Branta canadensis) have occurred over the past 50 years in eastern North America. Declines in numbers of subarctic-nesting geese wintering in southern states, and increases in numbers wintering in northern regions, have resulted in a northern shift in winter distributions. In contrast, numbers of temperate-nesting geese have increased throughout eastern North America. Management efforts to control overabundant temperate-nesting flocks have included the establishment of special early harvest seasons in September. However, the effect of early seasons on survival and harvest of subarctic-nesting populations has not been documented. Understanding the timing of migration movements and the fidelity of subarctic-nesting flocks to terminal winter refuges in the Southeast also is necessary to design early harvest seasons that target temperate-nesting flocks and protect subarctic-nesting populations. We used recoveries of marked geese to estimate survival and harvest rates before and after implementation of early harvest seasons within the Mississippi Flyway during 1976-1999. In addition, we used observations of neck-banded geese from the Southern James Bay Population (SJBP) to evaluate the hypothesis that subarctic-nesting geese arriving prior to mid-December on several key terminal winter refuges in the Southeast (early arriving migrants) were more likely to return to those refuges in subsequent years than were migrants, arriving after mid-December (late arriving migrants). September seasons during 1987-1994 were a minor source of mortality for subarctic-nesting populations and accounted for < 10% of their annual harvest mortality. The effectiveness of early seasons for increasing mortality of temperate-nesting flocks varied among the states we examined and was tempered by concurrent changes in state-specific harvest regulations during the regular harvest season. For SJBP Canada geese, annual fidelity to

  8. Do chimpanzee nests serve an anti-predatory function?

    PubMed

    Stewart, Fiona A; Pruetz, J D

    2013-06-01

    Sleep is a vulnerable state for animals as it compromises the ability to detect predators. The evolution of shelter construction in the great apes may have been a solution to the trade-off between restorative sleep and predation-risk, which allowed a large bodied ape to sleep recumbent in a safe, comfortable spot. In this article we review the evidence of predator pressure on great apes and specifically investigate the potential influence of predation-risk on chimpanzee nesting behavior by comparing nests between chimpanzees living in a habitat of several potential predators (Issa, Ugalla, Tanzania) and a habitat relatively devoid of predators (Fongoli, Senegal). Chimpanzees in Issa did not nest more frequently in forest vegetation than chimpanzees in Fongoli although forest vegetation is expected to provide greater opportunity for escape from terrestrial predators. Nor do chimpanzees in Issa nest in larger groups or aggregate together more than Fongoli chimpanzees, as would be expected if larger groups provide protection from or greater detection of predators. Nests in Issa also did not appear to provide greater opportunities for escape than nests in Fongoli. Chimpanzees in Issa nested more frequently within the same tree as other community members, which may indicate that these chimpanzees nest in greater proximity than chimpanzees in Fongoli. Finally, Issa chimpanzees built their nests proportionately higher and more peripherally within trees. The selection of high and peripheral nesting locations within trees may make Issa chimpanzees inaccessible to potential predators. Many factors influence nest site selection in chimpanzees, of which danger from terrestrial predators is likely to be one.

  9. Mourning Dove nesting habitat and nest success in Central Missouri

    USGS Publications Warehouse

    Drobney, R.D.; Schulz, J.H.; Sheriff, S.L.; Fuemmeler, W.J.

    1998-01-01

    Previous Mourning Dove (Zenaida macroura) nesting studies conducted in areas containing a mixture of edge and continuous habitats have focused on edge habitats. Consequently, little is known about the potential contribution of continuous habitats to dove production. In this study we evaluated the relative importance of these two extensive habitat types by monitoring the habitat use and nest success of 59 radio-marked doves during 1990-1991 in central Missouri. Of 83 nests initiated by our marked sample, most (81.9%) were located in edge habitats. Although continuous habitats were selected less as nest sites, the proportion of successful nests did not differ significantly from that in edge habitats. Our data indicate that continuous habitats should not be considered marginal nesting habitat. If the intensity of use and nest success that we observed are representative regionally or nationally, continuous habitats could contribute substantially to annual Mourning Dove production because of the high availability of these habitats throughout much of the Mourning Dove breeding range.

  10. Implementing nested conditional statements in SIMD machines

    NASA Technical Reports Server (NTRS)

    Middleton, David

    1989-01-01

    Single instruction, multiple data (SIMD) computers consist of a very large number of processors executing a common sequence of instructions. Maintaining the full speedup potential of such machines is most sensitive to conditional execution in their programs, regions of code where some processing elements (PEs) perform no useful work. Techniques are presented for efficiently implementing nested conditional statements, specifically if and case statements, in SIMD machines, while adding minimal specialized hardware.

  11. Primer design for PCR reactions in forensic biology.

    PubMed

    Elkins, Kelly M

    2015-01-01

    The polymerase chain reaction (PCR) is a popular method to copy DNA in vitro. Its invention revolutionized fields ranging from clinical medicine to anthropology, molecular biology, and forensic biology. The method employs one of many available heat-stable DNA polymerases in a reaction that is repeated many times in situ. The DNA polymerase reads a template DNA strand and using the components of the reaction mix, catalyzes the addition of free 2'-deoxynucleotide triphosphate nitrogenous bases to short segment of DNA that forms a complement with the template via Watson-Crick base pairing. This short segment of DNA is referred to as a PCR primer and it is essential to the success of the reaction. The most widely used application of PCR in forensic labs is the amplification of short tandem repeat (STR) loci used in DNA typing. The STRs are routinely evaluated in concert with 16 or more reactions, a multiplex, run in one test tube simultaneously. In a multiplex, it is essential that the primers work specifically and accurately on the intended reactions without hindering the other reactions. The primers, which are very specific, also can be used to probe single nucleotide polymorphisms (SNPs) in a DNA sequence of interest by single base extension. Primers are often designed using one of many available automated software packages. Here the process of manually designing PCR primers for forensic biology using no-cost software is described.

  12. Rapid, sensitive, and validated method for detection of Salmonella in food by an enrichment broth culture - nested PCR combination assay.

    PubMed

    Saroj, Sunil D; Shashidhar, R; Karani, Manisha; Bandekar, Jayant R

    2008-06-01

    A rapid nested PCR assay for detection of Salmonella from food was developed. The sensitivity of the assay developed was comparable to the traditional culture based methods with an advantage in reduction of assay time. The assay procedure with artificially contaminated samples was able to detect as low as 4CFU Salmonella/25g of food samples (sprout, carrot, cucumber and poultry meat). With two synthetic primers of 26 mer TS11 and 25 mer TS4, a 1.2kb fragment was amplified which served as a template for amplification of final 375bp product using TS11 and TS5 primers. No non-specific amplification from the native microbial flora of food samples was observed. The reaction generates a single band specific to Salmonella which allows the analyst to interpret data at ease and without any confusion. Enriched broth serves as template for the reaction which removes labour intensive DNA isolation procedures. In case of artificially contaminated samples, 6h enriched lactose broth can serve as template. However, for market samples where the organisms are under environmental stress, it is desirable to use template from Rappaport Vasiliadis medium. The assay also employes internal amplification control, which is amplified into a 300bp fragment and thus serves as positive control for the reaction and any possibility of false negative due to inhibitory action of food components on PCR reaction can be ruled out. PMID:18406104

  13. Single-tube nested PCR assay for the detection of avian botulism in cecal contents of chickens.

    PubMed

    Jang, Il; Lee, Jae-Il; Kwon, Yong-Kuk; Kang, Min-Su; Kim, Hye-Ryoung; Park, Ji-Young; Lee, Song-Hyun; Lee, Hee-Soo; Bae, You-Chan

    2015-10-01

    This paper describes a novel diagnostic method for the detection of avian botulism caused by Clostridium botulinum type C and C/D, using single-tube nested PCR assay. This assay was developed to overcome the disadvantages of bioassays used in experiments with mice. Three primer pairs including an antisense primer were designed to target the N-terminal of the toxin gene from C. botulinum types C and C/D. The specificity of the PCR assay was confirmed by using 33 bacterial strains and chicken cecal contents from farms that experienced botulism outbreaks. The detection limit for purified DNA was 1.1 fg/μl, and for bacterial spores was 4.3 spores/200 mg of cecal contents. While checking for specificity of the PCR assay, the reactions with the templates form C. botulinum type C and C/D which were tested became positive, but the rest of the reactions turned negative. However, the results for all clinical samples (n = 8) were positive. The PCR assay results for cecal samples obtained from 300 healthy chickens (150 Korean native chickens and 150 broilers) were all negative. This assay is rapid and straightforward and evades ethical issues associated with mouse bioassay. Moreover, it is more economical than real-time PCR.

  14. 30 CFR 56.6304 - Primer protection.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Primer protection. 56.6304 Section 56.6304 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL MINE... Primer protection. (a) Tamping shall not be done directly on a primer. (b) Rigid cartridges of...

  15. 30 CFR 56.6304 - Primer protection.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Primer protection. 56.6304 Section 56.6304 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL MINE... Primer protection. (a) Tamping shall not be done directly on a primer. (b) Rigid cartridges of...

  16. 30 CFR 56.6304 - Primer protection.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Primer protection. 56.6304 Section 56.6304 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL MINE... Primer protection. (a) Tamping shall not be done directly on a primer. (b) Rigid cartridges of...

  17. 30 CFR 75.1317 - Primer cartridges.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Primer cartridges. 75.1317 Section 75.1317... MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Explosives and Blasting § 75.1317 Primer cartridges. (a) Primer cartridges shall be primed and loaded only by a qualified person or a person working in...

  18. 30 CFR 75.1317 - Primer cartridges.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Primer cartridges. 75.1317 Section 75.1317... MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Explosives and Blasting § 75.1317 Primer cartridges. (a) Primer cartridges shall be primed and loaded only by a qualified person or a person working in...

  19. Using Primers to Motivate Your Students

    ERIC Educational Resources Information Center

    Graff, Dan

    2002-01-01

    Primers are used to motivate and uplift your class. They come in many different styles and can be used in a variety of ways. Making primers relevant to students helps them to learn and makes them feel appreciated and knowledgeable when they participate. Using primers in the classroom to make students feel valued brings much success.

  20. Landscape distribution of food and nesting sites affect larval diet and nest size, but not abundance of Osmia bicornis.

    PubMed

    Coudrain, Valérie; Rittiner, Sarah; Herzog, Felix; Tinner, Willy; Entling, Martin H

    2016-10-01

    Habitat fragmentation is a major threat for beneficial organisms and the ecosystem services they provide. Multiple-habitat users such as wild bees depend on both nesting and foraging habitat. Thus, they may be affected by the fragmentation of at least two habitat types. We investigated the effects of landscape-scale amount of and patch isolation from both nesting habitat (woody plants) and foraging habitat (specific pollen sources) on the abundance and diet of Osmia bicornis L. Trap-nests of O. bicornis were studied in 30 agricultural landscapes of the Swiss Plateau. Nesting and foraging habitats were mapped in a radius of 500 m around the sites. Pollen composition of larval diet changed as isolation to the main pollen source, Ranunculus, increased, suggesting that O. bicornis adapted its foraging strategy in function of the nest proximity to main pollen sources. Abundance of O. bicornis was neither related to isolation or amount of nesting habitat nor to isolation or abundance of food plants. Surprisingly, nests of O. bicornis contained fewer larvae in sites at forest edge compared to isolated sites, possibly due to higher parasitism risk. This study indicates that O. bicornis can nest in a variety of situations by compensating scarcity of its main larval food by exploiting alternative food sources.

  1. Landscape distribution of food and nesting sites affect larval diet and nest size, but not abundance of Osmia bicornis.

    PubMed

    Coudrain, Valérie; Rittiner, Sarah; Herzog, Felix; Tinner, Willy; Entling, Martin H

    2016-10-01

    Habitat fragmentation is a major threat for beneficial organisms and the ecosystem services they provide. Multiple-habitat users such as wild bees depend on both nesting and foraging habitat. Thus, they may be affected by the fragmentation of at least two habitat types. We investigated the effects of landscape-scale amount of and patch isolation from both nesting habitat (woody plants) and foraging habitat (specific pollen sources) on the abundance and diet of Osmia bicornis L. Trap-nests of O. bicornis were studied in 30 agricultural landscapes of the Swiss Plateau. Nesting and foraging habitats were mapped in a radius of 500 m around the sites. Pollen composition of larval diet changed as isolation to the main pollen source, Ranunculus, increased, suggesting that O. bicornis adapted its foraging strategy in function of the nest proximity to main pollen sources. Abundance of O. bicornis was neither related to isolation or amount of nesting habitat nor to isolation or abundance of food plants. Surprisingly, nests of O. bicornis contained fewer larvae in sites at forest edge compared to isolated sites, possibly due to higher parasitism risk. This study indicates that O. bicornis can nest in a variety of situations by compensating scarcity of its main larval food by exploiting alternative food sources. PMID:25973721

  2. Nest survival of forest birds in the Mississippi Alluvial Valley

    USGS Publications Warehouse

    Twedt, D.J.; Wilson, R.R.; Henne-Kerr, J.L.; Hamilton, R.B.

    2001-01-01

    vimns, 7%), eastern towhee (14%), indigo bunting (14%), and northern cardinal (17%) did not differ from nest success in cottonwood plantations that were coppiced from root sprouts following pulpwood harvest. Within bottomland hardwood forests, uneven-aged group-selection timber harvest reduced the combined daily nest survival of all species from 0.958 to 0.938, which reduced nest success by about 14%. Specifically, timber harvest reduced nest success of species that nest in the forest midstory and canopy, such as Acadian flycatcher (Empidonax virescens)--from 32% before harvest to 14% after harvest. Conversely, those species that nest primarily in the shrubby understory--such as northern cardinal--were not affected by timber harvest and maintained an overall nest success of about 33%. Thus, birds nesting in the understory of bottomland hardwood forests are not adversely impacted by selective timber harvest, but there is a short-term reduction in nest success for birds that nest in the canopy and midstory.

  3. Red-shouldered hawk nesting habitat preference in south Texas

    USGS Publications Warehouse

    Strobel, Bradley N.; Boal, Clint W.

    2010-01-01

    We examined nesting habitat preference by red-shouldered hawks Buteo lineatus using conditional logistic regression on characteristics measured at 27 occupied nest sites and 68 unused sites in 2005–2009 in south Texas. We measured vegetation characteristics of individual trees (nest trees and unused trees) and corresponding 0.04-ha plots. We evaluated the importance of tree and plot characteristics to nesting habitat selection by comparing a priori tree-specific and plot-specific models using Akaike's information criterion. Models with only plot variables carried 14% more weight than models with only center tree variables. The model-averaged odds ratios indicated red-shouldered hawks selected to nest in taller trees and in areas with higher average diameter at breast height than randomly available within the forest stand. Relative to randomly selected areas, each 1-m increase in nest tree height and 1-cm increase in the plot average diameter at breast height increased the probability of selection by 85% and 10%, respectively. Our results indicate that red-shouldered hawks select nesting habitat based on vegetation characteristics of individual trees as well as the 0.04-ha area surrounding the tree. Our results indicate forest management practices resulting in tall forest stands with large average diameter at breast height would benefit red-shouldered hawks in south Texas.

  4. Parallelized nested sampling

    NASA Astrophysics Data System (ADS)

    Henderson, R. Wesley; Goggans, Paul M.

    2014-12-01

    One of the important advantages of nested sampling as an MCMC technique is its ability to draw representative samples from multimodal distributions and distributions with other degeneracies. This coverage is accomplished by maintaining a number of so-called live samples within a likelihood constraint. In usual practice, at each step, only the sample with the least likelihood is discarded from this set of live samples and replaced. In [1], Skilling shows that for a given number of live samples, discarding only one sample yields the highest precision in estimation of the log-evidence. However, if we increase the number of live samples, more samples can be discarded at once while still maintaining the same precision. For computer code running only serially, this modification would considerably increase the wall clock time necessary to reach convergence. However, if we use a computer with parallel processing capabilities, and we write our code to take advantage of this parallelism to replace multiple samples concurrently, the performance penalty can be eliminated entirely and possibly reversed. In this case, we must use the more general equation in [1] for computing the expectation of the shrinkage distribution: E [- log t]= (N r-r+1)-1+(Nr-r+2)-1+⋯+Nr-1, for shrinkage t with Nr live samples and r samples discarded at each iteration. The equation for the variance Var (- log t)= (N r-r+1)-2+(Nr-r+2)-2+⋯+Nr-2 is used to find the appropriate number of live samples Nr to use with r > 1 to match the variance achieved with N1 live samples and r = 1. In this paper, we show that by replacing multiple discarded samples in parallel, we are able to achieve a more thorough sampling of the constrained prior distribution, reduce runtime, and increase precision.

  5. ecoPrimers: inference of new DNA barcode markers from whole genome sequence analysis

    PubMed Central

    Riaz, Tiayyba; Shehzad, Wasim; Viari, Alain; Pompanon, François; Taberlet, Pierre; Coissac, Eric

    2011-01-01

    Using non-conventional markers, DNA metabarcoding allows biodiversity assessment from complex substrates. In this article, we present ecoPrimers, a software for identifying new barcode markers and their associated PCR primers. ecoPrimers scans whole genomes to find such markers without a priori knowledge. ecoPrimers optimizes two quality indices measuring taxonomical range and discrimination to select the most efficient markers from a set of reference sequences, according to specific experimental constraints such as marker length or specifically targeted taxa. The key step of the algorithm is the identification of conserved regions among reference sequences for anchoring primers. We propose an efficient algorithm based on data mining, that allows the analysis of huge sets of sequences. We evaluate the efficiency of ecoPrimers by running it on three different sequence sets: mitochondrial, chloroplast and bacterial genomes. Identified barcode markers correspond either to barcode regions already in use for plants or animals, or to new potential barcodes. Results from empirical experiments carried out on a promising new barcode for analyzing vertebrate diversity fully agree with expectations based on bioinformatics analysis. These tests demonstrate the efficiency of ecoPrimers for inferring new barcodes fitting with diverse experimental contexts. ecoPrimers is available as an open source project at: http://www.grenoble.prabi.fr/trac/ecoPrimers. PMID:21930509

  6. Predator identity influences the effect of habitat management on nest predation.

    PubMed

    Lyons, Timothy P; Miller, James R; Debinski, Diane M; Engle, David M

    2015-09-01

    Predation is the leading cause of nest failure for many passerines and considerable effort is devoted to identifying the habitat characteristics and management practices that influence nest loss. The habitat components associated with nest loss are strongly influenced by the ecology of nest predators and differ among predator species as a result. Nevertheless, there is a tendency to generalize about the effects of habitat features and management on nest failure without considering how resulting patterns are influenced by nest predators. We examined how predator-specific patterns of nest loss differed among predators and in response to grassland management with fire and grazing by cattle (Bos taurus). We used video cameras to monitor and identify predators at nests of the Grasshopper Sparrow (Ammodramus savannarum), a species of conservation concern throughout its range. We observed predation by 15 different species that differed in their response to management and the habitat characteristics associated with nests they preyed on. Losses to mammals and snakes were more likely at nests with greater amounts of litter cover and tall fescue (Schedonorus phoenix). Mammals were less likely to prey on nests surrounded by greater forb cover. Nest predation by snakes was lower in burned areas, whereas predation by mammals and Brown-headed Cowbirds (Molothrus ater) was unaffected by the use of fire. Neither vegetation density at the nest, nor landscape context was related to nest loss by any predator taxon. Although there were many similarities, we identified important differences in the species composing the nest predator community between our. study and other published research. These differences are likely to be responsible for geographic variation in the influence of habitat features and management actions on nest success. Our results demonstrate the need for natural resource managers to incorporate knowledge of local nest predators and their ecology when developing

  7. Water based adhesive primers on aluminum substrates

    SciTech Connect

    Wightman, J.P.; Mori, S.

    1996-12-31

    The number of aluminum alloy bonding applications has been increasing recently in the automobile industry. Primer coating of aluminum substrates is one of the main processes used to promote bond performance. Solvent based organic primers have been used for a long time but environmental regulations now require the substitution of volatile organic compounds (VOC) by alternate materials such as water based adhesive primers. However, the bond strengths obtained with many water based primers are generally lower than for solvent based ones. Water based primers which have some reactive functional groups have been proposed recently but such primers require special treatment. This paper describes a study conducted to optimize bond strength using a water based adhesive as a primer in the adhesive bonding of anodized aluminum.

  8. Pre-nesting and nesting behavior of the Swainson's warbler

    USGS Publications Warehouse

    Meanley, B.

    1969-01-01

    The Swainson?s Warbler is one of the least known of southern birds. Although fairly common in some parts of its summer range, observations of its breeding biology have been made by very few persons. The present study was conducted mostly at Macon, Georgia; Pendleton Ferry, Arkansas; and Dismal Swamp, Virginia....In central Georgia and east-central Arkansas, Swainson?s Warblers usually arrive on their territories during the first two weeks in April. Territories in several localities ranged in size from 0.3 to 4.8 acres. A color-marked Arkansas male occupied the same territory for at least four months. Hostile encounters between territorial male Swainson?s Warblers usually take place along the boundary of adjacent territories. Paired males were more aggressive than unpaired males. Toward the end of an encounter one of the two males would usually perform a display in which the wing and tail feathers were spread and the tail vibrated. Following boundary encounters males drifted back onto their territories and usually sang unbroken courses of songs for several minutes.....During pre-nesting at Macon, a mated pair spent the day mostly on the ground within 20 feet of each other, often foragin g 3 to 4 feet apart. What may have been a form of courtship display, in which the male flew from a perch down to the female and either pecked her rump or pounced on her, occurred about three times each hour throughout the day. During this period the male sang less than at other times during the breeding season.....First nests are usually built by the first week in May. Although other investigators reported finding nests of this species outside of the defended territory, all nests that I have found were within the territory. The large, bulky nest of this species usually is placed 2-6 feet above the ground. It is built by the female from materials gathered close to the nest site; and takes two or three days to complete.....Three and occasionally four white eggs are laid. The female

  9. Detection of flaviviruses by reverse transcriptase-polymerase chain reaction with the universal primer set.

    PubMed

    Meiyu, F; Huosheng, C; Cuihua, C; Xiaodong, T; Lianhua, J; Yifei, P; Weijun, C; Huiyu, G

    1997-01-01

    Using a universal primer set designed to match the sequence of the NS1 gene of flaviviruses, the virus RNA of dengue (DEN), Japanese encephalitis (JEV), powassan and langat of Flaviviridae were successfully amplified by polymerase chain reaction (PCR) via cDNA; and with different internal primers, the serotypes of the dengue viruses were identified. Of the 78 clinically diagnosed dengue fever patients, 18 patients were positive for DEN 1, 48 patients for DEN 2 and 8 patients concurrently infected with DEN 4. Of the 52 patients admitted with Japanese encephalitis (JE), 45 were determined to be JEV infections. By nested PCR, we completed the identification of flaviviruses within 2 days. The results show that seven primers have a potential value for rapid clinical diagnosis of flavivirus infections.

  10. Sample Return Primer and Handbook

    NASA Technical Reports Server (NTRS)

    Barrow, Kirk; Cheuvront, Allan; Faris, Grant; Hirst, Edward; Mainland, Nora; McGee, Michael; Szalai, Christine; Vellinga, Joseph; Wahl, Thomas; Williams, Kenneth; Lee, Gentry; Duxbury, Thomas

    2007-01-01

    This three-part Sample Return Primer and Handbook provides a road map for conducting the terminal phase of a sample return mission. The main chapters describe element-by-element analyses and trade studies, as well as required operations plans, procedures, contingencies, interfaces, and corresponding documentation. Based on the experiences of the lead Stardust engineers, the topics include systems engineering (in particular range safety compliance), mission design and navigation, spacecraft hardware and entry, descent, and landing certification, flight and recovery operations, mission assurance and system safety, test and training, and the very important interactions with external support organizations (non-NASA tracking assets, landing site support, and science curation).

  11. Effect of primers on bonding agent polymerization.

    PubMed

    Hotta, M; Kondoh, K; Kamemizu, H

    1998-10-01

    The aim of the present study was to evaluate the effect of primers on the polymerization of bonding agent. We measured the degree of conversion (radical production) and mechanical properties (surface hardness and direct tensile strength) of various adhesives/primers mixed at different ratios and the effect of varying the visible-light curing time. With and without primer treatment, the tensile bond strength of adhesive resin to micacious glass ceramic and human enamel was measured. After the tensile bond test, using the Image Capture System, the failure patterns of adhesive resin bonded to micacious glass-ceramic were analysed. The results show that the mixtures containing the higher amounts of primer yielded a lower degree of conversion and inferior mechanical properties when compared with the mixtures containing a lower proportion of primer, except in the experimental bonding system. The adhesive/primer mixtures inhibited free radical polymerization. The value for the Knoop hardness number and the direct tensile strength of the adhesive/primer mixtures were significantly decreased compared with those of the adhesive bonding agent alone with no primer added. The tensile bond strength of adhesive resin bonded to micacious glass-ceramic or human enamel without primer treatment was significantly greater than that of adhesive resin with primer treatment in certain cases. Most of the fractures of ceramic surfaces were cohesive (within resins) and/or interface (at the ceramic surface) failure.

  12. A Tale of Tails: Dissecting the Enhancing Effect of Tailed Primers in Real-Time PCR

    PubMed Central

    Vandenbussche, Frank; Mathijs, Elisabeth; Lefebvre, David; De Clercq, Kris; Van Borm, Steven

    2016-01-01

    Non-specific tail sequences are often added to the 5’-terminus of primers to improve the robustness and overall performance of diagnostic assays. Despite the widespread use of tailed primers, the underlying working mechanism is not well understood. To address this problem, we conducted a detailed in vitro and in silico analysis of the enhancing effect of primer tailing on 2 well-established foot-and-mouth disease virus (FMDV) RT-qPCR assays using an FMDV reference panel. Tailing of the panFMDV-5UTR primers mainly affected the shape of the amplification curves. Modelling of the raw fluorescence data suggested a reduction of the amplification efficiency due to the accumulation of inhibitors. In depth analysis of PCR products indeed revealed the rapid accumulation of forward-primer derived artefacts. More importantly, tailing of the forward primer delayed artefacts formation and concomitantly restored the sigmoidal shape of the amplification curves. Our analysis also showed that primer tailing can alter utilisation patterns of degenerate primers and increase the number of primer variants that are able to participate in the reaction. The impact of tailed primers was less pronounced in the panFMDV-3D assay with only 5 out of 50 isolates showing a clear shift in Cq values. Sequence analysis of the target region of these 5 isolates revealed several mutations in the inter-primer region that extend an existing hairpin structure immediately downstream of the forward primer binding site. Stabilisation of the forward primer with either a tail sequence or cationic spermine units restored the sensitivity of the assay, which suggests that the enhancing effect in the panFMDV-3D assay is due to a more efficient extension of the forward primer. ur results show that primer tailing can alter amplification through various mechanisms that are determined by both the assay and target region. These findings expand our understanding of primer tailing and should enable a more targeted and

  13. UniPrimer: A Web-Based Primer Design Tool for Comparative Analyses of Primate Genomes

    PubMed Central

    Batnyam, Nomin; Lee, Jimin; Lee, Jungnam; Hong, Seung Bok; Oh, Sejong; Han, Kyudong

    2012-01-01

    Whole genome sequences of various primates have been released due to advanced DNA-sequencing technology. A combination of computational data mining and the polymerase chain reaction (PCR) assay to validate the data is an excellent method for conducting comparative genomics. Thus, designing primers for PCR is an essential procedure for a comparative analysis of primate genomes. Here, we developed and introduced UniPrimer for use in those studies. UniPrimer is a web-based tool that designs PCR- and DNA-sequencing primers. It compares the sequences from six different primates (human, chimpanzee, gorilla, orangutan, gibbon, and rhesus macaque) and designs primers on the conserved region across species. UniPrimer is linked to RepeatMasker, Primer3Plus, and OligoCalc softwares to produce primers with high accuracy and UCSC In-Silico PCR to confirm whether the designed primers work. To test the performance of UniPrimer, we designed primers on sample sequences using UniPrimer and manually designed primers for the same sequences. The comparison of the two processes showed that UniPrimer was more effective than manual work in terms of saving time and reducing errors. PMID:22693428

  14. Nest poaching in Neotropical parrots

    USGS Publications Warehouse

    Wright, T.F.; Toft, C.A.; Enkerlin-Hoeflich, E.; Gonzalez-Elizondo, J.; Albornoz, M.; Rodriguez-Ferraro, A.; Rojas-Suarez, F.; Sanz, V.; Trujillo, A.; Beissinger, S.R.; Berovides A., V.; Galvez A., X.; Brice, A.T.; Joyner, K.; Eberhard, J.; Gilardi, J.; Koenig, S.E.; Stoleson, S.; Martuscelli, P.; Meyers, J.M.; Renton, K.; Rodriguez, A.M.; Sosa-Asanza, A.C.; Vilella, F.J.; Wiley, J.W.

    2001-01-01

    Although the poaching of nestlings for the pet trade is thought to contribute to the decline of many species of parrots, its effects have been poorly demonstrated. We calculated rates of mortality due to nest poaching in 23 studies of Neotropical parrots, representing 4024 nesting attempts in 21 species and 14 countries. We also examined how poaching rates vary with geographic region, presence of active protection programs, conservation status and economic value of a species, and passage of the U.S. Wild Bird Conservation Act. The average poaching rate across all studies was 30% of all nests observed. Thirteen studies reported poaching rates of >20%, and four reported rates of >70%. Only six studies documented no nest poaching. Of these, four were conducted on islands in the Caribbean region, which had significantly lower poaching rates than the mainland Neotropics. The other two studies that showed no poaching were conducted on the two species with the lowest economic value in our sample (U.S. retail price). In four studies that allowed direct comparison between poaching at sites with active nest protection versus that at unprotected sites, poaching rates were significantly lower at protected sites, suggesting that active protection efforts can be effective in reducing nest poaching. In those studies conducted both before and after the passage of the U.S. Wild Bird Conservation Act, poaching rates were found to be significantly lower following its enactment than in the period before. This result supports the hypothesis that the legal and illegal parrot trades are positively related, rather than inversely related as has been suggested by avicultural interests. Overall, our study indicates that poaching of parrot nestlings for economic gain is a widespread and biologically significant source of nest mortality in Neotropical parrots.

  15. A comprehensive collection of experimentally validated primers for Polymerase Chain Reaction quantitation of murine transcript abundance

    PubMed Central

    Spandidos, Athanasia; Wang, Xiaowei; Wang, Huajun; Dragnev, Stefan; Thurber, Tara; Seed, Brian

    2008-01-01

    Background Quantitative polymerase chain reaction (QPCR) is a widely applied analytical method for the accurate determination of transcript abundance. Primers for QPCR have been designed on a genomic scale but non-specific amplification of non-target genes has frequently been a problem. Although several online databases have been created for the storage and retrieval of experimentally validated primers, only a few thousand primer pairs are currently present in existing databases and the primers are not designed for use under a common PCR thermal profile. Results We previously reported the implementation of an algorithm to predict PCR primers for most known human and mouse genes. We now report the use of that resource to identify 17483 pairs of primers that have been experimentally verified to amplify unique sequences corresponding to distinct murine transcripts. The primer pairs have been validated by gel electrophoresis, DNA sequence analysis and thermal denaturation profile. In addition to the validation studies, we have determined the uniformity of amplification using the primers and the technical reproducibility of the QPCR reaction using the popular and inexpensive SYBR Green I detection method. Conclusion We have identified an experimentally validated collection of murine primer pairs for PCR and QPCR which can be used under a common PCR thermal profile, allowing the evaluation of transcript abundance of a large number of genes in parallel. This feature is increasingly attractive for confirming and/or making more precise data trends observed from experiments performed with DNA microarrays. PMID:19108745

  16. Express Primer Tool for high-throughput gene cloning and expression

    2002-12-01

    A tool to assist in the design of primers for DNA amplification. The Express Primer web-based tool generates primer sequences specifically for the generation of expression clones for both lab scale and high-throughput projects. The application is designed not only to allow the user complete flexibility to specify primer design parameters but also to minimize the amount of manual intervention needed to generate a large number of primers for simultaneous amplification of multiple target genes.more » The Express Primer Tool enables the user to specify various experimental parameters (e.g. optimal Tm, Tm range, maximum Tm difference) for single or multiple candidate sequence(s) in FASTA format input as a flat text (ASCII) file. The application generates condidate primers, selects optimal primer pairs, and writes the forward and reverse primers pairs to an Excel file that is suitable for electronic submission to a synthesis facility. The program parameters emphasize high-throughput but allow for target atrition at various stages of the project.« less

  17. Localizing Tortoise Nests by Neural Networks.

    PubMed

    Barbuti, Roberto; Chessa, Stefano; Micheli, Alessio; Pucci, Rita

    2016-01-01

    The goal of this research is to recognize the nest digging activity of tortoises using a device mounted atop the tortoise carapace. The device classifies tortoise movements in order to discriminate between nest digging, and non-digging activity (specifically walking and eating). Accelerometer data was collected from devices attached to the carapace of a number of tortoises during their two-month nesting period. Our system uses an accelerometer and an activity recognition system (ARS) which is modularly structured using an artificial neural network and an output filter. For the purpose of experiment and comparison, and with the aim of minimizing the computational cost, the artificial neural network has been modelled according to three different architectures based on the input delay neural network (IDNN). We show that the ARS can achieve very high accuracy on segments of data sequences, with an extremely small neural network that can be embedded in programmable low power devices. Given that digging is typically a long activity (up to two hours), the application of ARS on data segments can be repeated over time to set up a reliable and efficient system, called Tortoise@, for digging activity recognition.

  18. Localizing Tortoise Nests by Neural Networks.

    PubMed

    Barbuti, Roberto; Chessa, Stefano; Micheli, Alessio; Pucci, Rita

    2016-01-01

    The goal of this research is to recognize the nest digging activity of tortoises using a device mounted atop the tortoise carapace. The device classifies tortoise movements in order to discriminate between nest digging, and non-digging activity (specifically walking and eating). Accelerometer data was collected from devices attached to the carapace of a number of tortoises during their two-month nesting period. Our system uses an accelerometer and an activity recognition system (ARS) which is modularly structured using an artificial neural network and an output filter. For the purpose of experiment and comparison, and with the aim of minimizing the computational cost, the artificial neural network has been modelled according to three different architectures based on the input delay neural network (IDNN). We show that the ARS can achieve very high accuracy on segments of data sequences, with an extremely small neural network that can be embedded in programmable low power devices. Given that digging is typically a long activity (up to two hours), the application of ARS on data segments can be repeated over time to set up a reliable and efficient system, called Tortoise@, for digging activity recognition. PMID:26985660

  19. Localizing Tortoise Nests by Neural Networks

    PubMed Central

    2016-01-01

    The goal of this research is to recognize the nest digging activity of tortoises using a device mounted atop the tortoise carapace. The device classifies tortoise movements in order to discriminate between nest digging, and non-digging activity (specifically walking and eating). Accelerometer data was collected from devices attached to the carapace of a number of tortoises during their two-month nesting period. Our system uses an accelerometer and an activity recognition system (ARS) which is modularly structured using an artificial neural network and an output filter. For the purpose of experiment and comparison, and with the aim of minimizing the computational cost, the artificial neural network has been modelled according to three different architectures based on the input delay neural network (IDNN). We show that the ARS can achieve very high accuracy on segments of data sequences, with an extremely small neural network that can be embedded in programmable low power devices. Given that digging is typically a long activity (up to two hours), the application of ARS on data segments can be repeated over time to set up a reliable and efficient system, called Tortoise@, for digging activity recognition. PMID:26985660

  20. KENO-VI Primer: A Primer for Criticality Calculations with SCALE/KENO-VI Using GeeWiz

    SciTech Connect

    Bowman, Stephen M

    2008-09-01

    The SCALE (Standardized Computer Analyses for Licensing Evaluation) computer software system developed at Oak Ridge National Laboratory is widely used and accepted around the world for criticality safety analyses. The well-known KENO-VI three-dimensional Monte Carlo criticality computer code is one of the primary criticality safety analysis tools in SCALE. The KENO-VI primer is designed to help a new user understand and use the SCALE/KENO-VI Monte Carlo code for nuclear criticality safety analyses. It assumes that the user has a college education in a technical field. There is no assumption of familiarity with Monte Carlo codes in general or with SCALE/KENO-VI in particular. The primer is designed to teach by example, with each example illustrating two or three features of SCALE/KENO-VI that are useful in criticality analyses. The primer is based on SCALE 6, which includes the Graphically Enhanced Editing Wizard (GeeWiz) Windows user interface. Each example uses GeeWiz to provide the framework for preparing input data and viewing output results. Starting with a Quickstart section, the primer gives an overview of the basic requirements for SCALE/KENO-VI input and allows the user to quickly run a simple criticality problem with SCALE/KENO-VI. The sections that follow Quickstart include a list of basic objectives at the beginning that identifies the goal of the section and the individual SCALE/KENO-VI features that are covered in detail in the sample problems in that section. Upon completion of the primer, a new user should be comfortable using GeeWiz to set up criticality problems in SCALE/KENO-VI. The primer provides a starting point for the criticality safety analyst who uses SCALE/KENO-VI. Complete descriptions are provided in the SCALE/KENO-VI manual. Although the primer is self-contained, it is intended as a companion volume to the SCALE/KENO-VI documentation. (The SCALE manual is provided on the SCALE installation DVD.) The primer provides specific examples of

  1. Nest use is influenced by the positions of nests and drinkers in aviaries.

    PubMed

    Lentfer, T L; Gebhardt-Henrich, S G; Fröhlich, E K F; von Borell, E

    2013-06-01

    The influence of the nest location and the placement of nipple drinkers on nest use by laying hens in a commercial aviary was assessed. Twenty pens in a laying hen house were equipped with the same commercial aviary system, but the pens differed in the nest location and the placement of nipple drinkers. Nests were placed along the walls in 10 pens, and nipple drinkers were installed in front of the nests in 5 of these pens. The other 10 pens were equipped with nests placed on a tier within the aviary (integrated nests). Nipple drinkers were installed in front of the nests in 5 of these pens. A total of 225 Lohmann Selected Leghorns were housed per pen. The hens were offered 4 nests per pen: 2 facing the service corridor of the laying hen house and 2 facing the outdoor area. The numbers of nest eggs and mislaid eggs were counted daily per pen. At 25, 36, and 43 wk of age, the nest platforms were videotaped and the behavior of laying hens in front of the nests was analyzed. The nest location affected the stationary and locomotive behaviors in front of the nests. Hens in front of the integrated nests and the nests with drinkers displayed more stationary behaviors than hens in front of wall-placed nests or nests without drinkers. No difference in the number of nest eggs could be detected, but the integration of the nests inside the aviary led to a more even distribution of hens while nest searching. In the pens with wall-placed nests, significantly more hens laid eggs in the nests at the wall near the service corridor than at the wall near the outdoor area. Due to this imbalance, crowding in front of the preferred nests occurred and pushing and agonistic interactions on the nest platforms were significantly more frequent. Placement of nipple drinkers in front of nests had no effect on the number of eggs laid in those nests.

  2. Successful nesting behavior of Puerto Rican parrots

    USGS Publications Warehouse

    Wilson, K.A.; Field, R.; Wilson, M.H.

    1995-01-01

    We analyzed nesting behavior of five pairs of the endangered Puerto Rican Parrot (Amazona vittata) during eight successful nesting attempts. Each stage of the nesting cycle (egg laying, incubation, early chick rearing, and late chick rearing) was characterized by distinct trends or levels of behavior. During egg laying, female attentiveness to tile nest increased, and male attentiveness decreased. Throughout incubation and the first several days of early chick rearing, females were highly attentive to their nests, whereas males rarely entered the nest cavities. Female attentiveness then began to decline. Male attentiveness to the nest was sporadic until chicks were 10-12 days old. when all males began to enter their nests at least once each day. During late chick rearing, both male and female attentiveness were erratic and highly variable. Biologists may be able to use these results to identify nest problems and the need for management intervention when patterns of nest attentiveness deviate from the limits described in this study..

  3. Nucleic acid amplification using modular branched primers

    SciTech Connect

    Ulanovsky, Levy; Raja, Mugasimangalam C.

    2001-01-01

    Methods and compositions expand the options for making primers for use in amplifying nucleic acid segments. The invention eliminates the step of custom synthesis of primers for Polymerase Chain Reactions (PCR). Instead of being custom-synthesized, a primer is replaced by a combination of several oligonucleotide modules selected from a pre-synthesized library. A modular combination of just a few oligonucleotides essentially mimics the performance of a conventional, custom-made primer by matching the sequence of the priming site in the template. Each oligonucleotide module has a segment that matches one of the stretches within the priming site.

  4. Great Lakes rivermouths: a primer for managers

    USGS Publications Warehouse

    Pebbles, Victoria; Larson, James; Seelbach, Paul; Pebbles, Victoria; Larson, James; Seelbach, Paul

    2013-01-01

    Between the North American Great Lakes and their tributaries are the places where the confluence of river and lake waters creates a distinct ecosystem: the rivermouth ecosystem. Human development has often centered around these rivermouths, in part, because they provide a rich array of ecosystem services. Not surprisingly, centuries of intense human activity have led to substantial pressures on, and alterations to, these ecosystems, often diminishing or degrading their ecological functions and associated ecological services. Many Great Lakes rivermouths are the focus of intense restoration efforts. For example, 36 of the active Great Lakes Areas of Concern (AOCs) are rivermouths or areas that include one or more rivermouths. Historically, research of rivermouth ecosystems has been piecemeal, focused on the Great Lakes proper or on the upper reaches of tributaries, with little direct study of the rivermouth itself. Researchers have been divided among disciplines, agencies and institutions; and they often work independently and use disparate venues to communicate their work. Management has also been fragmented with a focus on smaller, localized, sub-habitat units and socio-political or economic elements, rather than system-level consideration. This Primer presents the case for a more holistic approach to rivermouth science and management that can enable restoration of ecosystem services with multiple benefits to humans and the Great Lakes ecosystem. A conceptual model is presented with supporting text that describes the structures and processes common to all rivermouths, substantiating the case for treating these ecosystems as an identifiable class.1 Ecological services provided by rivermouths and changes in how humans value those services over time are illustrated through case studies of two Great Lakes rivermouths—the St. Louis River and the Maumee River. Specific ecosystem services are identified in italics throughout this Primer and follow definitions described

  5. A new PCR method: one primer amplification of PCR-CTPP products.

    PubMed

    Yin, Guang; Mitsuda, Yoko; Ezaki, Takayuki; Hamajima, Nobuyuki

    2012-10-01

    Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) is a convenient method for genotyping single nucleotide polymorphisms, saving time, and costs. It uses four primers for PCR; F1 and R1 for one allele, and F2 and R2 for the other allele, by which three different sizes of DNA are amplified; between F1 and R1, between F2 and R2, and between F1 and R2. To date, we have applied PCR-CTPP successfully for genotyping more than 60 polymorphisms. However, it is not rare that PCR does not produce balanced amplification of allele specific bands. Accordingly, the method was modified by attaching a common sequence at the 5' end of two-pair primers and adding another primer with the common sequence in PCR, in total five different primers in a tube for PCR. The modification allowed one primer amplification for the products of initial PCR with confronting two-pair primers, named as one primer amplification of PCR-CTPP products (OPA-CTPP). This article demonstrates an example for an A/G polymorphism of paraoxonase 1 (PON1) Gln192Arg (rs662). PCR-CTPP failed clear genotyping for the polymorphism, while OPA-CTPP successfully produced PCR products corresponding to the allele. The present example indicated that the OPA-CTPP would be useful in the case that PCR-CTPP failed to produce balanced PCR products specific to each allele.

  6. A Comprehensive Evaluation of PCR Primers to Amplify the nifH Gene of Nitrogenase

    PubMed Central

    Gaby, John Christian; Buckley, Daniel H.

    2012-01-01

    The nifH gene is the most widely sequenced marker gene used to identify nitrogen-fixing Bacteria and Archaea. Numerous PCR primers have been designed to amplify nifH, but a comprehensive evaluation of nifH PCR primers has not been performed. We performed an in silico analysis of the specificity and coverage of 51 universal and 35 group-specific nifH primers by using an aligned database of 23,847 nifH sequences. We found that there are 15 universal nifH primers that target 90% or more of nitrogen fixers, but that there are also 23 nifH primers that target less than 50% of nifH sequences. The nifH primers we evaluated vary in their phylogenetic bias and their ability to recover sequences from commonly sampled environments. In addition, many of these primers will amplify genes that do not mediate nitrogen fixation, and thus it would be advisable for researchers to screen their sequencing results for the presence of non-target genes before analysis. Universal primers that performed well in silico were tested empirically with soil samples and with genomic DNA from a phylogenetically diverse set of nitrogen-fixing strains. This analysis will be of great utility to those engaged in molecular analysis of nifH genes from isolates and environmental samples. PMID:22848735

  7. Hydrocarbons on harvester ant (Pogonomyrmex barbatus) middens guide foragers to the nest.

    PubMed

    Sturgis, Shelby J; Greene, Michael J; Gordon, Deborah M

    2011-05-01

    Colony-specific cuticular hydrocarbons are used by social insects in nestmate recognition. Here, we showed that hydrocarbons found on the mound of Pogonomyrmex barbatus nests facilitate the return of foragers to the nest. Colony-specific hydrocarbons, which ants use to distinguish nestmates from non-nestmates, are found on the midden pebbles placed on the nest mound. Midden hydrocarbons occur in a concentration gradient, growing stronger near the nest entrance, which is in the center of a 1-2 m diameter nest mound. Foraging behavior was disrupted when the gradient of hydrocarbons was altered experimentally. When midden material was diluted with artificial pebbles lacking the colony-specific hydrocarbons, the speed of returning foragers decreased significantly. The chemical environment of the nest mound contributes to the regulation of foraging behavior in harvester ants.

  8. Linear elastic fracture mechanics primer

    NASA Technical Reports Server (NTRS)

    Wilson, Christopher D.

    1992-01-01

    This primer is intended to remove the blackbox perception of fracture mechanics computer software by structural engineers. The fundamental concepts of linear elastic fracture mechanics are presented with emphasis on the practical application of fracture mechanics to real problems. Numerous rules of thumb are provided. Recommended texts for additional reading, and a discussion of the significance of fracture mechanics in structural design are given. Griffith's criterion for crack extension, Irwin's elastic stress field near the crack tip, and the influence of small-scale plasticity are discussed. Common stress intensities factor solutions and methods for determining them are included. Fracture toughness and subcritical crack growth are discussed. The application of fracture mechanics to damage tolerance and fracture control is discussed. Several example problems and a practice set of problems are given.

  9. A Practical Primer on Geostatistics

    USGS Publications Warehouse

    Olea, Ricardo A.

    2009-01-01

    significant methodological implications. HISTORICAL REMARKS As a discipline, geostatistics was firmly established in the 1960s by the French engineer Georges Matheron, who was interested in the appraisal of ore reserves in mining. Geostatistics did not develop overnight. Like other disciplines, it has built on previous results, many of which were formulated with different objectives in various fields. PIONEERS Seminal ideas conceptually related to what today we call geostatistics or spatial statistics are found in the work of several pioneers, including: 1940s: A.N. Kolmogorov in turbulent flow and N. Wiener in stochastic processing; 1950s: D. Krige in mining; 1960s: B. Mathern in forestry and L.S. Gandin in meteorology CALCULATIONS Serious applications of geostatistics require the use of digital computers. Although for most geostatistical techniques rudimentary implementation from scratch is fairly straightforward, coding programs from scratch is recommended only as part of a practice that may help users to gain a better grasp of the formulations. SOFTWARE For professional work, the reader should employ software packages that have been thoroughly tested to handle any sampling scheme, that run as efficiently as possible, and that offer graphic capabilities for the analysis and display of results. This primer employs primarily the package Stanford Geomodeling Software (SGeMS) - recently developed at the Energy Resources Engineering Department at Stanford University - as a way to show how to obtain results practically. This applied side of the primer should not be interpreted as the notes being a manual for the use of SGeMS. The main objective of the primer is to help the reader gain an understanding of the fundamental concepts and tools in geostatistics. ORGANIZATION OF THE PRIMER The chapters of greatest importance are those covering kriging and simulation. All other materials are peripheral and are included for better comprehension of th

  10. Nest- and colony-mate recognition in polydomous colonies of meat ants ( Iridomyrmex purpureus)

    NASA Astrophysics Data System (ADS)

    van Wilgenburg, E.; Ryan, D.; Morrison, P.; Marriott, P. J.; Elgar, M. A.

    2006-07-01

    Workers of polydomous colonies of social insects must recognize not only colony-mates residing in the same nest but also those living in other nests. We investigated the impact of a decentralized colony structure on colony- and nestmate recognition in the polydomous Australian meat ant ( Iridomyrmex purpureus). Field experiments showed that ants of colonies with many nests were less aggressive toward alien conspecifics than those of colonies with few nests. In addition, while meat ants were almost never aggressive toward nestmates, they were frequently aggressive when confronted with an individual from a different nest within the same colony. Our chemical analysis of the cuticular hydrocarbons of workers using a novel comprehensive two-dimensional gas chromatography technique that increases the number of quantifiable compounds revealed both colony- and nest-specific patterns. Combined, these data indicate an incomplete transfer of colony odor between the nests of polydomous meat ant colonies.

  11. Nest- and colony-mate recognition in polydomous colonies of meat ants (Iridomyrmex purpureus).

    PubMed

    van Wilgenburg, E; Ryan, D; Morrison, P; Marriott, P J; Elgar, M A

    2006-07-01

    Workers of polydomous colonies of social insects must recognize not only colony-mates residing in the same nest but also those living in other nests. We investigated the impact of a decentralized colony structure on colony- and nestmate recognition in the polydomous Australian meat ant (Iridomyrmex purpureus). Field experiments showed that ants of colonies with many nests were less aggressive toward alien conspecifics than those of colonies with few nests. In addition, while meat ants were almost never aggressive toward nestmates, they were frequently aggressive when confronted with an individual from a different nest within the same colony. Our chemical analysis of the cuticular hydrocarbons of workers using a novel comprehensive two-dimensional gas chromatography technique that increases the number of quantifiable compounds revealed both colony- and nest-specific patterns. Combined, these data indicate an incomplete transfer of colony odor between the nests of polydomous meat ant colonies.

  12. Changes in Rous Sarcoma Virus RNA Secondary Structure near the Primer Binding Site upon tRNATrp Primer Annealing

    PubMed Central

    Morris, Shannon; Leis, Jonathan

    1999-01-01

    Predicted secondary-structure elements encompassing the primer binding site in the 5′ untranslated region of Rous sarcoma virus (RSV) RNA play an integral role in multiple viral replications steps including reverse transcription, DNA integration, and RNA packaging (A. Aiyar, D. Cobrinik, Z. Ge, H. J. Kung, and J. Leis, J. Virol. 66:2464–2472, 1992; D. Cobrinik, A. Aiyar, Z. Ge, M. Katzman, H. Huang, and J. Leis, J. Virol. 65:3864–3872, 1991; J. T. Miller, Z. Ge, S. Morris, K. Das, and J. Leis, J. Virol. 71:7648–7656, 1997). These elements include the U5-Leader stem, U5-IR stem-loop, and U5-TΨC interaction region. Limited digestion of the 5′ untranslated region of wild-type and mutant RSV RNAs with structure- and/or sequence-specific RNases detects the presence of the U5-Leader stem and the U5-IR stem-loop. When a tRNATrp primer is annealed to wild-type RNAs in vitro, limited nuclease mapping indicates that the U5-IR stem becomes partially unwound. This is not observed when mutant RNAs with altered U5-IR stem-loop structures are substituted for wild-type RNAs. The U5-Leader stem also becomes destabilized when the tRNA primer is annealed to either wild-type or mutant RNA fragments. Nuclease mapping studies of tRNATrp, as well as the viral RNA, indicate that the U5-TΨC helix does form in vitro upon primer annealing. Collectively, these data suggest that the various structural elements near the RSV primer binding site undergo significant changes during the process of primer annealing. PMID:10400722

  13. Selection of more appropriate PCR primer pairs for improved efficiency in detecting Norwalk-like virus (NLV) RNA.

    PubMed

    Naitou, Hirotaka; Morita, Tamotsu

    2001-10-01

    A pair of primers, NV35 and NV36, and another pair of primers, NV81 and NV82/SM82, are commonly used for polymerase chain reaction (PCR) detection of Norwalk-like virus (NLV) genome RNA sequences in authorized test laboratories in Japan. However, the efficiency of NLV genome RNA detection with these primer pairs has been less than satisfactory. In the present study, we attempted to establish more appropriately matched primer pairs for improved detection of NLV genome RNA sequences using a combination of primers including NV35, NV36, NV81, NV82/SM82, SR33, and SRs (a mixture of 4 primers SR46, SR48, SR50, and SR52). We also evaluated appropriate primers for improved reverse transcription of NLV genome RNA. Stool samples used for detection of NLV included 18 samples collected from NLV-infected patients who ingested oysters (group 1) and 13 samples collected from those who did not ingest oysters (group 2). Reverse transcription of RNA genome with primer NV35 was less efficient compared with that with primer SR33 or NV81. When PCR products obtained with NV35 and NV36 as a pair of primers were subjected to gel electrophoresis, a strong extra band was detected compared with those obtained with other primer pairs. Since this extra band may represent heterodimeric or homodimeric hybrids, or intramolecular hybrids derived from these primers, this template-independent hybridization could lower the efficiency of primer-dependent polymerase reaction. Of 18 primer pairs, a pair of NV81 and SRs provided the best detection of PCR products following reverse transcription of NLV RNA with SR33 or NV81. The detection rate was 61% for both reverse transcription with SR33 and that with NV81. After reverse transcription using SR33 as a primer, nested PCR using a pair of NV81 and SRs following primary PCR using a pair of NV81 and NV82/SM82 increased the detection rate to 89% in group 1 and 100% in group 2.

  14. Power lines, roads, and avian nest survival: effects on predator identity and predation intensity

    PubMed Central

    DeGregorio, Brett A; Weatherhead, Patrick J; Sperry, Jinelle H

    2014-01-01

    1 Anthropogenic alteration of landscapes can affect avian nest success by influencing the abundance, distribution, and behavior of predators. Understanding avian nest predation risk necessitates understanding how landscapes affect predator distribution and behavior. 2 From a sample of 463 nests of 17 songbird species, we evaluated how landscape features (distance to forest edge, unpaved roads, and power lines) influenced daily nest survival. We also used video cameras to identify nest predators at 137 nest predation events and evaluated how landscape features influenced predator identity. Finally, we determined the abundance and distribution of several of the principal predators using surveys and radiotelemetry. 3 Distance to power lines was the best predictor of predator identity: predation by brown-headed cowbirds (Molothrus ater), corvids (Corvus sp. and Cyanocitta cristata), racers (Coluber constrictor), and coachwhips (Masticophis flagellum) increased with proximity to power lines, whereas predation by rat snakes (Elaphe obsoleta) and raptors decreased. In some cases, predator density may reliably indicate nest predation risk because racers, corvids, and cowbirds frequently used power line right-of-ways. 4 Of five bird species with enough nests to analyze individually, daily nest survival of only indigo buntings (Passerina cyanea) decreased with proximity to power lines, despite predation by most predators at our site being positively associated with power lines. For all nesting species combined, distance to unpaved road was the model that most influenced daily nest survival. This pattern is likely a consequence of rat snakes, the locally dominant nest predator (28% of predation events), rarely using power lines and associated areas. Instead, rat snakes were frequently associated with road edges, indicating that not all edges are functionally similar. 5 Our results suggest that interactions between predators and landscape features are likely to be specific to

  15. Host selection in the forest interior: cowbirds target ground-nesting species

    USGS Publications Warehouse

    Hahn, D.C.; Hatfield, J.S.

    2000-01-01

    We investigated patterns of cowbird host selection in a large (1300 ha), unfragmented forest in eastern New York in 1992-3 to determine whether cowbird parasitism rates can be attributed to species-specific traits or to other features associated with nest sites. Nest height was significantly associated with parasitism (P = 0.003) in this community of 23 species (n = 430 nests, 23% parasitized). Further analysis revealed that the difference in mean nest heights between parasitized and unparasitized nests was due to species identity, and within each species there was no difference in mean nest heights between parasitized and unparasitized nests. These results imply that during 1992-3 cowbirds in this forest specialized on species that have low nests and did not necessarily select low nests regardless of species. This was further supported by a negative association across all 23 species between mean nest height and parasitism rate (P = 0.03). Thus, although most of the forest-nesting species in this community experienced cowbird parasitism, there was a tendency for higher parasitism rates on low-nesting species such as the Ovenbird, Black-and-white Warbler, Louisiana Waterthrush, Veery, and Hermit Thrush. The Wood Thrush, a mid-range nester which is heavily parasitized in southern Illinois, experienced only 10% parasitism in our site and ranked 9th in parasitism rate, although it was the most abundant species in this forest in terms of the number of nests found. A long-term study is necessary to determine whether this cowbird population consistently parasitizes the ground-nesting species of this forest community more often than those nesting at higher levels or whether they periodically shift among hosts at different heights and in different habitats across the local landscape.

  16. In-planta detection and monitorization of endophytic colonization by a Beauveria bassiana strain using a new-developed nested and quantitative PCR-based assay and confocal laser scanning microscopy.

    PubMed

    Landa, B B; López-Díaz, C; Jiménez-Fernández, D; Montes-Borrego, M; Muñoz-Ledesma, F J; Ortiz-Urquiza, A; Quesada-Moraga, E

    2013-10-01

    Beauveria bassiana strain 04/01-Tip obtained from larvae of the opium poppy stem gall Iraella luteipes endophytically colonizes opium poppy plants and protect it against this pest. Development of a specific, rapid and sensitive technique that allows accurately determining the process and factors leading to the establishment of this strain in opium poppy plants would be essential to achieve its efficient control in a large field scale. For that purpose in the present study, species-specific primers that can be used in conventional or quantitative PCR protocols were developed for specifically identification and detection of B. bassiana in plant tissues. The combination of the designed BB.fw/BB.rv primer set with the universal ITS1-F/ITS4 primer set in a two-step nested-PCR approach, has allowed the amplification of up to 10fg of B. bassiana. This represented an increase in sensitivity of 10000- and 1000-fold of detection than when using the BB.fw/BB.rv primers in a single or single-tube semi-nested PCR approaches, respectively. The BB.fw and BB.rv primer set were subsequently optimized to be used in real time quantitative PCR assays and allowed to accurately quantify B. bassiana DNA in different plant DNA backgrounds (leaves and seeds) without losing accuracy and efficiency. The qPCR protocol was used to monitor the endophytic colonization of opium poppy leaves byB. bassiana after inoculation with the strain EABb 04/01-Tip, detecting as low as 26fg of target DNA in leaves and a decrease in fungal biomass over time. PCR quantification data were supported in parallel with CLMS by the monitoring of spatial and temporal patterns of leaf and stem colonization using a GFP-tagged transformant of the B. bassiana EABb 04/01-Tip strain, which enabled to demonstrate that B. bassiana effectively colonizes aerial tissues of opium poppy plants mainly through intercellular spaces and even leaf trichomes. A decline in endophytic colonization was also observed by the last sampling

  17. A cautionary note on the use of nested PCR for parasite screening--an example from avian blood parasites.

    PubMed

    Szöllsi, Eszter; Hellgren, Olof; Hasselquist, Dennis

    2008-04-01

    The use of new powerful nested polymerase chain reaction (PCR) techniques to identify and screen for prevalence of parasites has a huge potential. It allows for the detection and identification of low-intensity infections, but its high sensitivity and technical setup may also induce problems. Here, we report a cautionary note regarding misleading amplification of avian malaria species (Haemoproteus and Plasmodium) during Leucocytozoon spp. detection. We used a previously described nested PCR method for the molecular detection of avian malaria and Leucocytozoon spp. In the first step of the PCR protocol, these parasites are detected simultaneously; in the second PCR, Haemoproteus and Plasmodium spp. are separated from Leucocytozoon spp. However, in certain cases when a bird was infected with avian malaria, we obtained a slightly longer PCR product during the detection of Leucocytozoon spp. Our data imply that these "false" Leucocytozoon fragments are the consequences of strong amplification of certain malaria lineages in the first PCR, which can also be detected after the second PCR amplification that is specific to Leucocytozoon spp. parasites. Because these "false" Leucocytozoon fragments are slightly longer than the normal Leucocytozoon fragments, we suggest the use of well-separating agarose gels, several positive controls, and molecular standards to facilitate their separation. If one obtains a fragment that differs in length from the one expected for Leucocytozoon spp., sequencing is essential. More generally, in order to limit this type of problem with nested PCR protocols, we suggest that the first and the second primer pair be chosen so that they have different annealing temperatures.

  18. Unusual raptor nests around the world

    USGS Publications Warehouse

    Ellis, D.H.; Craig, T.; Craig, E.; Postupalsky, S.; LaRue, C.T.; Nelson, R.W.; Anderson, D.W.; Henny, C.J.; Watson, J.; Millsap, B.A.; Dawson, J.W.; Cole, K.L.; Martin, E.M.; Margalida, A.; Kung, P.

    2009-01-01

    From surveys in many countries, we report raptors using unusual nesting materials (e.g., paper money, rags, metal, antlers, and large bones) and unusual nesting situations. For example, we documented nests of Steppe Eagles Aquila nipalensis and Upland Buzzards Buteo hemilasius on the ground beside well-traveled roads, Saker Falcon Falco cherrug eyries in attics and a cistern, and Osprey Pandion haliaetus nests on the masts of boats and on a suspended automobile. Other records include a Golden Eagle A. chrysaetos nest 7.0 m in height, believed to be the tallest nest ever described, and, for the same species, we report nesting in rudimentary nests. Some nest sites are within a few meters of known predators or competitors. These unusual observations may be important in revealing the plasticity of a species' behavioral repertoire. ?? 2009 The Raptor Research Foundation, Inc.

  19. Techniques for identifying predators of goose nests

    USGS Publications Warehouse

    Anthony, R.M.; Grand, J.B.; Fondell, T.F.; Miller, David A.

    2006-01-01

    We used cameras and artificial eggs to identify nest predators of dusky Canada goose Branta canadensis occidentalis nests during 1997-2000. Cameras were set up at 195 occupied goose nests and 60 artificial nests. We placed wooden eggs and domestic goose eggs that were emptied and then filled with wax or foam in an additional 263 natural goose nests to identify predators from marks in the artificial eggs. All techniques had limitations, but each correctly identified predators and estimated their relative importance. Nests with cameras had higher rates of abandonment than natural nests, especially during laying. Abandonment rates were reduced by deploying artificial eggs late in laying and reducing time at nests. Predation rates for nests with cameras were slightly lower than for nests without cameras. Wax-filled artificial eggs caused mortality of embryos in natural nests, but were better for identifying predator marks at artificial nests. Use of foam-filled artificial eggs in natural nests was the most cost effective means of monitoring nest predation. ?? Wildlife Biology (2006).

  20. Nest success of northern bobwhite on managed and unmanaged landscapes in southeast Iowa

    USGS Publications Warehouse

    Potter, Lisa M.; Otis, David L.; Bogenschutz, Todd R.

    2011-01-01

    Range-wide declines in northern bobwhite populations (Colinus virginianus) have been attributed to concomitant loss of breeding habitat. Bobwhite management efforts to restore this habitat resource can be informed by empirical studies of associations between breeding success and multi-scale habitat attributes. We compared bobwhite nest success in 2 southern Iowa landscapes as a function of microhabitat and landscape composition. Lake Sugema Fish and Wildlife Area (LSWA) was managed to promote bobwhite recruitment, and Harrisburg Township (HT) was an adjacent landscape dominated by private agricultural production. Survival rate modeling based on telemetry data provided evidence for age-specific daily nest survival rate. Daily survival rates decreased as nest age increased, but the decline was more severe at HT. Nest survival at LSWA (S = 0.495, SE = 0.103) was nearly twice that on HT (S = 0.277, SE = 0.072). We found no evidence that habitat composition or spatial attributes within 210 m of a nest site significantly influenced nest success. Forb canopy at the nest site had a positive influence on nest success at HT but not at LSWA. We suggest nesting habitat with greater forb canopy cover will increase the opportunity for nesting success in landscapes with limited nesting habitat.

  1. 30 CFR 57.6304 - Primer protection.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Primer protection. 57.6304 Section 57.6304 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL MINE... Transportation-Surface and Underground § 57.6304 Primer protection. (a) Tamping shall not be done directly on...

  2. 30 CFR 57.6304 - Primer protection.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Primer protection. 57.6304 Section 57.6304 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL MINE... Transportation-Surface and Underground § 57.6304 Primer protection. (a) Tamping shall not be done directly on...

  3. 30 CFR 57.6304 - Primer protection.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Primer protection. 57.6304 Section 57.6304 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL MINE... Transportation-Surface and Underground § 57.6304 Primer protection. (a) Tamping shall not be done directly on...

  4. 30 CFR 57.6304 - Primer protection.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES Explosives... primer. (b) Rigid cartridges of explosives or blasting agents that are 4 inches (100 millimeters) in... of water to protect the primer from impact. Slit packages of prill, water gel, or emulsions are...

  5. 30 CFR 56.6304 - Primer protection.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-SURFACE METAL AND NONMETAL MINES Explosives Use § 56.6304 Primer protection. (a) Tamping shall not be done directly on a primer. (b) Rigid cartridges of explosives... impact. Slit packages of prill, water gel, or emulsions are not considered rigid cartridges and may...

  6. 30 CFR 56.6304 - Primer protection.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-SURFACE METAL AND NONMETAL MINES Explosives Use § 56.6304 Primer protection. (a) Tamping shall not be done directly on a primer. (b) Rigid cartridges of explosives... impact. Slit packages of prill, water gel, or emulsions are not considered rigid cartridges and may...

  7. 30 CFR 57.6304 - Primer protection.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES Explosives... primer. (b) Rigid cartridges of explosives or blasting agents that are 4 inches (100 millimeters) in... of water to protect the primer from impact. Slit packages of prill, water gel, or emulsions are...

  8. Nested-Fermi-liquid theory

    SciTech Connect

    Virosztek, A.; Ruvalds, J. )

    1990-09-01

    The susceptibility and quasiparticle self-energy are found to exhibit anomalous behavior in nested-Fermi-liquid (NFL) systems that have nearly parallel sections of the Fermi surface. Electron-electron scattering yields damping much stronger than the conventional electron-gas result and predicts a linear temperature variation of the resistivity. The susceptibility {chi}{sub NFL}{sup {prime}{prime}}({bold q},{omega}) for nested fermions is calculated at {bold q}{approx equal}{bold Q}, where {bold Q} is a typical nesting wave vector. The NFL susceptibility is linear in frequency up to a crossover region near {omega}{approx equal}4{ital T} where a saturation to a constant value occurs. The above features, as well as various theoretical constraints, are highly sensitive to the strength of the electron-electron coupling and to the degree of nesting. The relevance of the NFL results to superconducting oxides is briefly examined, with emphasis on the resistivity and the photoemission data, which supports the calculated damping {Gamma}({omega}{gt}{ital T}){approx equal}{alpha}{omega} with an intermediate on-site Coulomb coupling.

  9. Elementary maps on nest algebras

    NASA Astrophysics Data System (ADS)

    Li, Pengtong

    2006-08-01

    Let , be algebras and let , be maps. An elementary map of is an ordered pair (M,M*) such that for all , . In this paper, the general form of surjective elementary maps on standard subalgebras of nest algebras is described. In particular, such maps are automatically additive.

  10. Evidence of territoriality and species interactions from spatial point-pattern analyses of subarctic-nesting geese.

    PubMed

    Reiter, Matthew E; Andersen, David E

    2013-01-01

    Quantifying spatial patterns of bird nests and nest fate provides insights into processes influencing a species' distribution. At Cape Churchill, Manitoba, Canada, recent declines in breeding Eastern Prairie Population Canada geese (Branta canadensis interior) has coincided with increasing populations of nesting lesser snow geese (Chen caerulescens caerulescens) and Ross's geese (Chen rossii). We conducted a spatial analysis of point patterns using Canada goose nest locations and nest fate, and lesser snow goose nest locations at two study areas in northern Manitoba with different densities and temporal durations of sympatric nesting Canada and lesser snow geese. Specifically, we assessed (1) whether Canada geese exhibited territoriality and at what scale and nest density; and (2) whether spatial patterns of Canada goose nest fate were associated with the density of nesting lesser snow geese as predicted by the protective-association hypothesis. Between 2001 and 2007, our data suggest that Canada geese were territorial at the scale of nearest neighbors, but were aggregated when considering overall density of conspecifics at slightly broader spatial scales. The spatial distribution of nest fates indicated that lesser snow goose nest proximity and density likely influence Canada goose nest fate. Our analyses of spatial point patterns suggested that continued changes in the distribution and abundance of breeding lesser snow geese on the Hudson Bay Lowlands may have impacts on the reproductive performance of Canada geese, and subsequently the spatial distribution of Canada goose nests. PMID:24312520

  11. Evidence of territoriality and species interactions from spatial point-pattern analyses of subarctic-nesting geese.

    PubMed

    Reiter, Matthew E; Andersen, David E

    2013-01-01

    Quantifying spatial patterns of bird nests and nest fate provides insights into processes influencing a species' distribution. At Cape Churchill, Manitoba, Canada, recent declines in breeding Eastern Prairie Population Canada geese (Branta canadensis interior) has coincided with increasing populations of nesting lesser snow geese (Chen caerulescens caerulescens) and Ross's geese (Chen rossii). We conducted a spatial analysis of point patterns using Canada goose nest locations and nest fate, and lesser snow goose nest locations at two study areas in northern Manitoba with different densities and temporal durations of sympatric nesting Canada and lesser snow geese. Specifically, we assessed (1) whether Canada geese exhibited territoriality and at what scale and nest density; and (2) whether spatial patterns of Canada goose nest fate were associated with the density of nesting lesser snow geese as predicted by the protective-association hypothesis. Between 2001 and 2007, our data suggest that Canada geese were territorial at the scale of nearest neighbors, but were aggregated when considering overall density of conspecifics at slightly broader spatial scales. The spatial distribution of nest fates indicated that lesser snow goose nest proximity and density likely influence Canada goose nest fate. Our analyses of spatial point patterns suggested that continued changes in the distribution and abundance of breeding lesser snow geese on the Hudson Bay Lowlands may have impacts on the reproductive performance of Canada geese, and subsequently the spatial distribution of Canada goose nests.

  12. Nest sanitation elicits egg discrimination in cuckoo hosts.

    PubMed

    Yang, Canchao; Chen, Min; Wang, Longwu; Liang, Wei; Møller, Anders Pape

    2015-11-01

    Nest sanitation is a nearly universal behavior in birds, while egg discrimination is a more specific adaptation that has evolved to counter brood parasitism. These two behaviors are closely related with nest sanitation being the ancestral behavior, and it has been hypothesized to constitute a preadaptation for egg discrimination. However, previous studies found little evidence to support this hypothesis. Here, we conducted an empirical test of the association between nest sanitation and egg discrimination in the barn swallow (Hirundo rustica) by inserting a single non-mimetic model egg or a non-mimetic model egg plus half a peanut shell into host nests. Compared to the rejection rate of single model eggs, barn swallows significantly increased egg rejection frequency if a half peanut shell was simultaneously introduced. Our result for the first time shows the impact of nest sanitation on egg discrimination and demonstrates that nest sanitation can elicit egg discrimination in hosts of brood parasites. This study provided evidence for nest sanitation being a preadaptation to egg discrimination by facilitating egg rejection, thereby significantly advancing our understanding of avian cognition of foreign objects. Furthermore, we suggest that egg discrimination behavior in many accepters and intermediate rejecters may be lost or diluted. Such egg discrimination can be elicited and restored after nest sanitation, implying a sensitive and rapid phenotypic response to increased risk of parasitism. Our study offers a novel perspective for investigating the role of so-called intermediate rejecter individuals or species in the long-term coevolutionary cycle between brood parasites and their hosts. PMID:26160343

  13. Nest sanitation elicits egg discrimination in cuckoo hosts.

    PubMed

    Yang, Canchao; Chen, Min; Wang, Longwu; Liang, Wei; Møller, Anders Pape

    2015-11-01

    Nest sanitation is a nearly universal behavior in birds, while egg discrimination is a more specific adaptation that has evolved to counter brood parasitism. These two behaviors are closely related with nest sanitation being the ancestral behavior, and it has been hypothesized to constitute a preadaptation for egg discrimination. However, previous studies found little evidence to support this hypothesis. Here, we conducted an empirical test of the association between nest sanitation and egg discrimination in the barn swallow (Hirundo rustica) by inserting a single non-mimetic model egg or a non-mimetic model egg plus half a peanut shell into host nests. Compared to the rejection rate of single model eggs, barn swallows significantly increased egg rejection frequency if a half peanut shell was simultaneously introduced. Our result for the first time shows the impact of nest sanitation on egg discrimination and demonstrates that nest sanitation can elicit egg discrimination in hosts of brood parasites. This study provided evidence for nest sanitation being a preadaptation to egg discrimination by facilitating egg rejection, thereby significantly advancing our understanding of avian cognition of foreign objects. Furthermore, we suggest that egg discrimination behavior in many accepters and intermediate rejecters may be lost or diluted. Such egg discrimination can be elicited and restored after nest sanitation, implying a sensitive and rapid phenotypic response to increased risk of parasitism. Our study offers a novel perspective for investigating the role of so-called intermediate rejecter individuals or species in the long-term coevolutionary cycle between brood parasites and their hosts.

  14. Optimizing nest survival and female survival: Consequences of nest site selection for Canada Geese

    USGS Publications Warehouse

    Miller, David A.; Grand, J.B.; Fondell, T.F.; Anthony, R.M.

    2007-01-01

    We examined the relationship between attributes of nest sites used by Canada Geese (Branta canadensis) in the Copper River Delta, Alaska, and patterns in nest and female survival. We aimed to determine whether nest site attributes related to nest and female survival differed and whether nest site attributes related to nest survival changed within and among years. Nest site attributes that we examined included vegetation at and surrounding the nest, as well as associations with other nesting birds. Optimal nest site characteristics were different depending on whether nest survival or female survival was examined. Prior to 25 May, the odds of daily survival for nests in tall shrubs and on islands were 2.92 and 2.26 times greater, respectively, than for nests in short shrub sites. Bald Eagles (Halieaeetus leucocephalus) are the major predator during the early breeding season and their behavior was likely important in determining this pattern. After 25 May, when eagle predation is limited due to the availability of alternative prey, no differences in nest survival among the nest site types were found. In addition, nest survival was positively related to the density of other Canada Goose nests near the nest site. Although the number of detected mortalities for females was relatively low, a clear pattern was found, with mortality three times more likely at nest sites dominated by high shrub density within 50 m than at open sites dominated by low shrub density. The negative relationship of nest concealment and adult survival is consistent with that found in other studies of ground-nesting birds. Physical barriers that limited access to nest sites by predators and sites that allowed for early detection of predators were important characteristics of nest site quality for Canada Geese and nest site quality shifted within seasons, likely as a result of shifting predator-prey interactions.

  15. Electrostatic Discharge testing of propellants and primers

    SciTech Connect

    Berry, R.B.

    1994-02-01

    This report presents the results of testing of selected propellants and primers to Electrostatic Discharge (ESD) characteristic of the human body. It describes the tests and the fixturing built to accommodate loose material (propellants) and the packed energetic material of the primer. The results indicate that all powders passed and some primers, especially the electric primers, failed to pass established requirements which delineate insensitive energetic components. This report details the testing of components and materials to four ESD environments (Standard ESD, Severe ESD, Modified Standard ESD, and Modified Severe ESD). The purpose of this study was to collect data based on the customer requirements as defined in the Sandia Environmental Safety & Health (ES&H) Manual, Chapter 9, and to define static sensitive and insensitive propellants and primers.

  16. Selection of functional tRNA primers and primer binding site sequences from a retroviral combinatorial library: identification of new functional tRNA primers in murine leukemia virus replication

    PubMed Central

    Lund, Anders H.; Duch, Mogens; Pedersen, Finn Skou

    2000-01-01

    Retroviral reverse transcription is initiated from a cellular tRNA molecule and all known exogenous isolates of murine leukemia virus utilise a tRNAPro molecule. While several studies suggest flexibility in murine leukemia virus primer utilisation, studies on human immunodeficiency virus and avian retroviruses have revealed evidence of molecular adaptation towards the specific tRNA isoacceptor used as replication primer. In this study, murine leukemia virus tRNA utilisation is investigated by in vivo screening of a retroviral vector combinatorial library with randomised primer binding sites. While most of the selected primer binding sites are complementary to the 3′-end of tRNAPro, we also retrieved PBS sequences matching four other tRNA molecules and demonstrate that Akv murine leukemia virus vectors may efficiently replicate using tRNAArg(CCU), tRNAPhe(GAA) and a hitherto unknown human tRNASer(CGA). PMID:10637332

  17. SBE primer : multiplexing minisequencing-based genotyping

    SciTech Connect

    Kaderali, L.; Deshpande, A.; Uribe-Romeo, F. J.; Schliep, A.; Torney, D. C.

    2002-01-01

    Single-nucleotide polymorphism (SNP) analysis is a powerful tool for mapping and diagnosing disease-related alleles. Most of the known genetic diseases are caused by point mutations, and a growing number of SNPs will be routinely analyzed to diagnose genetic disorders. Mutation analysis by polymerase mediated single-base primer extension (minisequencing) can be massively parallelized using for example DNA microchips or flow cytometry with microspheres as solid support. By adding a unique oligonucleotide tag to the 5-inch end of the minisequencing primer and attaching the complementary anti-tag to the array or bead surface, the assay can be 'demultiplexed'. However, such high-throughput scoring of SNPs requires a high level of primer multiplexing in order to analyze multiple loci in one assay, thus enabling inexpensive and fast polymorphism scoring. Primers can be chosen from either the plus or the minus strand, and primers used in the same experiment must not bind to one another. To genotype a given number of polymorphic sites, the question is which primer to use for each SNP, and which primers to group into the same experiment. Furthermore, a crosshybridization-free tag/anti-tag code is required in order to sort the extended primers to the corresponding microspheres or chip spots. These problems pose challenging algorithmic questions. We present a computer program lo automate the design process for the assay. Oligonucleotide primers for the reaction are automatically selected by the software, a unique DNA tag/anti-tag system is generated, and the pairing of primers and DNA-Tags is automatically done in a way to avoid any crossreactivity. We report first results on a 45-plex genotyping assay, indicating that minisequencing can be adapted to be a powerful tool for high-throughput, massively parallel genotyping.

  18. Nested PCR-denaturing gradient gel electrophoresis analysis of human skin microbial diversity with age.

    PubMed

    Li, Wei; Han, Lei; Yu, Pengbo; Ma, Chaofeng; Wu, Xiaokang; Xu, Jiru

    2014-01-01

    To determine whether the composition and structure of skin microbiota differ with age, cutaneous bacteria were isolated from the axillary fossa of 37 healthy human adults in two age groups (old people and young adults). Bacterial genomic DNA was extracted and characterized by nested PCR-denaturing gradient gel electrophoresis (PCR-DGGE) with primers specifically targeting V3 region of the 16S rRNA gene. The excised gel bands were sequenced to identify bacterial categories. The total bacteria, Staphylococcus spp., Staphylococcus epidermidis and Corynebacterium spp. were further enumerated by quantitative PCR. There were no significant differences in the species diversity profiles between age groups. The similarity index was lower across age groups than that it was intra-group. This indicates that the composition of skin flora is more similar to others of the same age than across age groups. While Staphylococcus spp. and Corynebacterium spp. were the dominant bacteria in both groups, sequencing and quantitative PCR revealed that skin bacterial composition differed by age. The copy number of total bacteria and Corynebacterium spp. were significantly lower in younger subjects, whereas there were no statistical differences in the quantity of Staphylococcus spp. and Staphylococcus epidermidis. These results suggest that the skin flora undergo both quantitative and qualitative changes related to aging. PMID:24656938

  19. Climate change primer for respirologists.

    PubMed

    Takaro, Tim K; Henderson, Sarah B

    2015-01-01

    Climate change is already affecting the cardiorespiratory health of populations around the world, and these impacts are expected to increase. The present overview serves as a primer for respirologists who are concerned about how these profound environmental changes may affect their patients. The authors consider recent peer-reviewed literature with a focus on climate interactions with air pollution. They do not discuss in detail cardiorespiratory health effects for which the potential link to climate change is poorly understood. For example, pneumonia and influenza, which affect >500 million people per year, are not addressed, although clear seasonal variation suggests climate-related effects. Additionally, large global health impacts in low-resource countries, including migration precipitated by environmental change, are omitted. The major cardiorespiratory health impacts addressed are due to heat, air pollution and wildfires, shifts in allergens and infectious diseases along with respiratory impacts from flooding. Personal and societal choices about carbon use and fossil energy infrastructure should be informed by their impacts on health, and respirologists can play an important role in this discussion.

  20. Rapid Multiplex Nested PCR for Detection of Respiratory Viruses▿

    PubMed Central

    Lam, W. Y.; Yeung, Apple C. M.; Tang, Julian W.; Ip, Margaret; Chan, Edward W. C.; Hui, Mamie; Chan, Paul K. S.

    2007-01-01

    Respiratory tract infections can be caused by a heterogeneous group of viruses and bacteria that produce similar clinical presentations. Specific diagnosis therefore relies on laboratory investigation. This study developed and evaluated five groups of multiplex nested PCR assays that could simultaneously detect 21 different respiratory pathogens: influenza A virus (H1N1, H3N2, and H5N1); influenza B virus; parainfluenza virus types 1, 2, 3, 4a, and 4b; respiratory syncytial virus A and B; human rhinoviruses; human enteroviruses; human coronaviruses OC43 and 229E; severe acute respiratory syndrome coronavirus; human metapneumoviruses; Mycoplasma pneumoniae; Chlamydophila pneumoniae; Legionella pneumophila; and adenoviruses (A to F). These multiplex nested PCRs adopted fast PCR technology. The high speed of fast PCR (within 35 min) greatly improved the efficiency of these assays. The results show that these multiplex nested PCR assays are specific and more sensitive (100- to 1,000-fold) than conventional methods. Among the 303 clinical specimens tested, the multiplex nested PCR achieved an overall positive rate of 48.5% (95% confidence interval [CI], 42.9 to 54.1%), which was significantly higher than that of virus isolation (20.1% [95% CI, 15.6 to 24.6%]) and that of direct detection by immunofluorescence assay (13.5% [95% CI, 9.7 to 17.4%]). The improved sensitivity was partly due to the higher sensitivity of multiplex nested PCR than that of conventional methods in detecting cultivatable viruses. Moreover, the ability of the multiplex nested PCR to detect noncultivatable viruses, particularly rhinoviruses, coronavirus OC43, and metapneumoviruses, contributed a major gain (15.6%) in the overall positive rate. In conclusion, rapid multiplex nested PCR assays can improve the diagnostic yield for respiratory infections to allow prompt interventive actions to be taken. PMID:17804659

  1. Nest marking behavior and chemical composition of olfactory cues involved in nest recognition in Megachile rotundata.

    PubMed

    Guédot, Christelle; Buckner, James S; Hagen, Marcia M; Bosch, Jordi; Kemp, William P; Pitts-Singer, Theresa L

    2013-08-01

    In-nest observations of the solitary bee, Megachile rotundata (F.), revealed that nesting females apply olfactory cues to nests for nest recognition. On their way in and out of the nest, females drag the abdomen along the entire length of the nest, and sometimes deposit fluid droplets from the tip of the abdomen. The removal of bee-marked sections of the nest resulted in hesitation and searching behavior by females, indicating the loss of olfactory cues used for nest recognition. Chemical analysis of female cuticles and the deposits inside marked nesting tubes revealed the presence of hydrocarbons, wax esters, fatty aldehydes, and fatty alcohol acetate esters. Chemical compositions were similar across tube samples, but proportionally different from cuticular extracts. These findings reveal the importance of lipids as chemical signals for nest recognition and suggest that the nest-marking cues are derived from a source in addition to, or other than, the female cuticle.

  2. Nest marking behavior and chemical composition of olfactory cues involved in nest recognition in Megachile rotundata.

    PubMed

    Guédot, Christelle; Buckner, James S; Hagen, Marcia M; Bosch, Jordi; Kemp, William P; Pitts-Singer, Theresa L

    2013-08-01

    In-nest observations of the solitary bee, Megachile rotundata (F.), revealed that nesting females apply olfactory cues to nests for nest recognition. On their way in and out of the nest, females drag the abdomen along the entire length of the nest, and sometimes deposit fluid droplets from the tip of the abdomen. The removal of bee-marked sections of the nest resulted in hesitation and searching behavior by females, indicating the loss of olfactory cues used for nest recognition. Chemical analysis of female cuticles and the deposits inside marked nesting tubes revealed the presence of hydrocarbons, wax esters, fatty aldehydes, and fatty alcohol acetate esters. Chemical compositions were similar across tube samples, but proportionally different from cuticular extracts. These findings reveal the importance of lipids as chemical signals for nest recognition and suggest that the nest-marking cues are derived from a source in addition to, or other than, the female cuticle. PMID:23905742

  3. Nest predation increases with parental activity: Separating nest site and parental activity effects

    USGS Publications Warehouse

    Martin, T.E.; Scott, J.; Menge, C.

    2000-01-01

    Alexander Skutch hypothesized that increased parental activity can increase the risk of nest predation. We tested this hypothesis using ten open-nesting bird species in Arizona, USA. Parental activity was greater during the nestling than incubation stage because parents visited the nest frequently to feed their young during the nestling stage. However, nest predation did not generally increase with parental activity between nesting stages across the ten study species. Previous investigators have found similar results. We tested whether nest site effects might yield higher predation during incubation because the most obvious sites are depredated most rapidly. We conducted experiments using nest sites from the previous year to remove parental activity. Our results showed that nest sites have highly repeatable effects on nest predation risk; poor nest sites incurred rapid predation and caused predation rates to be greater during the incubation than nestling stage. This pattern also was exhibited in a bird species with similar (i.e. controlled) parental activity between nesting stages. Once nest site effects are taken into account, nest predation shows a strong proximate increase with parental activity during the nestling stage within and across species. Parental activity and nest sites exert antagonistic influences on current estimates of nest predation between nesting stages and both must be considered in order to understand current patterns of nest predation, which is an important source of natural selection.

  4. Decoration Increases the Conspicuousness of Raptor Nests

    PubMed Central

    Canal, David; Mulero-Pázmány, Margarita; Negro, Juan José; Sergio, Fabrizio

    2016-01-01

    Avian nests are frequently concealed or camouflaged, but a number of species builds noticeable nests or use conspicuous materials for nest decoration. In most cases, nest decoration has a role in mate choice or provides thermoregulatory or antiparasitic benefits. In territorial species however, decorations may serve additional or complementary functions, such as extended phenotypic signaling of nest-site occupancy and social status to potential intruders. The latter may benefit both signaler and receiver by minimizing the risk of aggressive interactions, especially in organisms with dangerous weaponry. Support for this hypothesis was recently found in a population of black kites (Milvus migrans), a territorial raptor that decorates its nest with white artificial materials. However, the crucial assumption that nest decorations increased nest-site visibility to conspecifics was not assessed, a key aspect given that black kite nests may be well concealed within the canopy. Here, we used an unmanned aircraft system to take pictures of black kite nests, with and without an experimentally placed decoration, from different altitudes and distances simulating the perspective of a flying and approaching, prospecting intruder. The pictures were shown to human volunteers through a standardized routine to determine whether detection rates varied according the nest decoration status and distance. Decorated nests consistently showed a higher detection frequency and a lower detection-latency, compared to undecorated versions of the same nests. Our results confirm that nest decoration in this species may act as a signaling medium that enhances nest visibility for aerial receivers, even at large distances. This finding complements previous work on this communication system, which showed that nest decoration was a threat informing trespassing conspecifics on the social dominance, territory quality and fighting capabilities of the signaler. PMID:27455066

  5. Decoration Increases the Conspicuousness of Raptor Nests.

    PubMed

    Canal, David; Mulero-Pázmány, Margarita; Negro, Juan José; Sergio, Fabrizio

    2016-01-01

    Avian nests are frequently concealed or camouflaged, but a number of species builds noticeable nests or use conspicuous materials for nest decoration. In most cases, nest decoration has a role in mate choice or provides thermoregulatory or antiparasitic benefits. In territorial species however, decorations may serve additional or complementary functions, such as extended phenotypic signaling of nest-site occupancy and social status to potential intruders. The latter may benefit both signaler and receiver by minimizing the risk of aggressive interactions, especially in organisms with dangerous weaponry. Support for this hypothesis was recently found in a population of black kites (Milvus migrans), a territorial raptor that decorates its nest with white artificial materials. However, the crucial assumption that nest decorations increased nest-site visibility to conspecifics was not assessed, a key aspect given that black kite nests may be well concealed within the canopy. Here, we used an unmanned aircraft system to take pictures of black kite nests, with and without an experimentally placed decoration, from different altitudes and distances simulating the perspective of a flying and approaching, prospecting intruder. The pictures were shown to human volunteers through a standardized routine to determine whether detection rates varied according the nest decoration status and distance. Decorated nests consistently showed a higher detection frequency and a lower detection-latency, compared to undecorated versions of the same nests. Our results confirm that nest decoration in this species may act as a signaling medium that enhances nest visibility for aerial receivers, even at large distances. This finding complements previous work on this communication system, which showed that nest decoration was a threat informing trespassing conspecifics on the social dominance, territory quality and fighting capabilities of the signaler. PMID:27455066

  6. Decoration Increases the Conspicuousness of Raptor Nests.

    PubMed

    Canal, David; Mulero-Pázmány, Margarita; Negro, Juan José; Sergio, Fabrizio

    2016-01-01

    Avian nests are frequently concealed or camouflaged, but a number of species builds noticeable nests or use conspicuous materials for nest decoration. In most cases, nest decoration has a role in mate choice or provides thermoregulatory or antiparasitic benefits. In territorial species however, decorations may serve additional or complementary functions, such as extended phenotypic signaling of nest-site occupancy and social status to potential intruders. The latter may benefit both signaler and receiver by minimizing the risk of aggressive interactions, especially in organisms with dangerous weaponry. Support for this hypothesis was recently found in a population of black kites (Milvus migrans), a territorial raptor that decorates its nest with white artificial materials. However, the crucial assumption that nest decorations increased nest-site visibility to conspecifics was not assessed, a key aspect given that black kite nests may be well concealed within the canopy. Here, we used an unmanned aircraft system to take pictures of black kite nests, with and without an experimentally placed decoration, from different altitudes and distances simulating the perspective of a flying and approaching, prospecting intruder. The pictures were shown to human volunteers through a standardized routine to determine whether detection rates varied according the nest decoration status and distance. Decorated nests consistently showed a higher detection frequency and a lower detection-latency, compared to undecorated versions of the same nests. Our results confirm that nest decoration in this species may act as a signaling medium that enhances nest visibility for aerial receivers, even at large distances. This finding complements previous work on this communication system, which showed that nest decoration was a threat informing trespassing conspecifics on the social dominance, territory quality and fighting capabilities of the signaler.

  7. Improved PCR primers for the detection and identification of arbuscular mycorrhizal fungi.

    PubMed

    Lee, Jaikoo; Lee, Sangsun; Young, J Peter W

    2008-08-01

    A set of PCR primers that should amplify all subgroups of arbuscular mycorrhizal fungi (AMF, Glomeromycota), but exclude sequences from other organisms, was designed to facilitate rapid detection and identification directly from field-grown plant roots. The small subunit rRNA gene was targeted for the new primers (AML1 and AML2) because phylogenetic relationships among the Glomeromycota are well understood for this gene. Sequence comparisons indicate that the new primers should amplify all published AMF sequences except those from Archaeospora trappei. The specificity of the new primers was tested using 23 different AMF spore morphotypes from trap cultures and Miscanthus sinensis, Glycine max and Panax ginseng roots sampled from the field. Non-AMF DNA of 14 plants, 14 Basidiomycota and 18 Ascomycota was also tested as negative controls. Sequences amplified from roots using the new primers were compared with those obtained using the established NS31 and AM1 primer combination. The new primers have much better specificity and coverage of all known AMF groups. PMID:18631176

  8. Primer on spontaneous heating and pyrophoricity

    SciTech Connect

    Not Available

    1994-12-01

    This primer was prepared as an information resource for personnel responsible for operation of DOE nuclear facilities. It has sections on combustion principles, spontaneous heating/ignition of hydrocarbons and organics, pyrophoric gases and liquids, pyrophoric nonmetallic solids, pyrophoric metals (including Pu and U), and accident case studies. Although the information in this primer is not all-encompassing, it should provide the reader with a fundamental knowledge level sufficient to recognize most spontaneous combustion hazards and how to prevent ignition and widespread fires. This primer is provided as an information resource only, and is not intended to replace any fire protection or hazardous material training.

  9. Woodcock nesting habitat in northern Wisconsin

    USGS Publications Warehouse

    Gregg, L.E.; Hale, J.B.

    1977-01-01

    Of 32 woodcock nests studied in northern Wisconsin, 29 were in forest stands dominated by aspen, and 3 were in northern hardwoods. Well-drained, upland nest sites near the brushy edges of poorly stocked poletimber stands were apparently preferred. More than 30 woody plant species were found at the 32 nest sites. Hazel was the most important shrub species noted.

  10. Nesting behavior of the poo-uli

    USGS Publications Warehouse

    Kepler, C.B.; Pratt, T.K.; Ecton, A.M.; Engilis, A.; Fluetsch, K.M.

    1996-01-01

    We describe two sequential nestings of a pair of Poo-uli (Melamprosops phaeosoma), a Hawaiian honeycreeper nearing extinction. Similarities to nesting of most other honeycreepers included: nest site in ohia lehua (Metrosideros polymorpha Gaud.) canopy; breeding in March through June; monogamous breeding system with the putative male helping build the nest, feeding the putative female throughout each nesting event, and feeding the chicks, but not incubating or brooding; and complete nest sanitation. Notable differences were the paucity of songs and calls by the parents and inclusion of snails in the diet of nestlings. Clutch size was probably two eggs for both nests. High winds, rain, or both influenced parental behavior: the female stayed longer on the nest and took shorter recesses in poor weather. Weather did not affect rates at which the male fed the female on the nest; however, the feeding rate increased from the egg to the chick stage probably because food was passed on to the chicks. At nest #2, parents fed young chicks (<14 days old) more often in good than in poor weather; data were insufficient for old chicks. Weather is usually poor throughout the year in the relictual range of the Poo-uli and is likely to impact nesting success. The first nest failed in poor weather. The second fledged a single young 21 days old. Diet of nestlings appeared to consist of a higher proportion of insect larvae than that of older birds, which are reported to eat mostly snails.

  11. Teaching Ecological Concepts with Mud Dauber Nests.

    ERIC Educational Resources Information Center

    Matthews, Robert W.; Matthews, Janice R.

    1999-01-01

    Contends that mud dauber nests--which are widely available, safe, inexpensive, and easy to use--offer a novel and highly motivating way to teach ecological concepts to life science students at many grade levels. Presents background information for teachers, details classroom-tested methods for nest dissection, provides keys to nest contents, and…

  12. Primers-4-Yeast: a comprehensive web tool for planning primers for Saccharomyces cerevisiae.

    PubMed

    Yofe, Ido; Schuldiner, Maya

    2014-02-01

    The budding yeast Saccharomyces cerevisiae is a key model organism of functional genomics, due to its ease and speed of genetic manipulations. In fact, in this yeast, the requirement for homologous sequences for recombination purposes is so small that 40 base pairs (bp) are sufficient. Hence, an enormous variety of genetic manipulations can be performed by simply planning primers with the correct homology, using a defined set of transformation plasmids. Although designing primers for yeast transformations and for the verification of their correct insertion is a common task in all yeast laboratories, primer planning is usually done manually and a tool that would enable easy, automated primer planning for the yeast research community is still lacking. Here we introduce Primers-4-Yeast, a web tool that allows primers to be designed in batches for S. cerevisiae gene-targeting transformations, and for the validation of correct insertions. This novel tool enables fast, automated, accurate primer planning for large sets of genes, introduces consistency in primer planning and is therefore suggested to serve as a standard in yeast research. Primers-4-Yeast is available at: http://www.weizmann.ac.il/Primers-4-Yeast

  13. MRPrimer: a MapReduce-based method for the thorough design of valid and ranked primers for PCR

    PubMed Central

    Kim, Hyerin; Kang, NaNa; Chon, Kang-Wook; Kim, Seonho; Lee, NaHye; Koo, JaeHyung; Kim, Min-Soo

    2015-01-01

    Primer design is a fundamental technique that is widely used for polymerase chain reaction (PCR). Although many methods have been proposed for primer design, they require a great deal of manual effort to generate feasible and valid primers, including homology tests on off-target sequences using BLAST-like tools. That approach is inconvenient for many target sequences of quantitative PCR (qPCR) due to considering the same stringent and allele-invariant constraints. To address this issue, we propose an entirely new method called MRPrimer that can design all feasible and valid primer pairs existing in a DNA database at once, while simultaneously checking a multitude of filtering constraints and validating primer specificity. Furthermore, MRPrimer suggests the best primer pair for each target sequence, based on a ranking method. Through qPCR analysis using 343 primer pairs and the corresponding sequencing and comparative analyses, we showed that the primer pairs designed by MRPrimer are very stable and effective for qPCR. In addition, MRPrimer is computationally efficient and scalable and therefore useful for quickly constructing an entire collection of feasible and valid primers for frequently updated databases like RefSeq. Furthermore, we suggest that MRPrimer can be utilized conveniently for experiments requiring primer design, especially real-time qPCR. PMID:26109350

  14. TSUNAMI Primer: A Primer for Sensitivity/Uncertainty Calculations with SCALE

    SciTech Connect

    Rearden, Bradley T; Mueller, Don; Bowman, Stephen M; Busch, Robert D.; Emerson, Scott

    2009-01-01

    This primer presents examples in the application of the SCALE/TSUNAMI tools to generate k{sub eff} sensitivity data for one- and three-dimensional models using TSUNAMI-1D and -3D and to examine uncertainties in the computed k{sub eff} values due to uncertainties in the cross-section data used in their calculation. The proper use of unit cell data and need for confirming the appropriate selection of input parameters through direct perturbations are described. The uses of sensitivity and uncertainty data to identify and rank potential sources of computational bias in an application system and TSUNAMI tools for assessment of system similarity using sensitivity and uncertainty criteria are demonstrated. Uses of these criteria in trending analyses to assess computational biases, bias uncertainties, and gap analyses are also described. Additionally, an application of the data adjustment tool TSURFER is provided, including identification of specific details of sources of computational bias.

  15. Collection of mammal manure and other Debris by nesting Burrowing Owls

    USGS Publications Warehouse

    Smith, M.D.; Conway, C.J.

    2011-01-01

    Burrowing Owls (Athene cunicularia) routinely collect and scatter dry manure of mammals around their nesting burrows. Recent studies have suggested this behavior attracts insect prey to the nesting burrow. However, some Burrowing Owls do not use manure, but instead, collect and scatter other materials (e.g., grass, moss, paper, plastic) around their nesting burrow in a similar fashion. Use of these materials seemingly contradicts the prey-attraction hypothesis. Using observational and experimental methods, we tested whether Burrowing Owls preferred manure to other materials commonly found at nesting burrows in eastern Washington. We found a wide variety of materials at nests, but grass and manure were the most common materials. The amount of manure present at nests was negatively correlated with the amount of other materials, and with the distance to the nearest source of manure. Burrowing Owls showed no preference between horse manure and grass divots at experimental supply stations that we placed near nesting burrows. They did prefer these two materials to carpet pieces and aluminum foil (both materials that are often found at Burrowing Owl nests). Our results did not support the premise that Burrowing Owls specifically seek out manure when lining their nesting burrows. The unusual behavior of collecting and scattering mammal manure and other debris at Burrowing Owl nests may serve functions other than (or in addition to) prey attraction and alternative hypotheses need further testing before the function of this behavior is certain. ?? 2011 The Raptor Research Foundation, Inc.

  16. Nest Material Shapes Eggs Bacterial Environment.

    PubMed

    Ruiz-Castellano, Cristina; Tomás, Gustavo; Ruiz-Rodríguez, Magdalena; Martín-Gálvez, David; Soler, Juan José

    2016-01-01

    Selective pressures imposed by pathogenic microorganisms to embryos have selected in hosts for a battery of antimicrobial lines of defenses that includes physical and chemical barriers. Due to the antimicrobial properties of volatile compounds of green plants and of chemicals of feather degrading bacteria, the use of aromatic plants and feathers for nest building has been suggested as one of these barriers. However, experimental evidence suggesting such effects is scarce in the literature. During two consecutive years, we explored experimentally the effects of these nest materials on loads of different groups of bacteria (mesophilic bacteria, Enterobacteriaceae, Staphylococcus and Enterococcus) of eggshells in nests of spotless starlings (Sturnus unicolor) at the beginning and at the end of the incubation period. This was also explored in artificial nests without incubation activity. We also experimentally increased bacterial density of eggs in natural and artificial nests and explored the effects of nest lining treatments on eggshell bacterial load. Support for the hypothetical antimicrobial function of nest materials was mainly detected for the year and location with larger average values of eggshell bacterial density. The beneficial effects of feathers and plants were more easily detected in artificial nests with no incubation activity, suggesting an active role of incubation against bacterial colonization of eggshells. Pigmented and unpigmented feathers reduced eggshell bacterial load in starling nests and artificial nest boxes. Results from artificial nests allowed us to discuss and discard alternative scenarios explaining the detected association, particularly those related to the possible sexual role of feathers and aromatic plants in starling nests. All these results considered together confirm the antimicrobial functionality mainly of feathers but also of plants used as nest materials, and highlight the importance of temporally and geographically

  17. Nest Material Shapes Eggs Bacterial Environment

    PubMed Central

    Ruiz-Castellano, Cristina; Tomás, Gustavo; Ruiz-Rodríguez, Magdalena; Martín-Gálvez, David; Soler, Juan José

    2016-01-01

    Selective pressures imposed by pathogenic microorganisms to embryos have selected in hosts for a battery of antimicrobial lines of defenses that includes physical and chemical barriers. Due to the antimicrobial properties of volatile compounds of green plants and of chemicals of feather degrading bacteria, the use of aromatic plants and feathers for nest building has been suggested as one of these barriers. However, experimental evidence suggesting such effects is scarce in the literature. During two consecutive years, we explored experimentally the effects of these nest materials on loads of different groups of bacteria (mesophilic bacteria, Enterobacteriaceae, Staphylococcus and Enterococcus) of eggshells in nests of spotless starlings (Sturnus unicolor) at the beginning and at the end of the incubation period. This was also explored in artificial nests without incubation activity. We also experimentally increased bacterial density of eggs in natural and artificial nests and explored the effects of nest lining treatments on eggshell bacterial load. Support for the hypothetical antimicrobial function of nest materials was mainly detected for the year and location with larger average values of eggshell bacterial density. The beneficial effects of feathers and plants were more easily detected in artificial nests with no incubation activity, suggesting an active role of incubation against bacterial colonization of eggshells. Pigmented and unpigmented feathers reduced eggshell bacterial load in starling nests and artificial nest boxes. Results from artificial nests allowed us to discuss and discard alternative scenarios explaining the detected association, particularly those related to the possible sexual role of feathers and aromatic plants in starling nests. All these results considered together confirm the antimicrobial functionality mainly of feathers but also of plants used as nest materials, and highlight the importance of temporally and geographically

  18. Excavated substrate modulates growth instability during nest building in ants

    PubMed Central

    Toffin, Etienne; Kindekens, Jonathan; Deneubourg, Jean-Louis

    2010-01-01

    In social insects, the nests of the same species can show a large difference in size and shape. Despite these large variations, the nests share the same substructures, some appearing during nest growth. In ants, the interplay between nest size and digging activity leads to two successive morphological transitions from circular to branched shapes (budding along the perimeter of the circular cavity and tunnelling of the galleries). Like several other self-organized collective behaviours, this phenomenon, as well as the entire nest-digging process, is thought to be modulated by environmental properties. The present study investigates the effect of excavated substrate on the nest morphogenesis and the morphological transitions by using two materials with different cohesions. Here, we show that the two morphological transitions occur more frequently with a cohesive substrate than with a granular one: 96 per cent of cohesive experiments showed both transitions, whereas only 50 per cent did in granular experiments. We found that transitions and excavation cessation follow area–response thresholds: the shape transitions take place and the digging activity stops when the dug area reaches the corresponding threshold values. The shape transition thresholds are lower with the cohesive substrate and that of stopping digging is independent of nest shape and material. According to simulations, the experimental frequencies of transitions found their origin in the competition between transitions and activity cessation and in the difference between the transition threshold values of each substrate. Our results demonstrate how the substrate properties modulate the collective response and lead to various patterns. Considering the non-specific mechanisms at work, such effects of substrate coarseness have their counterparts in various collective behaviours, generating alternative patterns to colonize and exploit the environment. PMID:20410036

  19. Multiplexing Short Primers for Viral Family PCR

    SciTech Connect

    Gardner, S N; Hiddessen, A L; Hara, C A; Williams, P L; Wagner, M; Colston, B W

    2008-06-26

    We describe a Multiplex Primer Prediction (MPP) algorithm to build multiplex compatible primer sets for large, diverse, and unalignable sets of target sequences. The MPP algorithm is scalable to larger target sets than other available software, and it does not require a multiple sequence alignment. We applied it to questions in viral detection, and demonstrated that there are no universally conserved priming sequences among viruses and that it could require an unfeasibly large number of primers ({approx}3700 18-mers or {approx}2000 10-mers) to generate amplicons from all sequenced viruses. We then designed primer sets separately for each viral family, and for several diverse species such as foot-and-mouth disease virus, hemagglutinin and neuraminidase segments of influenza A virus, Norwalk virus, and HIV-1.

  20. Use of labeled primers for differential display

    SciTech Connect

    Paunesku, T.; Woloschak, G.E.

    1995-01-01

    Two artifacts introduced in using differential display technology are (1) random priming from dT present from affinity purification of PolyA+ RNA and (2) hybridization of the arbitrary primer to template target sequences on both cDNA strands. We have developed a method eliminating both problems. By separately using 5`-end-labeled (T){sub 12}XY and arbitrary primers to label bands and comparing two differential display patterns, we can detect only those products incorporating the (T){sub 12}XY primer on the 3` ends and the arbitrary primer on 5` ends. Those bands that are generated randomly in the PCR are readily detectable and can be ignored.

  1. Nest-site selection by sage thrashers in southeastern Idaho. [Oreoscoptes montanus

    SciTech Connect

    Petersen, K.L. ); Best, L.B. )

    1991-09-01

    Nest sites selected by Sage Thrashers (Oreoscoptes montanus) were characterized and compared with available habitat. The study area, consisting of 25 ha of sagebrush shrubsteppe on the upper Snake River plain 11 km south of Howe, Idaho, is administered by the U.S. Department of Energy as part of the Idaho National Engineering Laboratory (INEL). Microhabitats within 5 m of nests had taller and more aggregated shrubs and less bare ground than the study area in general. Big sagebrush (Artemisia tridentata wyomingensis) plants used for nesting were taller than average available shrubs, had greater foliage density, were more often living, and more frequently had branches and foliage within 30 cm of the ground. Nest placement was specific with respect to relative nest height and distance from the top and perimeter of the support shrub. Sage Thrashers disproportionately used easterly exposures and underused westerly exposures for their nests.

  2. Protective Coats For Zinc-Rich Primers

    NASA Technical Reports Server (NTRS)

    Macdowell, Louis G, III

    1993-01-01

    Report describes tests of topcoats for inorganic zinc-rich primers on carbon steel. Topcoats intended to provide additional protection against corrosion in acidic, salty seacoast-air/rocket-engine-exhaust environment of Space Shuttle launch site. Tests focused on polyurethane topcoats on epoxy tie coats on primers. Part of study involved comparison between "high-build" coating materials and thin-film coating materials.

  3. A Dozen Primers on Important Information Standards

    ERIC Educational Resources Information Center

    Dempsey, Kathy, Comp.

    2007-01-01

    This is a compilation of 12 primers on important information standards and protocols. These primers are: (1) Atom; (2) COinS; (3) MADS; (4) MARC 21/MARCXML; (5) MIX; (6) MXG; (7) OpenSearch; (8) PREMIS; (9) RESTful HTTP; (10) unAPI; (11) XMPP (aka Jabber); and (12) ZeeRex. The Atom Syndication Format defines a new XML-based syndication format for…

  4. [Screening of peafowl microsatellite primers and analysis of genetic diversity].

    PubMed

    Bao, Wen-Bin; Chen, Guo-Hong; Shu, Jing-Ting; Xu, Qi; Li, Hui-Fang

    2006-10-01

    The applicability of chicken microsatellite primers to peafowl population was analyzed in the present paper, and the results showed 14 of 29 pairs of microsatellite primers from chicken could amplify peafowl DNA and produce specific allele patterns. A mean of 1.71 alleles was found for each locus. Seven pairs were highly polymorphic, and MCW0080 and MCW0098 were ideal markers for peafowl. Genetic diversity analysis within and between the green peafowl and the blue peafowl populations demonstrated that the expected heterozygosity of two peafowl populations were 0.2482 and 0.2744, respectively. The inbreeding index (FST), Reynolds' genetic distance and gene flow between the two populations were 0.078, 0.0603 and 3.896 respectively. These results indicate that the heterozygosity and the genetic diversity of these two peafowl populations were very low, and suggest a tendency towards intermixing.

  5. [Screening of peafowl microsatellite primers and analysis of genetic diversity].

    PubMed

    Bao, Wen-Bin; Chen, Guo-Hong; Shu, Jing-Ting; Xu, Qi; Li, Hui-Fang

    2006-10-01

    The applicability of chicken microsatellite primers to peafowl population was analyzed in the present paper, and the results showed 14 of 29 pairs of microsatellite primers from chicken could amplify peafowl DNA and produce specific allele patterns. A mean of 1.71 alleles was found for each locus. Seven pairs were highly polymorphic, and MCW0080 and MCW0098 were ideal markers for peafowl. Genetic diversity analysis within and between the green peafowl and the blue peafowl populations demonstrated that the expected heterozygosity of two peafowl populations were 0.2482 and 0.2744, respectively. The inbreeding index (FST), Reynolds' genetic distance and gene flow between the two populations were 0.078, 0.0603 and 3.896 respectively. These results indicate that the heterozygosity and the genetic diversity of these two peafowl populations were very low, and suggest a tendency towards intermixing. PMID:17035182

  6. COMplementary Primer ASymmetric PCR (COMPAS-PCR) Applied to the Identification of Salmo salar, Salmo trutta and Their Hybrids

    PubMed Central

    2016-01-01

    Avoiding complementarity between primers when designing a PCR assay constitutes a central rule strongly anchored in the mind of the molecular scientist. 3’-complementarity will extend the primers during PCR elongation using one another as template, consequently disabling further possible involvement in traditional target amplification. However, a 5’-complementarity will leave the primers unchanged during PCR cycles, albeit sequestered to one another, therefore also suppressing target amplification. We show that 5’-complementarity between primers may be exploited in a new PCR method called COMplementary-Primer-Asymmetric (COMPAS)-PCR, using asymmetric primer concentrations to achieve target PCR amplification. Moreover, such a design may paradoxically reduce spurious non-target amplification by actively sequestering the limiting primer. The general principles were demonstrated using 5S rDNA direct repeats as target sequences to design a species-specific assay for identifying Salmo salar and Salmo trutta using almost fully complementary primers overlapping the same target sequence. Specificity was enhanced by using 3’-penultimate point mutations and the assay was further developed to enable identification of S. salar x S. trutta hybrids by High Resolution Melt analysis in a 35 min one-tube assay. This small paradigm shift, using highly complementary primers for PCR, should help develop robust assays that previously would not be considered. PMID:27783658

  7. Nest materials as a source of genetic data for avian ecological studies

    USGS Publications Warehouse

    Pearce, J.M.; Fields, R.L.; Scribner, K.T.

    1997-01-01

    We examined the utility of feathers and egg shell membranes, deposited in the nests of Spectacled Eiders (Somateria fischeri), as a source of DNA for genetic studies at both the population and individual level. The potential for feather DNA contamination as a result of female behavioral interactions (e.g. nest parasitism), reuse of nest sites from previous years, or other unknown occurrences was acknowledged and specifically tested. DNA was successfully extracted from both feathers and egg shell membranes and waterfowl microsatellite loci were used to construct individual genotypes. We found no difference in the genotypes obtained from nest feathers or blood of the incubating female. Detection of nest feather contamination was possible with as little as one feather when samples from multiple females were intentionally mixed. Triplicate DNA extractions from 33 nests provided a means of detecting contamination in 3 nests. Egg membranes proved a viable source of offspring DNA and can contribute valuable data to investigations of parentage when assayed jointly with maternal feather DNA. Nest materials provide an efficient, non-invasive method of genetic sampling that can be readily incorporated into field research. However, the natural history traits and mating strategies of a species must be considered during sample collection to identify the possible sources of nest materials (e.g., paternal, maternal, parasite, etc.). Specific experiments should also be designed to test sampling assumptions.

  8. Nest Predation Deviates from Nest Predator Abundance in an Ecologically Trapped Bird.

    PubMed

    Hollander, Franck A; Van Dyck, Hans; San Martin, Gilles; Titeux, Nicolas

    2015-01-01

    In human-modified environments, ecological traps may result from a preference for low-quality habitat where survival or reproductive success is lower than in high-quality habitat. It has often been shown that low reproductive success for birds in preferred habitat types was due to higher nest predator abundance. However, between-habitat differences in nest predation may only weakly correlate with differences in nest predator abundance. An ecological trap is at work in a farmland bird (Lanius collurio) that recently expanded its breeding habitat into open areas in plantation forests. This passerine bird shows a strong preference for forest habitat, but it has a higher nest success in farmland. We tested whether higher abundance of nest predators in the preferred habitat or, alternatively, a decoupling of nest predator abundance and nest predation explained this observed pattern of maladaptive habitat selection. More than 90% of brood failures were attributed to nest predation. Nest predator abundance was more than 50% higher in farmland, but nest predation was 17% higher in forest. Differences between nest predation on actual shrike nests and on artificial nests suggested that parent shrikes may facilitate nest disclosure for predators in forest more than they do in farmland. The level of caution by parent shrikes when visiting their nest during a simulated nest predator intrusion was the same in the two habitats, but nest concealment was considerably lower in forest, which contributes to explaining the higher nest predation in this habitat. We conclude that a decoupling of nest predator abundance and nest predation may create ecological traps in human-modified environments.

  9. Nest Predation Deviates from Nest Predator Abundance in an Ecologically Trapped Bird

    PubMed Central

    Hollander, Franck A.; Van Dyck, Hans; San Martin, Gilles; Titeux, Nicolas

    2015-01-01

    In human-modified environments, ecological traps may result from a preference for low-quality habitat where survival or reproductive success is lower than in high-quality habitat. It has often been shown that low reproductive success for birds in preferred habitat types was due to higher nest predator abundance. However, between-habitat differences in nest predation may only weakly correlate with differences in nest predator abundance. An ecological trap is at work in a farmland bird (Lanius collurio) that recently expanded its breeding habitat into open areas in plantation forests. This passerine bird shows a strong preference for forest habitat, but it has a higher nest success in farmland. We tested whether higher abundance of nest predators in the preferred habitat or, alternatively, a decoupling of nest predator abundance and nest predation explained this observed pattern of maladaptive habitat selection. More than 90% of brood failures were attributed to nest predation. Nest predator abundance was more than 50% higher in farmland, but nest predation was 17% higher in forest. Differences between nest predation on actual shrike nests and on artificial nests suggested that parent shrikes may facilitate nest disclosure for predators in forest more than they do in farmland. The level of caution by parent shrikes when visiting their nest during a simulated nest predator intrusion was the same in the two habitats, but nest concealment was considerably lower in forest, which contributes to explaining the higher nest predation in this habitat. We conclude that a decoupling of nest predator abundance and nest predation may create ecological traps in human-modified environments. PMID:26624619

  10. Use of labeled primers for differential display

    SciTech Connect

    Paunesku, T.; Woloschak, G.E.

    1995-02-01

    The differential display of eukaryotic cDNAs using PCR allows for determination of mRNA species differentially expressed when comparing two similar cell populations. This procedure uses a (T){sub 12}XY oligonucleotide as the 3 ft primer and an arbitrary 8-10-mer as the 5 ft primer. Labeling occurs by inclusion of {alpha}[{sup 33}P]-dATP in the PCR reaction. Two artifacts caused by this approach are (1) random printing from dT present from affinity purification of PolyA+RNA and (2) hybridization of the arbitrary primer to template target sequences on both cDNA strands. In this work, we have developed an approach for both eliminating smearing and identifying nonspecific bands on sequencing gels. By separately using 5 ft-end-labeled (T){sub 12}XY and arbitrary primers to label bands and comparing two differential display patterns rather than including labeled nucleotides in the PCR reaction itself, we can detect only those products incorporating the M{sub 12}XY primer on the 3 ft ends and the arbitrary primer on 5 ft ends. Those bands that are generated randomly in the PCR reaction are readily detectable and can be ignored. If on the other hand, one is interested only in a diagnostic banding pattern for differential display, benefit can be derived from the simplicity of the pattern obtained when labeled (T){sub 12}XY is used.

  11. Breeding biology and nesting success of palila

    USGS Publications Warehouse

    Pletschet, S.M.; Kelly, J.F.

    1990-01-01

    We studied the breeding biology of Palila (Loxioides bailleui ) at 85 nests from 20 April to 14 September 1988. Eggs were laid over a 139-day period and incubation averaged 16.6 days. The female incubated 85.2% of daylight hours and males fed incubating females. Modal clutch size was 2 (x super(-) = 2.0) and an average of 1.4 nestlings fledged per successful nest. Nestlings were in the nest an average of 25.3 days. Both females and males fed nestlings with the rate of feeding decreasing as the nestlings grew older. Palila nesting success was 25%, reduced primarily by hatching failure and depredation of nestlings. Hatching failure, due to inviable eggs or desertion, occurred in 41% of nests with eggs (55% of nest mortality). Egg depredation was rare (5% of nest mortality). Inbreeding and low food availability are postulated as the major causes for poor hatching success.

  12. Quantifying avian nest survival along an urbanization gradient using citizen- and scientist-generated data.

    PubMed

    Ryder, Thomas B; Reitsma, Robert; Evans, Brian; Marra, Peter P

    2010-03-01

    Despite the increasing pace of urbanization little is known about the factors that limit bird populations (i.e., population-level processes) within the urban/suburban land-use matrix. Here, we report rates of nest survival within the matrix of an urban land-use gradient in the greater Washington, D.C., USA, area for five common songbirds using data collected by scientists and citizens as part of a project called Neighborhood Nestwatch. Using program MARK, we modeled the effects of species, urbanization at multiple spatial scales (canopy cover and impervious surface), and observer (citizen vs. scientist) on nest survival of four open-cup and one cavity-nesting species. In addition, artificial nests were used to determine the relative impacts of specific predators along the land-use gradient. Our results suggest that predation on nests within the land-use matrix declines with urbanization but that there are species-specific differences. Moreover, variation in nest survival among species was best explained by urbanization metrics measured at larger "neighborhood" spatial scales (e.g., 1000 m). Trends were supported by data from artificial nests and suggest that variable predator communities (avian vs. mammalian) are one possible mechanism to explain differential nest survival. In addition, we assessed the quality of citizen science data and show that citizens had no negative effect on nest survival and provided estimates of nest survival comparable to Smithsonian biologists. Although birds nesting within the urban matrix experienced higher nest survival, individuals also faced a multitude of other challenges such as contaminants and invasive species, all of which could reduce adult survival.

  13. Archiving California’s historical duck nesting data

    USGS Publications Warehouse

    Ackerman, Joshua T.; Herzog, Mark P.; Brady, Caroline; Eadie, John M.; Yarris, Greg S.

    2015-07-14

    With the conclusion of this project, most duck nest data have been entered, but all nest-captured hen data and other breeding waterfowl data that were outside the scope of this project have still not been entered and electronically archived. Maintaining an up-to-date archive will require additional resources to archive and enter the new duck nest data each year in an iterative process. Further, data proofing should be conducted whenever possible, and also should be considered an iterative process as there was sometimes missing data that could not be filled in without more direct knowledge of specific projects. Despite these disclaimers, this duck data archive represents a massive and useful dataset to inform future research and management questions.

  14. Archiving California’s historical duck nesting data

    USGS Publications Warehouse

    Ackerman, Joshua T.; Herzog, Mark P.; Brady, Caroline; Eadie, John M.; Yarris, Greg S.

    2015-01-01

    With the conclusion of this project, most duck nest data have been entered, but all nest-captured hen data and other breeding waterfowl data that were outside the scope of this project have still not been entered and electronically archived. Maintaining an up-to-date archive will require additional resources to archive and enter the new duck nest data each year in an iterative process. Further, data proofing should be conducted whenever possible, and also should be considered an iterative process as there was sometimes missing data that could not be filled in without more direct knowledge of specific projects. Despite these disclaimers, this duck data archive represents a massive and useful dataset to inform future research and management questions.

  15. ABCs: A Basic Guide to Early Care and Education in New York City. Child Care Primer Series, 2002.

    ERIC Educational Resources Information Center

    Child Care, Inc., New York, NY.

    This primer presents key facts about child care and early education in New York City and delineates issues related to the current status of child care and early education. Specifically, the primer provides information on the number of children served by child care programs, Head Start, the Universal Prekindergarten program, preschool special…

  16. Waterbird nest density and nest survival in rice fields of southwestern Louisiana

    USGS Publications Warehouse

    Pierluissi, S.; King, Sammy L.; Kaller, Michael D.

    2010-01-01

    Rice fields in southwestern Louisiana provide breeding habitat for several waterbird species; however, little is known about nest density, nest survival and the importance of landscape context of rice fields in determining breeding activity. In 2004, 42 rice fields were searched for nests, and 40 were searched in 2005. Land uses surrounding rice fields, including irrigation canals, trees, crawfish ponds, rice, fallow and soybean fields, were examined to determine influence on nest density and survival. Nest densities were 13.5-16.0 nests/km2 for Purple Gallinules (Porphyrio martinica), 3.0-13.7 nests/km2 for Fulvous Whistling Ducks (Dendrocygna bicolor), 2.6-2.8 nests/km2 for Common Moorhens (Gallinula chloropus), 0.3-0.92 nests/km2 for Least Bitterns (Ixobrychus exilisi) and 0-0.6 nests/km2 for Mottled Ducks (Anas fulvigula). Nest survival was 52-79% for Purple Gallinules and 39-43% for Fulvous Whistling Ducks. Apparent nest success of Common Moorhens was 73-75%, 83% for Least Bitterns and 33% for Mottled Ducks. Purple Gallinule and Common Moorhen nest densities were highest in fields with a larger proportion of irrigation canals surrounding rice fields. Purple Gallinule nest densities were greater in fields devoid of trees and landscapes dominated by rice fields and pasture, rather than landscapes containing soybean fields and residential areas. Fulvous Whistling Duck nest densities were higher in agriculturally-dominated landscapes with few trees.

  17. Home improvement: C57BL/6J mice given more naturalistic nesting materials build better nests.

    PubMed

    Hess, Sarah E; Rohr, Stephanie; Dufour, Brett D; Gaskill, Brianna N; Pajor, Edmond A; Garner, Joseph P

    2008-11-01

    Environmental enrichment of laboratory mice can improve the quality of research, but debate arises over the means of enrichment and its ability to be used in a sterile environment. One important form of enrichment is nesting material. Mice in the wild build dome-shaped, complex, multilayered nests, but this behavior is not seen in the laboratory, perhaps due to inappropriate nesting material rather than the nest-building ability of the mice. Here we focus on the use of naturalistic nesting materials to test whether they improve nest quality through the use of a 'naturalistic nest score' system; we also focus on materials that can be sterilized and easily used in existing housing systems. We first determined whether C57BL/6J mice build naturalistic nests when given shredded paper strips. We then compared these shredded paper strips with other commonly used nesting enrichments (facial tissues and compressed cotton squares). Nests were scored for 6 d. We found that the shredded paper strips allowed the mice to build higher quality nests than those built with any of the other materials. Nests built with tissues were of intermediate quality, and nests built with compressed cotton squares were of poor quality, similar to those built by the control group. These results suggest that C57BL/6J mice given appropriate nesting materials can build nests similar to those built by their wild counterparts.

  18. Comparative in silico analysis of PCR primers suited for diagnostics and cloning of ammonia monooxygenase genes from ammonia-oxidizing bacteria.

    PubMed

    Junier, Pilar; Kim, Ok-Sun; Molina, Verónica; Limburg, Petra; Junier, Thomas; Imhoff, Johannes F; Witzel, Karl-Paul

    2008-04-01

    Over recent years, several PCR primers have been described to amplify genes encoding the structural subunits of ammonia monooxygenase (AMO) from ammonia-oxidizing bacteria (AOB). Most of them target amoA, while amoB and amoC have been neglected so far. This study compared the nucleotide sequence of 33 primers that have been used to amplify different regions of the amoCAB operon with alignments of all available sequences in public databases. The advantages and disadvantages of these primers are discussed based on the original description and the spectrum of matching sequences obtained. Additionally, new primers to amplify the almost complete amoCAB operon of AOB belonging to Betaproteobacteria (betaproteobacterial AOB), a primer pair for DGGE analysis of amoA and specific primers for gammaproteobacterial AOB, are also described. The specificity of these new primers was also evaluated using the databases of the sequences created during this study. PMID:18248438

  19. Development of a thermostabilized, one-step, nested, tetraplex PCR assay for simultaneous identification and differentiation of Entamoeba species, Entamoeba histolytica and Entamoeba dispar from stool samples.

    PubMed

    Foo, Phiaw Chong; Chan, Yean Yean; See Too, Wei Cun; Tan, Zi Ning; Wong, Weng Kin; Lalitha, Pattabhiraman; Lim, Boon Huat

    2012-09-01

    Entamoeba histolytica is the only Entamoeba species that causes amoebiasis in humans. Approximately 50 million people are infected, with 100, 000 deaths annually in endemic countries. Molecular diagnosis of Entamoeba histolytica is important to differentiate it from the morphologically identical Entamoeba dispar to avoid unnecessary medication. Conventional molecular diagnostic tests require trained personnel, cold-chain transportation and/or are storage-dependent, which make them user-unfriendly. The aim of this study was to develop a thermostabilized, one-step, nested, tetraplex PCR assay for the detection of Entamoeba histolytica, Entamoeba dispar and Entamoeba species in cold-chain-free and ready-to-use form. The PCR test was designed based on the Entamoeba small subunit rRNA (SSU-rRNA) gene, which detects the presence of any Entamoeba species, and simultaneously can be used to differentiate Entamoeba histolytica from Entamoeba dispar. In addition, a pair of primers was designed to serve as an internal amplification control to help identify inhibitors in the samples. All PCR reagents together with the designed primers were thermostabilized by lyophilization and were stable at 24 °C for at least 6 months. The limit of detection of the tetraplex PCR was found to be 39 pg DNA or 1000 cells for Entamoeba histolytica and 78 pg DNA or 1000 cells for Entamoeba dispar, and the specificity was 100 %. In conclusion, this cold-chain-free, thermostabilized, one-step, nested, multiplex PCR assay was found to be efficacious in differentiating Entamoeba histolytica from other non-pathogenic Entamoeba species.

  20. Detection and Quantification of the Entomopathogenic Fungal Endophyte Beauveria bassiana in Plants by Nested and Quantitative PCR.

    PubMed

    Garrido-Jurado, Inmaculada; Landa, Blanca B; Quesada-Moraga, Enrique

    2016-01-01

    The described protocol allows detecting as low as 10 fg the entomopathogenic fungal endophyte Beauveria bassiana in host plants by using a two-step nested PCR with the ITS1F/ITS4 and BB.fw and BB.rv primer pairs. On the other hand, a qPCR protocol using BB.fw and BB.rv primers is also available allowing the quantification of up to 26 fg of B. bassiana DNA per 20 ng of leaf DNA. PMID:27565498

  1. Multiplex Degenerate Primer Design for Targeted Whole Genome Amplification of Many Viral Genomes

    DOE PAGES

    Gardner, Shea N.; Jaing, Crystal J.; Elsheikh, Maher M.; Peña, José; Hysom, David A.; Borucki, Monica K.

    2014-01-01

    Background . Targeted enrichment improves coverage of highly mutable viruses at low concentration in complex samples. Degenerate primers that anneal to conserved regions can facilitate amplification of divergent, low concentration variants, even when the strain present is unknown. Results . A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping amplicons across multiple whole genomes is described. The new script, run_tiled_primers, is part of the PriMux software. Primers were designed for each segment of South American hemorrhagic fever viruses, tick-borne encephalitis, Henipaviruses, Arenaviruses, Filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Japanese encephalitis virus.more » Each group is highly diverse with as little as 5% genome consensus. Primer sets were computationally checked for nontarget cross reactions against the NCBI nucleotide sequence database. Primers for murine hepatitis virus were demonstrated in the lab to specifically amplify selected genes from a laboratory cultured strain that had undergone extensive passage in vitro and in vivo. Conclusions . This software should help researchers design multiplex sets of primers for targeted whole genome enrichment prior to sequencing to obtain better coverage of low titer, divergent viruses. Applications include viral discovery from a complex background and improved sensitivity and coverage of rapidly evolving strains or variants in a gene family.« less

  2. Insight into the Human DNA Primase Interaction with Template-Primer.

    PubMed

    Baranovskiy, Andrey G; Zhang, Yinbo; Suwa, Yoshiaki; Gu, Jianyou; Babayeva, Nigar D; Pavlov, Youri I; Tahirov, Tahir H

    2016-02-26

    DNA replication in almost all organisms depends on the activity of DNA primase, a DNA-dependent RNA polymerase that synthesizes short RNA primers of defined size for DNA polymerases. Eukaryotic and archaeal primases are heterodimers consisting of small catalytic and large accessory subunits, both of which are necessary for the activity. The mode of interaction of primase subunits with substrates during the various steps of primer synthesis that results in the counting of primer length is not clear. Here we show that the C-terminal domain of the large subunit (p58C) plays a major role in template-primer binding and also defines the elements of the DNA template and the RNA primer that interact with p58C. The specific mode of interaction with a template-primer involving the terminal 5'-triphosphate of RNA and the 3'-overhang of DNA results in a stable complex between p58C and the DNA/RNA duplex. Our results explain how p58C participates in RNA synthesis and primer length counting and also indicate that the binding site for initiating NTP is located on p58C. These findings provide notable insight into the mechanism of primase function and are applicable for DNA primases from other species.

  3. Human DNA polymerase α in binary complex with a DNA:DNA template-primer

    PubMed Central

    Coloma, Javier; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.

    2016-01-01

    The Polα/primase complex assembles the short RNA-DNA fragments for priming of lagging and leading strand DNA replication in eukaryotes. As such, the Polα polymerase subunit encounters two types of substrates during primer synthesis: an RNA:DNA helix and a DNA:DNA helix. The engagement of the polymerase subunit with the DNA:DNA helix has been suggested as the of basis for primer termination in eukaryotes. However, there is no structural information on how the Polα polymerase subunit actually engages with a DNA:DNA helix during primer synthesis. We present here the first crystal structure of human Polα polymerase subunit in complex with a DNA:DNA helix. Unexpectedly, we find that portion of the DNA:DNA helix in contact with the polymerase is not in a B-form but in a hybrid A-B form. Almost all of the contacts observed previously with an RNA primer are preserved with a DNA primer – with the same set of polymerase residues tracking the sugar-phosphate backbone of the DNA or RNA primer. Thus, rather than loss of specific contacts, the free energy cost of distorting DNA from B- to hybrid A-B form may augur the termination of primer synthesis in eukaryotes. PMID:27032819

  4. Multiplex Degenerate Primer Design for Targeted Whole Genome Amplification of Many Viral Genomes

    PubMed Central

    Gardner, Shea N.; Jaing, Crystal J.; Elsheikh, Maher M.; Peña, José; Hysom, David A.; Borucki, Monica K.

    2014-01-01

    Background. Targeted enrichment improves coverage of highly mutable viruses at low concentration in complex samples. Degenerate primers that anneal to conserved regions can facilitate amplification of divergent, low concentration variants, even when the strain present is unknown. Results. A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping amplicons across multiple whole genomes is described. The new script, run_tiled_primers, is part of the PriMux software. Primers were designed for each segment of South American hemorrhagic fever viruses, tick-borne encephalitis, Henipaviruses, Arenaviruses, Filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Japanese encephalitis virus. Each group is highly diverse with as little as 5% genome consensus. Primer sets were computationally checked for nontarget cross reactions against the NCBI nucleotide sequence database. Primers for murine hepatitis virus were demonstrated in the lab to specifically amplify selected genes from a laboratory cultured strain that had undergone extensive passage in vitro and in vivo. Conclusions. This software should help researchers design multiplex sets of primers for targeted whole genome enrichment prior to sequencing to obtain better coverage of low titer, divergent viruses. Applications include viral discovery from a complex background and improved sensitivity and coverage of rapidly evolving strains or variants in a gene family. PMID:25157264

  5. Nested reverse transcriptase-polymerase chain reaction (RT-PCR) for typing ruminant pestiviruses: bovine viral diarrhea viruses and border disease virus.

    PubMed Central

    Fulton, R W; d'Offay, J M; Saliki, J T; Burge, L J; Helman, R G; Confer, A W; Bolin, S R; Ridpath, J F

    1999-01-01

    A nested reverse transcription (RT) polymerase chain reaction (PCR) assay was evaluated for differentiating reference bovine viral diarrhea virus (BVDV) strains, BVDV from diagnostic accessions, modified-live virus (MLV) BVDV strains in bovine viral vaccines, and a reference border disease virus (BDV). The detection level of this assay was compared to viral infection in cell culture. The PCR assay was used to distinguish 3 ruminant pestiviruses, types 1 and 2 BVDV, and type 3 BDV. The consensus (first) PCR assay detected all 3 ruminant pestiviruses, a result of the shared sequence homology. The consensus PCR product was subjected to a second (nested) PCR which used type-specific primers. The nested PCR was able to differentiate the 3 ruminant pestiviruses. Viral stocks of BVDV were diluted 10-fold and processed for the 2-step PCR assay. The sensitivity of this 2-step PCR assay was compared to viral infectivity in cell culture based on identical volumes of the system tested (cell culture assay and processing for RNA). The RT-PCR type-specific assay differentiated BVDV laboratory reference strains (12), diagnostic laboratory isolates (15), 2 MLV BVDV vaccine strains, and a BDV strain. The 30 ruminant pestiviruses typed included: (1) 27 reference strains and diagnostic laboratory isolates; 18 cytopathic (CP) type 1 strains, 3 CP type 2 strains, 3 noncytopathic (NCP) type 1 strains, and 3 NCP type 2 strains; (2) 2 MLV strains, type 1; and (3) 1 CP BDV type 3. The PCR assay had a detection limit of 10 TCID50/0.025 mL of virus when 3 separate BVDV were tested. This 2 step RT-PCR assay would be useful for the typing of ruminant pestiviruses, particularly BVDV isolates from the diagnostic laboratory. Images Figure 1. Figure 2. Figure 3. PMID:10534007

  6. DNest3: Diffusive Nested Sampling

    NASA Astrophysics Data System (ADS)

    Brewer, Brendon

    2016-04-01

    DNest3 is a C++ implementation of Diffusive Nested Sampling (ascl:1010.029), a Markov Chain Monte Carlo (MCMC) algorithm for Bayesian Inference and Statistical Mechanics. Relative to older DNest versions, DNest3 has improved performance (in terms of the sampling overhead, likelihood evaluations still dominate in general) and is cleaner code: implementing new models should be easier than it was before. In addition, DNest3 is multi-threaded, so one can run multiple MCMC walkers at the same time, and the results will be combined together.

  7. Concentric Nested Toroidal Inflatable Structures

    NASA Technical Reports Server (NTRS)

    Johnson, Christopher J.; Raboin, Jasen L.; Spexarth, Gary R.

    2010-01-01

    Assemblies comprising multiple limited- height toroidal inflatable structures nested in a concentric arrangement have been invented to obtain more design flexibility than can be obtained in single taller, wider toroidal inflatable structures (see figure). Originally intended for use as containers for habitats for humans in outer space or on remote planets, these and related prior inflatable structures could also be useful on Earth as lightweight, compactly stowable, portable special-purpose buildings that could be transported to remote locations and there inflated to full size and shape. In the case of a single inflatable toroidal structure, one important source of lack of design flexibility is the fact that an increase in outer diameter (which is sometimes desired) is necessarily accompanied by an increase in height (which is sometimes undesired). Increases in diameter and height can also cause difficulty in utilization of the resulting larger volume, in that it can become necessary to partition the volume by means of walls and floors, and features (e.g., stairs or ladders) must be added to enable vertical movement between floors. Moreover, ascending and descending between floors in a gravitational environment could pose unacceptable difficulty for the inhabitants under some circumstances. Another source of lack of design flexibility in a single toroidal inflatable structure is that for a given inflation pressure, an increase in the outer diameter of the structure necessarily entails an increase in the maximum stress in the structure. Because it is necessary to keep the maximum stress within the load-bearing capability of the structural materials, consistent with other aspects of the design, this may translate to a limit on the outer diameter. In an assembly comprising concentric nested toroidal structures, an increase in outer diameter does not necessarily entail an increase in height or a maximum stress in excess of the load-bearing capability of the structural

  8. Nested-cone transformer antenna

    DOEpatents

    Ekdahl, C.A.

    1991-05-28

    A plurality of conical transmission lines are concentrically nested to form an output antenna for pulsed-power, radio-frequency, and microwave sources. The diverging conical conductors enable a high power input density across a bulk dielectric to be reduced below a breakdown power density at the antenna interface with the transmitting medium. The plurality of cones maintain a spacing between conductors which minimizes the generation of high order modes between the conductors. Further, the power input feeds are isolated at the input while enabling the output electromagnetic waves to add at the transmission interface. Thus, very large power signals from a pulse rf, or microwave source can be radiated. 6 figures.

  9. Nested-cone transformer antenna

    DOEpatents

    Ekdahl, Carl A.

    1991-01-01

    A plurality of conical transmission lines are concentrically nested to form n output antenna for pulsed-power, radio-frequency, and microwave sources. The diverging conical conductors enable a high power input density across a bulk dielectric to be reduced below a breakdown power density at the antenna interface with the transmitting medium. The plurality of cones maintain a spacing between conductors which minimizes the generation of high order modes between the conductors. Further, the power input feeds are isolated at the input while enabling the output electromagnetic waves to add at the transmission interface. Thus, very large power signals from a pulse rf, or microwave source can be radiated.

  10. Nested and Dynamic Contract Boundaries

    NASA Astrophysics Data System (ADS)

    Strickland, T. Stephen; Felleisen, Matthias

    Previous work on software contracts assumes fixed and statically known boundaries between the parties to a contract. Implementations of contract monitoring systems rely on this assumption to explain the nature of contract violations and to assign blame to violators. In this paper, we explain how to implement arbitrary, nested, and dynamic contract boundaries with two examples. First, we add nestable contract regions to a static, first-order module system. Second, we show that even a dynamic, higher-order, and hierarchical module system can be equipped with software contracts that support precise blame assignment.

  11. PrimerDesign-M: A multiple-alignment based multiple-primer design tool for walking across variable genomes

    SciTech Connect

    Yoon, Hyejin; Leitner, Thomas

    2014-12-17

    Analyses of entire viral genomes or mtDNA requires comprehensive design of many primers across their genomes. In addition, simultaneous optimization of several DNA primer design criteria may improve overall experimental efficiency and downstream bioinformatic processing. To achieve these goals, we developed PrimerDesign-M. It includes several options for multiple-primer design, allowing researchers to efficiently design walking primers that cover long DNA targets, such as entire HIV-1 genomes, and that optimizes primers simultaneously informed by genetic diversity in multiple alignments and experimental design constraints given by the user. PrimerDesign-M can also design primers that include DNA barcodes and minimize primer dimerization. PrimerDesign-M finds optimal primers for highly variable DNA targets and facilitates design flexibility by suggesting alternative designs to adapt to experimental conditions.

  12. PrimerDesign-M: A multiple-alignment based multiple-primer design tool for walking across variable genomes

    DOE PAGES

    Yoon, Hyejin; Leitner, Thomas

    2014-12-17

    Analyses of entire viral genomes or mtDNA requires comprehensive design of many primers across their genomes. In addition, simultaneous optimization of several DNA primer design criteria may improve overall experimental efficiency and downstream bioinformatic processing. To achieve these goals, we developed PrimerDesign-M. It includes several options for multiple-primer design, allowing researchers to efficiently design walking primers that cover long DNA targets, such as entire HIV-1 genomes, and that optimizes primers simultaneously informed by genetic diversity in multiple alignments and experimental design constraints given by the user. PrimerDesign-M can also design primers that include DNA barcodes and minimize primer dimerization. PrimerDesign-Mmore » finds optimal primers for highly variable DNA targets and facilitates design flexibility by suggesting alternative designs to adapt to experimental conditions.« less

  13. Beam shaping for laser initiated optical primers

    NASA Astrophysics Data System (ADS)

    Lizotte, Todd E.

    2008-08-01

    Remington was one of the first firearm manufacturing companies to file a patent for laser initiated firearms, in 1969. Nearly 40 years later, the development of laser initiated firearms has not become a mainstream technology in the civilian market. Requiring a battery is definitely a short coming, so it is easy to see how such a concept would be problematic. Having a firearm operate reliably and the delivery of laser energy in an efficient manner to ignite the shock-sensitive explosive primer mixtures is a tall task indeed. There has been considerable research on optical element based methods of transferring or compressing laser energy to ignite primer charges, including windows, laser chip primers and various lens shaped windows to focus the laser energy. The focusing of laser light needs to achieve igniting temperatures upwards of >400°C. Many of the patent filings covering this type of technology discuss simple approaches where a single point of light might be sufficient to perform this task. Alternatively a multi-point method might provide better performance, especially for mission critical applications, such as precision military firearms. This paper covers initial design and performance test of the laser beam shaping optics to create simultaneous multiple point ignition locations and a circumferential intense ring for igniting primer charge compounds. A simple initial test of the ring beam shaping technique was evaluated on a standard large caliber primer to determine its effectiveness on igniting the primer material. Several tests were conducted to gauge the feasibility of laser beam shaping, including optic fabrication and mounting on a cartridge, optic durability and functional ignition performance. Initial data will be presented, including testing of optically elements and empirical primer ignition / burn analysis.

  14. Estimating nest success: when Mayfield wins

    USGS Publications Warehouse

    Johnson, D.H.; Shaffer, T.L.

    1990-01-01

    The Apparent estimator of nest success may be severely biased because unsuccessful nests are less likely to be found than are successful nests. The Mayfield estimator is a preferred alternative. The situation is somewhat different for nests in colonies or on islands because of greater visibility of nests, higher synchrony of nesting, and often higher hatch rates than dispersed mainland nests. Also, destruction is more likely to occur catastrophically, which violates an assumption of the Mayfield method that the mortality rate is constant. By simulation we investigated the performance of the Apparent and Mayfield estimators under a variety of circumstances.We found that when mortality rate was constant, the Mayfield estimator generally performed well regardless of whether or not nesting was synchronous or whether mortality was high or low. The Apparent estimator required more searches and higher detectability of nests. When mortality was mostly catastrophic, the Mayfield method performed poorly. The Apparent method was better, but high levels of detectability were needed for accurate estimates. We reached similar conclusions for attempts to estimate the number of nests initiated.

  15. Nest guarding from observation blinds: strategy for improving Puerto Rican parrot nest success

    USGS Publications Warehouse

    Lindsey, G.D.

    1992-01-01

    The effectiveness of 17 yr of nestguarding from observation blinds for increasing reproductive success of the endangered Puerto Rican Parrot (Amazona vittata) is described. As personnel and time allowed, active nests were guarded part-time during the nest site exploration and selection s stage of the breeding cycle, and part-time to full-time when a nest contained eggs or chicks. Biologists identified nine categories of threat to the success of parrot nests. Since 1973, a minimum of 20 nests, which otherwise would have failed, successfully produced fledglings as a direct result of nest guarding and intervention. Nest success averaged 66% with nest guarding compared to an estimated 38% without guarding. Nest guarding from blinds can help maintain a wild population of a critically endangered species while other management techniques are being developed to stimulate population growth.

  16. Nest Mosquito Trap quantifies contact rates between nesting birds and mosquitoes

    PubMed Central

    Caillouët, Kevin A.; Riggan, Anna E.; Rider, Mark; Bulluck, Lesley P.

    2012-01-01

    Accurate estimates of host-vector contact rates are required for precise determination of arbovirus transmission intensity. We designed and tested a novel mosquito collection device, the Nest Mosquito Trap (NMT), to collect mosquitoes as they attempt to feed on unrestrained nesting birds in artificial nest boxes. In the laboratory, the NMT collected nearly one-third of the mosquitoes introduced to the nest boxes. We then used these laboratory data to estimate our capture efficiency of field-collected bird-seeking mosquitoes collected over 66 trap nights. We estimated that 7.5 mosquitoes per trap night attempted to feed on nesting birds in artificial nest boxes. Presence of the NMT did not have a negative effect on avian nest success when compared to occupied nest boxes that were not sampled with the trap. Future studies using the NMT may elucidate the role of nestlings in arbovirus transmission and further refine estimates of nesting bird and vector contact rates. PMID:22548555

  17. Comparative evaluation of the GP5+/6+, MY09/11 and PGMY09/11 primer sets for HPV detection by PCR in oral squamous cell carcinomas.

    PubMed

    Erhart, Sibele Morais Miyata; Rivero, Elena Riet Correa; Bazzo, Maria Luiza; Onofre, Alexandre Sherlley Casimiro

    2016-02-01

    The aim of this study was to evaluate the use of GP5+/6+, MY09/11 and PGMY09/11 primer sets for the detection of human papillomavirus (HPV) DNA by single step polymerase chain reaction (PCR) and nested PCR in formalin-fixed and paraffin-embedded (FFPE) tissues from oral squamous cell carcinomas (OSCCs). DNA extracted from FFPE tissues were tested for amplification of the human beta globin gene with PCO3/4 primers. Positive samples for this gene were tested for HPV DNA using single step PCR with GP5+/6+, MY09/11 and PGMY09/11 primer sets. All negative samples at single step PCR with MY09/11 and PGMY09/11 were subjected to a further PCR with GP5+/6+ primers using the non-amplified product in the previously reactions (nested PCR) as samples. Among 26 samples, 23 were positive for the human beta globin gene and were considered viable for HPV DNA detection by PCR. Single step PCR with GP5+/6+ and MY09/11 primers and MY/GP+ nested PCR did not amplify HPV DNA in any samples. PGMY09/11 primers detected HPV DNA in 13.0% of OSCC cases and this rate was raise to 17.4% with the use of PGMY/GP+ nested PCR. According to our results the PGMY/GP+ nested PCR is the most appropriate primer set for the detection of HPV DNA using FFPE samples from OSCC.

  18. Studies on nest construction and nest microclimate of the Baya weaver, Ploceus philippinus (Linn.).

    PubMed

    Asokan, S; Ali, A Mohamed Samsoor; Nagarajan, R

    2008-05-01

    The nest construction pattern at different stages of nest and variations in the nest microclimate, i.e., temperature and light intensity were assessed in different nests of Baya weaver (Ploceus philippinus) between November 2002 and March 2003 in Nagapattinam and Tiruvarur District of Tamil Nadu, India. The Baya weaver constructed nests in palm (Borassus flabellifer), coconut (Cocos nucifera) and date palm trees (Phoneix psuilla) and majority of the nests were found in the solitary palm. The male bird only involved in the construction and took 18 days to construct a single nest. The birds spent different amount of working hours (in terms of days) for completing various stages of nests viz., wad, ring and helmet stage and in which the 'helmet stage took a maximum of eight days. Furthermore, totally eight active nests were selected and once in a week the variations in the nest microclimate was investigated with reference to atmospheric temperature and light intensity (two active nests) across day throughout the study period. The mean temperature of the nests ranged from 25 degrees C to 29 degrees C and light intensity varied between 25 Lux and 625 Lux. The analysis of variance (ANOVA and ANCOVA) indicated that the nest microclimate varied among the nests in different hr of a day PMID:18972698

  19. Remediation using nested, horizontal wells

    SciTech Connect

    Desantis, P.J.; Andrilenas, J.S.; Cheng, S.; Esler, C.; Miller, R.S.; Lew, K.V.

    1995-11-01

    A pair of nested, horizontal wells (one for vapor extraction, one for groundwater extraction) were utilized to remediate a mixed aromatic volatile organic compound (AVOC) and halogenated volatile organic compound (HVOC) soil and groundwater plume. The project site is an operating gasoline service station located in Portland, Oregon. The site has low permeability soils, a thin unconfined aquifer, with a relatively steep groundwater gradient. Each of the nested horizontal wells was drilled using the continuous borehole directional drilling method. The wells are each 110 meters long employing 73 meters of pre-packed well screen. The groundwater extraction well was pumped via vacuum-enhanced methods utilizing a two-pump configuration to eliminate the need for installation of a pump within the horizontal well and to increase both flow and radius of influence in fine-grained soils. Two groundwater models, a 2D analytical model and a finite element model, were utilized to analyze the potential and actual performance of the well. Of the two models used, the finite element model has produced results closely matching the actual performance of the well. After one year of operation, both AVOCs and HVOCs concentrations have been reduced by between 70 to 100%. All but two downgradient site wells have met cleanup standards for AVOCs; all have met substantial compliance for HVOCs.

  20. Chemical profiles of body surfaces and nests from six Bornean stingless bee species.

    PubMed

    Leonhardt, Sara Diana; Blüthgen, Nico; Schmitt, Thomas

    2011-01-01

    Stingless bees (Apidae: Meliponini) are the most diverse group of Apid bees and represent common pollinators in tropical ecosystems. Like honeybees they live in large eusocial colonies and rely on complex chemical recognition and communication systems. In contrast to honeybees, their ecology and especially their chemical ecology have received only little attention, particularly in the Old World. We previously have analyzed the chemical profiles of six paleotropical stingless bee species from Borneo and revealed the presence of species-specific cuticular terpenes- an environmentally derived compound class so far unique among social insects. Here, we compared the bees' surface profiles to the chemistry of their nest material. Terpenes, alkanes, and alkenes were the dominant compound groups on both body surfaces and nest material. However, bee profiles and nests strongly differed in their chemical composition. Body surfaces thus did not merely mirror nests, rendering a passive compound transfer from nests to bees unlikely. The difference between nests and bees was particularly pronounced when all resin-derived compounds (terpenes) were excluded and only genetically determined compounds were considered. When terpenes were included, bee profiles and nest material still differed, because whole groups of terpenes (e.g., sesquiterpenes) were found in nest material of some species, but missing in their chemical profile, indicating that bees are able to influence the terpene composition both in their nests and on their surfaces. PMID:21165680

  1. Chemical profiles of body surfaces and nests from six Bornean stingless bee species.

    PubMed

    Leonhardt, Sara Diana; Blüthgen, Nico; Schmitt, Thomas

    2011-01-01

    Stingless bees (Apidae: Meliponini) are the most diverse group of Apid bees and represent common pollinators in tropical ecosystems. Like honeybees they live in large eusocial colonies and rely on complex chemical recognition and communication systems. In contrast to honeybees, their ecology and especially their chemical ecology have received only little attention, particularly in the Old World. We previously have analyzed the chemical profiles of six paleotropical stingless bee species from Borneo and revealed the presence of species-specific cuticular terpenes- an environmentally derived compound class so far unique among social insects. Here, we compared the bees' surface profiles to the chemistry of their nest material. Terpenes, alkanes, and alkenes were the dominant compound groups on both body surfaces and nest material. However, bee profiles and nests strongly differed in their chemical composition. Body surfaces thus did not merely mirror nests, rendering a passive compound transfer from nests to bees unlikely. The difference between nests and bees was particularly pronounced when all resin-derived compounds (terpenes) were excluded and only genetically determined compounds were considered. When terpenes were included, bee profiles and nest material still differed, because whole groups of terpenes (e.g., sesquiterpenes) were found in nest material of some species, but missing in their chemical profile, indicating that bees are able to influence the terpene composition both in their nests and on their surfaces.

  2. Primers on Special Education and Charter Schools: Compilation of Full Primer Set

    ERIC Educational Resources Information Center

    Ahearn, Eileen M.; Giovannetti, Elizabeth A.; Lange, Cheryl M.; Rhim, Lauren Morando; Warren, Sandra Hopfengardner

    2004-01-01

    This set of primers for charter school authorizers; charter school operators and state-level administrators has been developed to provide background information and resources for the "builders" of charter schools and policymakers to facilitate the successful inclusion of students with disabilities in charter schools. The primers open with a…

  3. Mitigation effectiveness for improving nesting success of greater sage-grouse influenced by energy development

    PubMed Central

    Kirol, Christopher P.; Sutphin, Andrew L.; Bond, Laura; Fuller, Mark R.; Maechtle, Thomas L.

    2015-01-01

    Sagebrush (Artemisia spp.) habitats being developed for oil and gas reserves are inhabited by sagebrush obligate species—including the greater sage-grouse (Centrocercus urophasianus; sage-grouse) that is currently being considered for protection under the U.S. Endangered Species Act. Numerous studies suggest increasing oil and gas development may exacerbate species extinction risks. Therefore, there is a great need for effective on-site mitigation to reduce impacts to co-occurring wildlife such as sage-grouse. Nesting success is a primary factor in avian productivity and declines in nesting success are also thought to be an important contributor to population declines in sage-grouse. From 2008 to 2011 we monitored 296 nests of radio-marked female sage-grouse in a natural gas (NG) field in the Powder River Basin, Wyoming, USA and compared nest survival in mitigated and non-mitigated development areas and relatively unaltered areas to determine if specific mitigation practices were enhancing nest survival. Nest survival was highest in relatively unaltered habitats followed by mitigated, and then non-mitigated NG areas. Reservoirs used for holding NG discharge water had the greatest support as having a direct relationship to nest survival. Within a 5 km2 area surrounding a nest, the probability of nest failure increased by about 15% for every 1.5 km increase in reservoir water edge. Reducing reservoirs was a mitigation focus and sage-grouse nesting in mitigated areas were exposed to almost half of the amount of water edge compared to those in non-mitigated areas. Further, we found that an increase in sagebrush cover was positively related to nest survival. Consequently, mitigation efforts focused on reducing reservoir construction and reducing surface disturbance, especially when the surface disturbance results in sagebrush removal, are important to enhancing sage-grouse nesting success. PMID:26366042

  4. Mitigation effectiveness for improving nesting success of greater sage-grouse influenced by energy development

    USGS Publications Warehouse

    Kirol, Christopher P.; Sutphin, Andrew L.; Bond, Laura S.; Fuller, Mark R.; Maechtle, Thomas L.

    2015-01-01

    Sagebrush Artemisia spp. habitats being developed for oil and gas reserves are inhabited by sagebrush obligate species — including the greater sage-grouse Centrocercus urophasianus (sage-grouse) that is currently being considered for protection under the U.S. Endangered Species Act. Numerous studies suggest increasing oil and gas development may exacerbate species extinction risks. Therefore, there is a great need for effective on-site mitigation to reduce impacts to co-occurring wildlife such as sage-grouse. Nesting success is a primary factor in avian productivity and declines in nesting success are also thought to be an important contributor to population declines in sage-grouse. From 2008 to 2011 we monitored 296 nests of radio-marked female sage-grouse in a natural gas (NG) field in the Powder River Basin, Wyoming, USA, and compared nest survival in mitigated and non-mitigated development areas and relatively unaltered areas to determine if specific mitigation practices were enhancing nest survival. Nest survival was highest in relatively unaltered habitats followed by mitigated, and then non-mitigated NG areas. Reservoirs used for holding NG discharge water had the greatest support as having a direct relationship to nest survival. Within a 5-km2 area surrounding a nest, the probability of nest failure increased by about 15% for every 1.5 km increase in reservoir water edge. Reducing reservoirs was a mitigation focus and sage-grouse nesting in mitigated areas were exposed to almost half of the amount of water edge compared to those in non-mitigated areas. Further, we found that an increase in sagebrush cover was positively related to nest survival. Consequently, mitigation efforts focused on reducing reservoir construction and reducing surface disturbance, especially when the surface disturbance results in sagebrush removal, are important to enhancing sage-grouse nesting success.

  5. Touchdown nested multiplex PCR detection of Phytophthora cinnamomi and P. cambivora from French and English chestnut grove soils.

    PubMed

    Langrell, Stephen R H; Morel, Olivier; Robin, Cécile

    2011-07-01

    Soil borne Phytophthora cinnamomi and Phytophthora cambivora are considered the most pathogenic species associated with chestnut (Castanea sativa) decline in Europe. Mapping their incidence and distribution from nursery and plantation soils may offer valuable information for limiting spread. As conventional biological baiting and taxonomic confirmation is generally time consuming, labour, logistically and space intensive, we have focused on the development of a specific touchdown nested multiplex Polymerase Chain Reaction (PCR) approach for the simultaneous detection of both species direct from soil. Pre-existing and novel primers, based on Internal Transcribed Spacer (ITS) sequences, have been evaluated for their specificity and use in a multiplex capacity in various combinations. Coupled to this we have modified a mechanical lysis procedure for DNA extraction from up to 10 g of chestnut under storey soils (ranging from 0.5 to 25 μg DNA g(-1)fresh soil). Using serial dilutions and/or polyvinylpolypyrrolidone chromatography purification, both species have been successfully detected, in artificially and naturally infected soils. Levels of assay detection are comparable to other Phytophthora species where PCR based diagnostic systems have been reported. A qualitative evaluation of this approach against conventional baiting is presented.

  6. Establishment of a porcine parvovirus (PPV) DNA standard and evaluation of a new lightcycler nested-PCR assay for detection of PPV.

    PubMed

    Prikhod'ko, Grigori G; Reyes, Herbert; Vasilyeva, Irina; Busby, Thomas F

    2003-07-01

    Porcine parvovirus (PPV) is a major causative agent in a syndrome of reproductive failure in swine. In validation (viral clearance) studies, PPV is a model of non-enveloped viruses and is widely used instead of human parvovirus B19, which causes a variety of illnesses including erythema infectiosum (fifth disease) in children and hydrops fetalis in pregnant women. To improve the sensitivity of current PCR-based assays for detection of PPV and to standardize the quantification of PPV, we have developed a lightcycler (LC) nested-PCR (nPCR) assay and constructed a PPV DNA standard evaluated in the LC nPCR assay. The PPV DNA standard, a plasmid termed pPPV, encodes a 3.3 kb PPV NADL-2 genome fragment. One genome copy equivalent (gce) of PPV equals 6.7 attograms of pPPV. The LC nPCR assay is a simple and specific method developed for detection of PPV strains but not any other viruses including members of Parvoviridae. The first 25-cycle PCR with outer primers chose by comparative analysis of 12 primers in 21 different combinations and a second 45-cycle PCR with inner primers amplify 286 and 251 bp fragments of PPV genome, respectively, for 40 min with a sensitivity of approximately 100 gce per assay (ml). By using the LC nPCR assay for analysis of PPV samples with known infectivity, we found that one 50% tissue culture infectious dose (TCID(50)) equals 1.93 +/- 0.24 log(10) gce. PMID:12821192

  7. A Primer on Campus Networks.

    ERIC Educational Resources Information Center

    Charp, Sylvia; Hines, Duffy

    1988-01-01

    Networking trends have accelerated the convergence of computers and communications. With this and the increasing need to share resources and information, educators are faced with many considerations concerning vendor selection, equipment compatibility, performance criteria, wire and cable specifications, and installation of selection systems to…

  8. PRIMEGENSw3: a web-based tool for high-throughput primer and probe design.

    PubMed

    Kushwaha, Garima; Srivastava, Gyan Prakash; Xu, Dong

    2015-01-01

    Highly specific and efficient primer and probe design has been a major hurdle in many high-throughput techniques. Successful implementation of any PCR or probe hybridization technique depends on the quality of primers and probes used in terms of their specificity and cross-hybridization. Here we describe PRIMEGENSw3, a set of web-based utilities for high-throughput primer and probe design. These utilities allow users to select genomic regions and to design primer/probe for selected regions in an interactive, user-friendly, and automatic fashion. The system runs the PRIMEGENS algorithm in the back-end on the high-performance server with the stored genomic database or user-provided custom database for cross-hybridization check. Cross-hybridization is checked not only using BLAST but also by checking mismatch positions and energy calculation of potential hybridization hits. The results can be visualized online and also can be downloaded. The average success rate of primer design using PRIMEGENSw3 is ~90 %. The web server also supports primer design for methylated sequences, which is used in epigenetic studies. Stand-alone version of the software is also available for download at the website.

  9. Primerize: automated primer assembly for transcribing non-coding RNA domains

    PubMed Central

    Tian, Siqi; Yesselman, Joseph D.; Cordero, Pablo; Das, Rhiju

    2015-01-01

    Customized RNA synthesis is in demand for biological and biotechnological research. While chemical synthesis and gel or chromatographic purification of RNA is costly and difficult for sequences longer than tens of nucleotides, a pipeline of primer assembly of DNA templates, in vitro transcription by T7 RNA polymerase and kit-based purification provides a cost-effective and fast alternative for preparing RNA molecules. Nevertheless, designing template primers that optimize cost and avoid mispriming during polymerase chain reaction currently requires expert inspection, downloading specialized software or both. Online servers are currently not available or maintained for the task. We report here a server named Primerize that makes available an efficient algorithm for primer design developed and experimentally tested in our laboratory for RNA domains with lengths up to 300 nucleotides. Free access: http://primerize.stanford.edu. PMID:25999345

  10. Copper accumulation by stickleback nests containing spiggin.

    PubMed

    Pinho, G L L; Martins, C M G; Barber, I

    2016-07-01

    The three-spined stickleback is a ubiquitous fish of marine, brackish and freshwater ecosystems across the Northern hemisphere that presents intermediate sensitivity to copper. Male sticklebacks display a range of elaborate reproductive behaviours that include nest construction. To build the nests, each male binds nesting material together using an endogenous glycoprotein nesting glue, known as 'spiggin'. Spiggin is a cysteine-rich protein and, therefore, potentially binds heavy metals present in the environment. The aim of this study was to investigate the capacity of stickleback nests to accumulate copper from environmental sources. Newly built nests, constructed by male fish from polyester threads in laboratory aquaria, were immersed in copper solutions ranging in concentration from 21.1-626.6 μg Cu L(-1). Bundles of polyester threads from aquaria without male fish were also immersed in the same copper solutions. After immersion, nests presented higher amounts of copper than the thread bundles, indicating a higher capacity of nests to bind this metal. A significant, positive correlation between the concentration of copper in the exposure solution and in the exposed nests was identified, but there was no such relationship for thread bundles. Since both spiggin synthesis and male courtship behaviour are under the control of circulating androgens, we predicted that males with high courtship scores would produce and secrete high levels of the spiggin protein. In the present study, nests built by high courtship score males accumulated more copper than those built by low courtship score males. Considering the potential of spiggin to bind metals, the positive relationship between fish courtship and spiggin secretion seems to explain the higher amount of copper on the nests from the fish showing high behaviour scores. Further work is now needed to determine the consequences of the copper binding potential of spiggin in stickleback nests for the health and survival of

  11. Climate Change, Health, and Communication: A Primer.

    PubMed

    Chadwick, Amy E

    2016-01-01

    Climate change is one of the most serious and pervasive challenges facing us today. Our changing climate has implications not only for the ecosystems upon which we depend, but also for human health. Health communication scholars are well-positioned to aid in the mitigation of and response to climate change and its health effects. To help theorists, researchers, and practitioners engage in these efforts, this primer explains relevant issues and vocabulary associated with climate change and its impacts on health. First, this primer provides an overview of climate change, its causes and consequences, and its impacts on health. Then, the primer describes ways to decrease impacts and identifies roles for health communication scholars in efforts to address climate change and its health effects.

  12. Climate Change, Health, and Communication: A Primer.

    PubMed

    Chadwick, Amy E

    2016-01-01

    Climate change is one of the most serious and pervasive challenges facing us today. Our changing climate has implications not only for the ecosystems upon which we depend, but also for human health. Health communication scholars are well-positioned to aid in the mitigation of and response to climate change and its health effects. To help theorists, researchers, and practitioners engage in these efforts, this primer explains relevant issues and vocabulary associated with climate change and its impacts on health. First, this primer provides an overview of climate change, its causes and consequences, and its impacts on health. Then, the primer describes ways to decrease impacts and identifies roles for health communication scholars in efforts to address climate change and its health effects. PMID:26580230

  13. Nest predation risk influences a cavity-nesting passerine during the post-hatching care period.

    PubMed

    Yoon, Jongmin; Kim, Byung-Su; Joo, Eun-Jin; Park, Shi-Ryong

    2016-01-01

    Some nest predators visually assess parental activities to locate a prey nest, whereas parents modify fitness-related traits to reduce the probability of nest predation, and/or nestlings fledge early to escape the risky nest environment. Here, we experimentally tested if the parental and fledging behaviours of oriental tits (Parus minor) that bred in the nest-box varied with cavity conditions associated with nest predation risk during the nestling period. The entrance of experimental nest-boxes was enlarged to create a long-term risk soon after clutch competition. A short-term risk, using simulated playbacks with a coexisting control bird and avian nest predator sound, was simultaneously applied to the nest-boxes whether or not the long-term risk existed. We found that the parents reduced their hourly feeding trips, and the nestlings fledged early with the long-term risk, although the nest mortality of the two nest-box types was low and did not differ. While this study presents a portion of prey-predator interactions with the associated uncertainties, our results highlight that the entrance size of cavities for small hole-nesting birds may play an important role in determining their fitness-related traits depending upon the degree of perceived risk of nest predation. PMID:27553176

  14. Nest predation risk influences a cavity-nesting passerine during the post-hatching care period

    PubMed Central

    Yoon, Jongmin; Kim, Byung-Su; Joo, Eun-Jin; Park, Shi-Ryong

    2016-01-01

    Some nest predators visually assess parental activities to locate a prey nest, whereas parents modify fitness-related traits to reduce the probability of nest predation, and/or nestlings fledge early to escape the risky nest environment. Here, we experimentally tested if the parental and fledging behaviours of oriental tits (Parus minor) that bred in the nest-box varied with cavity conditions associated with nest predation risk during the nestling period. The entrance of experimental nest-boxes was enlarged to create a long-term risk soon after clutch competition. A short-term risk, using simulated playbacks with a coexisting control bird and avian nest predator sound, was simultaneously applied to the nest-boxes whether or not the long-term risk existed. We found that the parents reduced their hourly feeding trips, and the nestlings fledged early with the long-term risk, although the nest mortality of the two nest-box types was low and did not differ. While this study presents a portion of prey–predator interactions with the associated uncertainties, our results highlight that the entrance size of cavities for small hole-nesting birds may play an important role in determining their fitness-related traits depending upon the degree of perceived risk of nest predation. PMID:27553176

  15. Nests and nest sites of the San Miguel Island Song Sparrow

    USGS Publications Warehouse

    Kern, Michael D.; Sogge, Mark K.; Kern, Robert B.; Van Riper, Charles

    1993-01-01

    Nests and nest sites of the San Miguel Island (SMI) Song Sparrow (Melospiza melodia micronyx) are described; nests are compared with those of 16 other races of Song Sparrows. Bush lupins (Lupinus albifrons), coyote brush (Baccharis pilularis) and golden bush (Haplopappus venetus) were the shrubs used most commonly as nest sites by Song Sparrows on SMI. As a result of its location, the nest was effectively concealed from gray foxes (Urocyon littoralis), the major predator of this sparrow. Nest and nest site also moderated the combined chilling effects of cool air temperatures and strong northwesterly winds on the eggs and nestlings. Even in the absence of these moderating effects of the nest site, the energetic cost of incubation, estimated at 41-53% of the sparrow's resting metabolic rate, was modest. Twenty-nine percent of the canopy above the nest was open and as much as 73% of the nest cup was in the sun at midday, a time when surface temperatures of foliage, nest and nestlings sometimes exceeded 40 C. Whereas this exposure did not apparently reduce fledging success, it may explain why the incidence of addled eggs was so high in this population of Song Sparrows compared to others. Significant differences existed among races of Song Sparrows in the size, porosity and insulation of the nest. In most cases, these differences were not related to the latitude of the races' nesting areas.

  16. [Structure characteristics of natural nests and its implication to artificial nest frame design for Ciconia boyciana].

    PubMed

    Wei, Yi-qing; Cui, Guo-fa

    2014-12-01

    Artificial nest can improve the breeding success of birds in the field, and it has been proved to be more effective to endangered species. We surveyed the structure characteristics of natural nest and the status of the use of artificial nests for oriental white stork, Ciconia boyciana, in Honghe National Nature Reserve, Heilongjiang Province. Differences were investigated among the structure characteristics of the used and unused artificial nests, and natural nests based on one-way ANOVA. It was observed that significant differences in the diameter of nest branch, the vertical an- gle between nest branch, the height of the jointthe height of the nest above ground exited in different nest types. On account of the structure characteristics of the natural nests of C. boyciana, the suitable diameter of nest pillar for artificial nest frame should be 15.0-25.0 cm with the height of 5.0-12.0 m, which would be better if they were constructed by some acid-resistant materials, e.g., cement. The number of nest stands should be 3-4 individuals with the diameter of 9.0-12.0 cm, the vertical angle of 45 degrees-60 degrees, and the length of 90.0-140.0 cm.

  17. The design and function of birds' nests.

    PubMed

    Mainwaring, Mark C; Hartley, Ian R; Lambrechts, Marcel M; Deeming, D Charles

    2014-10-01

    All birds construct nests in which to lay eggs and/or raise offspring. Traditionally, it was thought that natural selection and the requirement to minimize the risk of predation determined the design of completed nests. However, it is becoming increasingly apparent that sexual selection also influences nest design. This is an important development as while species such as bowerbirds build structures that are extended phenotypic signals whose sole purpose is to attract a mate, nests contain eggs and/or offspring, thereby suggesting a direct trade-off between the conflicting requirements of natural and sexual selection. Nest design also varies adaptively in order to both minimize the detrimental effects of parasites and to create a suitable microclimate for parents and developing offspring in relation to predictable variation in environmental conditions. Our understanding of the design and function of birds' nests has increased considerably in recent years, and the evidence suggests that nests have four nonmutually exclusive functions. Consequently, we conclude that the design of birds' nests is far more sophisticated than previously realized and that nests are multifunctional structures that have important fitness consequences for the builder/s. PMID:25505520

  18. The design and function of birds' nests.

    PubMed

    Mainwaring, Mark C; Hartley, Ian R; Lambrechts, Marcel M; Deeming, D Charles

    2014-10-01

    All birds construct nests in which to lay eggs and/or raise offspring. Traditionally, it was thought that natural selection and the requirement to minimize the risk of predation determined the design of completed nests. However, it is becoming increasingly apparent that sexual selection also influences nest design. This is an important development as while species such as bowerbirds build structures that are extended phenotypic signals whose sole purpose is to attract a mate, nests contain eggs and/or offspring, thereby suggesting a direct trade-off between the conflicting requirements of natural and sexual selection. Nest design also varies adaptively in order to both minimize the detrimental effects of parasites and to create a suitable microclimate for parents and developing offspring in relation to predictable variation in environmental conditions. Our understanding of the design and function of birds' nests has increased considerably in recent years, and the evidence suggests that nests have four nonmutually exclusive functions. Consequently, we conclude that the design of birds' nests is far more sophisticated than previously realized and that nests are multifunctional structures that have important fitness consequences for the builder/s.

  19. Blue jays nest in an unusual structure

    USGS Publications Warehouse

    Muths, Erin L.; Lyons, Curtis P.; Sedgwick, James A.

    2007-01-01

    We describe a successful Blue Jay (Cyanocitta cristata) nest in an unusual structure on the side of a building.  The nest was located near the edge of the species' range along the Front Range of the Rocky Mountains in Colorado.  The nest was completely obvious, suggesting that the structure itself provided adequate cover and sercurity for the jays.  Blue Jays appear to be declining in some areas of the United States such as the Southeast.  Structures such as the one we describe may be more useful in attracting Blue Jays than the nesting platforms available commercially.

  20. The design and function of birds' nests

    PubMed Central

    Mainwaring, Mark C; Hartley, Ian R; Lambrechts, Marcel M; Deeming, D Charles

    2014-01-01

    All birds construct nests in which to lay eggs and/or raise offspring. Traditionally, it was thought that natural selection and the requirement to minimize the risk of predation determined the design of completed nests. However, it is becoming increasingly apparent that sexual selection also influences nest design. This is an important development as while species such as bowerbirds build structures that are extended phenotypic signals whose sole purpose is to attract a mate, nests contain eggs and/or offspring, thereby suggesting a direct trade-off between the conflicting requirements of natural and sexual selection. Nest design also varies adaptively in order to both minimize the detrimental effects of parasites and to create a suitable microclimate for parents and developing offspring in relation to predictable variation in environmental conditions. Our understanding of the design and function of birds' nests has increased considerably in recent years, and the evidence suggests that nests have four nonmutually exclusive functions. Consequently, we conclude that the design of birds' nests is far more sophisticated than previously realized and that nests are multifunctional structures that have important fitness consequences for the builder/s. PMID:25505520

  1. Do seasonal patterns of rat snake (Pantherophis obsoletus) and black racer (Coluber constrictor) activity predict avian nest predation?

    PubMed

    DeGregorio, Brett A; Weatherhead, Patrick J; Ward, Michael P; Sperry, Jinelle H

    2016-04-01

    Avian nest success often varies seasonally and because predation is the primary cause of nest failure, seasonal variation in predator activity has been hypothesized to explain seasonal variation in nest success. Despite the fact that nest predator communities are often diverse, recent evidence from studies of snakes that are nest predators has lent some support to the link between snake activity and nest predation. However, the strength of the relationship has varied among studies. Explaining this variation is difficult, because none of these studies directly identified nest predators, the link between predator activity and nest survival was inferred. To address this knowledge gap, we examined seasonal variation in daily survival rates of 463 bird nests (of 17 bird species) and used cameras to document predator identity at 137 nests. We simultaneously quantified seasonal activity patterns of two local snake species (N = 30 individuals) using manual (2136 snake locations) and automated (89,165 movements detected) radiotelemetry. Rat snakes (Pantherophis obsoletus), the dominant snake predator at the site (~28% of observed nest predations), were most active in late May and early June, a pattern reported elsewhere for this species. When analyzing all monitored nests, we found no link between nest predation and seasonal activity of rat snakes. When analyzing only nests with known predator identities (filmed nests), however, we found that rat snakes were more likely to prey on nests during periods when they were moving the greatest distances. Similarly, analyses of all monitored nests indicated that nest survival was not linked to racer activity patterns, but racer-specific predation (N = 17 nests) of filmed nests was higher when racers were moving the greatest distances. Our results suggest that the activity of predators may be associated with higher predation rates by those predators, but that those effects can be difficult to detect when nest predator communities

  2. Nested trampoline resonators for optomechanics

    NASA Astrophysics Data System (ADS)

    Weaver, M. J.; Pepper, B.; Luna, F.; Buters, F. M.; Eerkens, H. J.; Welker, G.; Perock, B.; Heeck, K.; de Man, S.; Bouwmeester, D.

    2016-01-01

    Two major challenges in the development of optomechanical devices are achieving a low mechanical and optical loss rate and vibration isolation from the environment. We address both issues by fabricating trampoline resonators made from low pressure chemical vapor deposition Si3N4 with a distributed Bragg reflector mirror. We design a nested double resonator structure with 80 dB of mechanical isolation from the mounting surface at the inner resonator frequency, and we demonstrate up to 45 dB of isolation at lower frequencies in agreement with the design. We reliably fabricate devices with mechanical quality factors of around 400 000 at room temperature. In addition, these devices were used to form optical cavities with finesse up to 181 000 ± 1000. These promising parameters will enable experiments in the quantum regime with macroscopic mechanical resonators.

  3. Automatic blocking of nested loops

    NASA Technical Reports Server (NTRS)

    Schreiber, Robert; Dongarra, Jack J.

    1990-01-01

    Blocked algorithms have much better properties of data locality and therefore can be much more efficient than ordinary algorithms when a memory hierarchy is involved. On the other hand, they are very difficult to write and to tune for particular machines. The reorganization is considered of nested loops through the use of known program transformations in order to create blocked algorithms automatically. The program transformations used are strip mining, loop interchange, and a variant of loop skewing in which invertible linear transformations (with integer coordinates) of the loop indices are allowed. Some problems are solved concerning the optimal application of these transformations. It is shown, in a very general setting, how to choose a nearly optimal set of transformed indices. It is then shown, in one particular but rather frequently occurring situation, how to choose an optimal set of block sizes.

  4. Arctic nesting geese: alaskan populations

    USGS Publications Warehouse

    Hupp, Jerry W.; Stehn, Robert A.; Ely, Craig R.; Derksen, Dirk V.

    1995-01-01

    While data for some areas are lacking, populations of greater white-fronted geese (Anser albifrons frontalis) and medium-sized Canada geese (Branta canadensis) in interior and northern Alaska appear stable or have increased (King and Derksen 1986). Although only a small number of lesser snow geese (Chen caerulescens caerulescens) nest in Alaska, substantial populations occur in Canada and Russia. Populations of Pacific black brant (B. bernicla nigricans), emperor geese (C. canagica), greater white-fronted geese, and cackling Canada geese (B.c. minima) on the Yukon-Kuskokwim Delta (YKD) of western Alaska have declined from their historical numbers and are the focus of special management efforts (USFWS 1989). In addition, populations of tule white-fronted geese (A.a. gambeli), Aleutian Canada geese (B.c. leucopareia), Vancouver Canada Geese (B.c. fulva), and dusky Canada geese (B.c. occidentalis) are of special concern because of their limited geographic distributions and small numbers.

  5. A new set of primers directed to 18S rRNA gene for molecular identification of Cryptosporidium spp. and their performance in the detection and differentiation of oocysts shed by synanthropic rodents.

    PubMed

    Silva, Sheila O S; Richtzenhain, Leonardo J; Barros, Iracema N; Gomes, Alessandra M M C; Silva, Aristeu V; Kozerski, Noemila D; de Araújo Ceranto, Jaqueline B; Keid, Lara B; Soares, Rodrigo M

    2013-11-01

    Cryptosporidium spp. are cosmopolitan protozoa that infect fishes, reptiles, amphibians, birds and mammals. More than 20 species are recognized within this genus. Rodents are a group of abundant and ubiquitous organisms that have been considered reservoirs of Cryptosporidium for humans and livestock. The aim of this study was to design specific primers for the gene encoding 18S rRNA, potentially capable of amplifying any species or genotype of Cryptosporidium spp. and evaluate the diagnostic attributes of the nested-PCR based on such probes. The primers were designed to amplify the shortest segment as possible to maximize the sensitivity of the test, but preserving the discriminatory potential of the amplified sequences for phylogenetic inferences. The nested-PCR standardized in this study (nPCR-SH) was compared in terms of sensitivity with another similar assay (nPCR-XIAO) that has been largely used for the detection and identification of Cryptosporidium spp. worldwide. We also aimed to molecularly characterize samples of Cryptosporidum spp. isolated from synanthropic rodents using these probes. Forty-five rodents were captured in urban areas of the municipality of Umuarama, Paraná State, Brazil. Fecal samples were submitted to three molecular tests (nested-PCRs), two of them targeted to the 18S rDNA gene (nPCR-SH and nPCR-XIAO) and the third targeted to the gene encoding actin (nPCR-actin). The nPCR-SH was tested positive on samples of Cryptosporidum parvum, Cryptosporidum andersoni, Cryptosporidum meleagridis, Cryptosporidum hominis, Cryptosporidum canis, and Cryptosporidum serpentis. Sixteen samples of rodents were positive by nPCR-SH, six by nPCR-XIAO and five by nPCR-actin. Sequencing of amplified fragments allowed the identification of Cryptosporidum muris in three samples of Rattus rattus, and two genotypes of Cryptosporidium, the genotypes mouse II and III. Cryptosporidium genotype mouse II was found in one sample of Mus musculus and genotype mouse III

  6. A simple and rapid method for detection of Trypanosoma evansi in the dromedary camel using a nested polymerase chain reaction

    PubMed Central

    Aradaib, Imadeldin E; Majid, Ali A

    2006-01-01

    A nested polymerase chain reaction (nPCR)-based assay, was developed and evaluated for rapid detection of Trypanosoma evansi in experimentally infected mice and naturally infected camels (Camelus dromedarius). Four oligonucleotide primers (TE1, TE2, TE3 and TE4), selected from nuclear repetitive gene of T. evansi, were designed and used for PCR amplifications. The first amplification, using a pair of outer primers TE1 and TE2, produced a 821-bp primary PCR product from T. evansi DNA. The second amplification, using nested (internal) pair of primers TE3 and TE4, produced a 270-bp PCR product. T. evansi DNAs extracted from blood samples of experimentally infected mice and naturally infected Sudanese breed of dromedary camels were detected by this nested PCR-based assay. The nested primers TE3 and TE4 increased the sensitivity of the PCR assay and as little as 10 fg of T. evansi DNA (equivalent to a single copy of the putative gene of the parasite) was amplified and visualized onto ethidium bromide-stained agarose gels. Amplification products were not detected when the PCR-based assay was applied to DNA from other blood parasites including Thieleria annulata, Babesia bigemina or nucleic acid free samples. Application of this nPCR-based assay to clinical samples resulted in direct detection of T. evansi from a variety of tissue samples collected from experimentally infected mice and blood from naturally infected camels. The described nPCR-based assay provides a valuable tool to study the epidemiology of T. evansi infection in camels and other susceptible animal populations. PMID:16712737

  7. Novel wide-range quantitative nested real-time PCR assay for Mycobacterium tuberculosis DNA: development and methodology.

    PubMed

    Takahashi, Teruyuki; Tamura, Masato; Asami, Yukihiro; Kitamura, Eiko; Saito, Kosuke; Suzuki, Tsukasa; Takahashi, Sachiko Nonaka; Matsumoto, Koichi; Sawada, Shigemasa; Yokoyama, Eise; Takasu, Toshiaki

    2008-05-01

    Previously, we designed an internally controlled quantitative nested real-time (QNRT) PCR assay for Mycobacterium tuberculosis DNA in order to rapidly diagnose tuberculous meningitis. This technique combined the high sensitivity of nested PCR with the accurate quantification of real-time PCR. In this study, we attempted to improve the original QNRT-PCR assay and newly developed the wide-range QNRT-PCR (WR-QNRT-PCR) assay, which is more accurate and has a wider detection range. For use as an internal-control "calibrator" to measure the copy number of M. tuberculosis DNA, an original new-mutation plasmid (NM-plasmid) was developed. It had artificial random nucleotides in five regions annealing specific primers and probes. The NM-plasmid demonstrated statistically uniform amplifications (F = 1.086, P = 0.774) against a range (1 to 10(5)) of copy numbers of mimic M. tuberculosis DNA and was regarded as appropriate for use as a new internal control in the WR-QNRT-PSR assay. In addition, by the optimization of assay conditions in WR-QNRT-PCR, two-step amplification of target DNA was completely consistent with the standard curve of this assay. Due to the development of the NM-plasmid as the new internal control, significantly improved quantitative accuracy and a wider detection range were realized with the WR-QNRT-PCR assay. In the next study, we will try to use this novel assay method with actual clinical samples and examine its clinical usefulness.

  8. Florida Harvester Ant Nest Architecture, Nest Relocation and Soil Carbon Dioxide Gradients

    PubMed Central

    Tschinkel, Walter R.

    2013-01-01

    Colonies of the Florida harvester ant, Pogonomyrmex badius, excavate species-typical subterranean nests up the 3 m deep with characteristic vertical distribution of chamber area/shape, spacing between levels and vertical arrangement of the ants by age and brood stage. Colonies excavate and occupy a new nest about once a year, and doing so requires that they have information about the depth below ground. Careful excavation and mapping of vacated and new nests revealed that there was no significant difference between the old and new nests in any measure of nest size, shape or arrangement. Colonies essentially built a replicate of the just-vacated nest (although details differed), and they did so in less than a week. The reason for nest relocation is not apparent. Tschinkel noted that the vertical distribution of chamber area, worker age and brood type was strongly correlated to the soil carbon dioxide gradient, and proposed that this gradient serves as a template for nest excavation and vertical distribution. To test this hypothesis, the carbon dioxide gradient of colonies that were just beginning to excavate a new nest was eliminated by boring 6 vent holes around the forming nest, allowing the soil CO2 to diffuse into the atmosphere and eliminating the gradient. Sadly, neither the nest architecture nor the vertical ant distribution of vented nests differed from either unvented control or from their own vacated nest. In a stronger test, workers excavated a new nest under a reversed carbon dioxide gradient (high concentration near the surface, low below). Even under these conditions, the new and old nests did not differ significantly, showing that the soil carbon dioxide gradient does not serve as a template for nest construction or vertical worker distribution. The possible importance of soil CO2 gradients for soil-dwelling animals is discussed. PMID:23555829

  9. Adaptive nest clustering and density-dependent nest survival in dabbling ducks

    USGS Publications Warehouse

    Ringelman, Kevin M.; Eadie, John M.; Ackerman, Joshua T.

    2014-01-01

    Density-dependent population regulation is observed in many taxa, and understanding the mechanisms that generate density dependence is especially important for the conservation of heavily-managed species. In one such system, North American waterfowl, density dependence is often observed at continental scales, and nest predation has long been implicated as a key factor driving this pattern. However, despite extensive research on this topic, it remains unclear if and how nest density influences predation rates. Part of this confusion may have arisen because previous studies have studied density-dependent predation at relatively large spatial and temporal scales. Because the spatial distribution of nests changes throughout the season, which potentially influences predator behavior, nest survival may vary through time at relatively small spatial scales. As such, density-dependent nest predation might be more detectable at a spatially- and temporally-refined scale and this may provide new insights into nest site selection and predator foraging behavior. Here, we used three years of data on nest survival of two species of waterfowl, mallards and gadwall, to more fully explore the relationship between local nest clustering and nest survival. Throughout the season, we found that the distribution of nests was consistently clustered at small spatial scales (˜50–400 m), especially for mallard nests, and that this pattern was robust to yearly variation in nest density and the intensity of predation. We demonstrated further that local nest clustering had positive fitness consequences – nests with closer nearest neighbors were more likely to be successful, a result that is counter to the general assumption that nest predation rates increase with nest density.

  10. Florida harvester ant nest architecture, nest relocation and soil carbon dioxide gradients.

    PubMed

    Tschinkel, Walter R

    2013-01-01

    Colonies of the Florida harvester ant, Pogonomyrmex badius, excavate species-typical subterranean nests up the 3 m deep with characteristic vertical distribution of chamber area/shape, spacing between levels and vertical arrangement of the ants by age and brood stage. Colonies excavate and occupy a new nest about once a year, and doing so requires that they have information about the depth below ground. Careful excavation and mapping of vacated and new nests revealed that there was no significant difference between the old and new nests in any measure of nest size, shape or arrangement. Colonies essentially built a replicate of the just-vacated nest (although details differed), and they did so in less than a week. The reason for nest relocation is not apparent. Tschinkel noted that the vertical distribution of chamber area, worker age and brood type was strongly correlated to the soil carbon dioxide gradient, and proposed that this gradient serves as a template for nest excavation and vertical distribution. To test this hypothesis, the carbon dioxide gradient of colonies that were just beginning to excavate a new nest was eliminated by boring 6 vent holes around the forming nest, allowing the soil CO2 to diffuse into the atmosphere and eliminating the gradient. Sadly, neither the nest architecture nor the vertical ant distribution of vented nests differed from either unvented control or from their own vacated nest. In a stronger test, workers excavated a new nest under a reversed carbon dioxide gradient (high concentration near the surface, low below). Even under these conditions, the new and old nests did not differ significantly, showing that the soil carbon dioxide gradient does not serve as a template for nest construction or vertical worker distribution. The possible importance of soil CO2 gradients for soil-dwelling animals is discussed.

  11. Spawning chronology, nest site selection and nest success of smallmouth bass during benign streamflow conditions

    USGS Publications Warehouse

    Dauwalter, D.C.; Fisher, W.L.

    2007-01-01

    We documented the nesting chronology, nest site selection and nest success of smallmouth bass Micropterus dolomieu in an upstream (4th order) and downstream (5th order) reach of Baron Fork Creek, Oklahoma. Males started nesting in mid-Apr. when water temperatures increased to 16.9 C upstream, and in late-Apr. when temperatures increased to 16.2 C downstream. Streamflows were low (77% upstream to 82% downstream of mean Apr. streamflow, and 12 and 18% of meanjun. streamflow; 47 and 55 y of record), and decreased throughout the spawning period. Larger males nested first upstream, as has been observed in other populations, but not downstream. Upstream, progeny in 62 of 153 nests developed to swim-up stage. Downstream, progeny in 31 of 73 nests developed to swim-up. Nesting densities upstream (147/km) and downstream (100/km) were both higher than any densities previously reported. Males selected nest sites with intermediate water depths, low water velocity and near cover, behavior that is typical of smallmouth bass. Documented nest failures resulted from human disturbance, angling, and longear sunfish predation. Logistic exposure models showed that water velocity at the nest was negatively related and length of the guarding male was positively related to nest success upstream. Male length and number of degree days were both positively related to nest success downstream. Our results, and those of other studies, suggest that biological factors account for most nest failures during benign (stable, low flow) streamflow conditions, whereas nest failures attributed to substrate mobility or nest abandonment dominate when harsh streamflow conditions (spring floods) coincide with the spawning season.

  12. Frog Foam Nest Protein Diversity and Synthesis.

    PubMed

    Hissa, Denise Cavalcante; Bezerra, Walderly Melgaço; Freitas, Cléverson Diniz Teixeira De; Ramos, Márcio Viana; Lopes, José Luiz De Souza; Beltramini, Leila Maria; Roberto, Igor Joventino; Cascon, Paulo; Melo, Vânia Maria Maciel

    2016-08-01

    Some amphibian species have developed a breeding strategy in which they deposit their eggs in stable foam nests to protect their eggs and larvae. The frog foam nests are rich in proteins (ranaspumin), especially surfactant proteins, involved in the production of the foam nest. Despite the ecological importance of the foam nests for evolution and species conservation, the biochemical composition, the long-term stability and even the origin of the components are still not completely understood. Recently we showed that Lv-RSN-1, a 23.5-kDa surfactant protein isolated from the nest of the frog Leptodacylus vastus, presents a structural conformation distinct from any protein structures yet reported. So, in the current study we aimed to reveal the protein composition of the foam nest of L. vastus and further characterize the Lv-RSN-1. Proteomic analysis showed the foam nest contains more than 100 of proteins, and that Lv-RSN-1 comprises 45% of the total proteins, suggesting a key role in the nest construction and stability. We demonstrated by Western blotting that Lv-RSN-1 is mainly produced only by the female in the pars convoluta dilata, which highlights the importance of the female preservation for conservation of species that depend on the production of foam nests in the early stages of development. Overall, our results showed the foam nest of L. vastus is composed of a great diversity of proteins and that besides Lv-RSN-1, the main protein in the foam, other proteins must have a coadjuvant role in building and stability of the nest.

  13. Frog Foam Nest Protein Diversity and Synthesis.

    PubMed

    Hissa, Denise Cavalcante; Bezerra, Walderly Melgaço; Freitas, Cléverson Diniz Teixeira De; Ramos, Márcio Viana; Lopes, José Luiz De Souza; Beltramini, Leila Maria; Roberto, Igor Joventino; Cascon, Paulo; Melo, Vânia Maria Maciel

    2016-08-01

    Some amphibian species have developed a breeding strategy in which they deposit their eggs in stable foam nests to protect their eggs and larvae. The frog foam nests are rich in proteins (ranaspumin), especially surfactant proteins, involved in the production of the foam nest. Despite the ecological importance of the foam nests for evolution and species conservation, the biochemical composition, the long-term stability and even the origin of the components are still not completely understood. Recently we showed that Lv-RSN-1, a 23.5-kDa surfactant protein isolated from the nest of the frog Leptodacylus vastus, presents a structural conformation distinct from any protein structures yet reported. So, in the current study we aimed to reveal the protein composition of the foam nest of L. vastus and further characterize the Lv-RSN-1. Proteomic analysis showed the foam nest contains more than 100 of proteins, and that Lv-RSN-1 comprises 45% of the total proteins, suggesting a key role in the nest construction and stability. We demonstrated by Western blotting that Lv-RSN-1 is mainly produced only by the female in the pars convoluta dilata, which highlights the importance of the female preservation for conservation of species that depend on the production of foam nests in the early stages of development. Overall, our results showed the foam nest of L. vastus is composed of a great diversity of proteins and that besides Lv-RSN-1, the main protein in the foam, other proteins must have a coadjuvant role in building and stability of the nest. PMID:27460953

  14. Nest marking behavior and chemical composition of olfactory cues involved in nest recognition in Megachile rotundata

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The present study examines the use of olfactory cues for nest recognition by Megachile rotundata (Fabricius) (Hymenoptera: Megachilidae), an economically important pollinator of seed alfalfa throughout western North America. In-nest observations revealed that nesting females drag their abdomen alon...

  15. Nesting phenology of marine turtles: insights from a regional comparative analysis on green turtle (Chelonia mydas).

    PubMed

    Dalleau, Mayeul; Ciccione, Stéphane; Mortimer, Jeanne A; Garnier, Julie; Benhamou, Simon; Bourjea, Jérôme

    2012-01-01

    Changes in phenology, the timing of seasonal activities, are among the most frequently observed responses to environmental disturbances and in marine species are known to occur in response to climate changes that directly affects ocean temperature, biogeochemical composition and sea level. We examined nesting seasonality data from long-term studies at 8 green turtle (Chelonia mydas) rookeries that include 21 specific nesting sites in the South-West Indian Ocean (SWIO). We demonstrated that temperature drives patterns of nesting seasonality at the regional scale. We found a significant correlation between mean annual Sea Surface Temperature (SST) and dates of peak nesting with rookeries exposed to higher SST having a delayed nesting peak. This supports the hypothesis that temperature is the main factor determining peak nesting dates. We also demonstrated a spatial synchrony in nesting activity amongst multiple rookeries in the northern part of the SWIO (Aldabra, Glorieuses, Mohéli, Mayotte) but not with the eastern and southern rookeries (Europa, Tromelin), differences which could be attributed to females with sharply different adult foraging conditions. However, we did not detect a temporal trend in the nesting peak date over the study period or an inter-annual relation between nesting peak date and SST. The findings of our study provide a better understanding of the processes that drive marine species phenology. The findings will also help to predict their ability to cope with climate change and other environmental perturbations. Despite demonstrating this spatial shift in nesting phenology, no trend in the alteration of nesting dates over more than 20 years was found.

  16. Nesting Phenology of Marine Turtles: Insights from a Regional Comparative Analysis on Green Turtle (Chelonia mydas)

    PubMed Central

    Dalleau, Mayeul; Ciccione, Stéphane; Mortimer, Jeanne A.; Garnier, Julie; Benhamou, Simon; Bourjea, Jérôme

    2012-01-01

    Changes in phenology, the timing of seasonal activities, are among the most frequently observed responses to environmental disturbances and in marine species are known to occur in response to climate changes that directly affects ocean temperature, biogeochemical composition and sea level. We examined nesting seasonality data from long-term studies at 8 green turtle (Chelonia mydas) rookeries that include 21 specific nesting sites in the South-West Indian Ocean (SWIO). We demonstrated that temperature drives patterns of nesting seasonality at the regional scale. We found a significant correlation between mean annual Sea Surface Temperature (SST) and dates of peak nesting with rookeries exposed to higher SST having a delayed nesting peak. This supports the hypothesis that temperature is the main factor determining peak nesting dates. We also demonstrated a spatial synchrony in nesting activity amongst multiple rookeries in the northern part of the SWIO (Aldabra, Glorieuses, Mohéli, Mayotte) but not with the eastern and southern rookeries (Europa, Tromelin), differences which could be attributed to females with sharply different adult foraging conditions. However, we did not detect a temporal trend in the nesting peak date over the study period or an inter-annual relation between nesting peak date and SST. The findings of our study provide a better understanding of the processes that drive marine species phenology. The findings will also help to predict their ability to cope with climate change and other environmental perturbations. Despite demonstrating this spatial shift in nesting phenology, no trend in the alteration of nesting dates over more than 20 years was found. PMID:23056527

  17. Internet Primer: Workshop Design and Objectives.

    ERIC Educational Resources Information Center

    Laverty, Corinne Y. C.

    1996-01-01

    Outlines the design, objectives, and evaluation of an introductory Internet workshop offered with library instruction classes in an electronic classroom at Queens University (Kingston, Ontario, Canada). Presents teaching tips and frequently-asked questions. The Internet primer handouts are appended. (AEF)

  18. Microsatellite primers for red drum (Sciaenops ocellatus)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this note, we document polymerase-chain-reaction (PCR) primer pairs for 101, nuclear-encoded microsatellites designed and developed from a red drum (Sciaenops ocellatus) genomic library. The 101 microsatellites (Genbank Accession Numbers EU015882-EU015982) were amplified successfully and used to...

  19. Theme: A Primer for Agricultural Education.

    ERIC Educational Resources Information Center

    Vaughn, Paul, Ed.; And Others

    1998-01-01

    Includes "A Primer for Agricultural Education" (Vaughn); "What Are the Goals and Purposes of Ag Ed?" (Case, Whitaker); "Basics of Supervised Experience" (Lee); "The FFA [Future Farmers of America]: Why Do We Have It?" (Case, Whitaker); "The Council: Providing Visionary Leadership" (Daniel, Vaughn); "Ag Communications" (Lockaby, Vernon); and…

  20. Microsatellite primer resource for Populus developed from

    SciTech Connect

    Yin, Tongming; Yang, Xiaohan; Gunter, Lee E; Tuskan, Gerald A; Wullschleger, Stan D; Huang, Prof. Minren; Li, Shuxian; Zhang, Xinye

    2008-01-01

    In this study, 148 428 simple sequence repeat (SSR) primer pairs were designed from the unambiguously mapped sequence scaffolds of the Nisqually-1 genome. The physical position of the priming sites were identified along each of the 19 Populus chromosomes, and it was specified whether the priming sequences belong to intronic, intergenic, exonic or UTR regions. A subset of 150 SSR loci were amplified and a high amplification success rate (72%) was obtained in P. tremuloides, which belongs to a divergent subgenus of Populus relative to Nisqually-1. PCR reactions showed that the amplification success rate of exonic primer pairs was much higher than that of the intronic/intergenic primer pairs. Applying ANOVA and regression analyses to the flanking sequences of microsatellites, the repeat lengths, the GC contents of the repeats, the repeat motif numbers, the repeat motif length and the base composition of the repeat motif, it was determined that only the base composition of the repeat motif and the repeat motif length significantly affect the microsatellite variability in P. tremuloides samples. The SSR primer resource developed in this study provides a database for selecting highly transferable SSR markers with known physical position in the Populus genome and provides a comprehensive genetic tool to extend the genome sequence of Nisqually-1 to genetic studies in different Populus species.

  1. A Primer on Simulation and Gaming.

    ERIC Educational Resources Information Center

    Barton, Richard F.

    In a primer intended for the administrative professions, for the behavioral sciences, and for education, simulation and its various aspects are defined, illustrated, and explained. Man-model simulation, man-computer simulation, all-computer simulation, and analysis are discussed as techniques for studying object systems (parts of the "real…

  2. Issues Primer. EEE708 Negotiated Study Program.

    ERIC Educational Resources Information Center

    Jennings, Leonie

    This issues primer is structured around a series of 20 contemporary concerns in the changing world of work and training in Australia in the early 1990s. It is part of the study materials for the one-semester distance education unit, Negotiated Study Program, in the Open Campus Program at Deakin University (Australia). Information on each issue is…

  3. A Push-pull Protocol to Reduce Colonization of Bird Nest Boxes by Honey Bees.

    PubMed

    Efstathion, Caroline A; Kern, William H

    2016-01-01

    Introduction of the invasive Africanized honey bee (AHB) into the Neotropics is a serious problem for many cavity nesting birds, specifically parrots. These bees select cavities that are suitable nest sites for birds, resulting in competition. The difficulty of removing bees and their defensive behavior makes a prevention protocol necessary. Here, we describe a push-pull integrated pest management protocol to deter bees from inhabiting bird boxes by applying a bird safe insecticide, permethrin, to repel bees from nest boxes, while simultaneously attracting them to pheromone-baited swarm traps. Shown here is an example experiment using Barn Owl nest boxes. This protocol successfully reduced colonization of Barn Owl nest boxes by Africanized honey bees. This protocol is flexible, allowing adjustments to accommodate a wide range of bird species and habitats. This protocol could benefit conservation efforts where AHB are located. PMID:27685258

  4. 7 CFR 29.1037 - Nested.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... INSPECTION Standards Official Standard Grades for Flue-Cured Tobacco (u.s. Types 11, 12, 13, 14 and Foreign Type 92) § 29.1037 Nested. Any lot of Types 11-14 tobacco which has been loaded, packed or arranged to conceal tobacco of inferior grade, quality or condition. Nested includes: (a) Any lot of tobacco...

  5. 7 CFR 29.1037 - Nested.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... INSPECTION Standards Official Standard Grades for Flue-Cured Tobacco (u.s. Types 11, 12, 13, 14 and Foreign Type 92) § 29.1037 Nested. Any lot of Types 11-14 tobacco which has been loaded, packed or arranged to conceal tobacco of inferior grade, quality or condition. Nested includes: (a) Any lot of tobacco...

  6. 7 CFR 29.1037 - Nested.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... INSPECTION Standards Official Standard Grades for Flue-Cured Tobacco (u.s. Types 11, 12, 13, 14 and Foreign Type 92) § 29.1037 Nested. Any lot of Types 11-14 tobacco which has been loaded, packed or arranged to conceal tobacco of inferior grade, quality or condition. Nested includes: (a) Any lot of tobacco...

  7. 7 CFR 29.1037 - Nested.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... INSPECTION Standards Official Standard Grades for Flue-Cured Tobacco (u.s. Types 11, 12, 13, 14 and Foreign Type 92) § 29.1037 Nested. Any lot of Types 11-14 tobacco which has been loaded, packed or arranged to conceal tobacco of inferior grade, quality or condition. Nested includes: (a) Any lot of tobacco...

  8. 7 CFR 29.1037 - Nested.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... INSPECTION Standards Official Standard Grades for Flue-Cured Tobacco (u.s. Types 11, 12, 13, 14 and Foreign Type 92) § 29.1037 Nested. Any lot of Types 11-14 tobacco which has been loaded, packed or arranged to conceal tobacco of inferior grade, quality or condition. Nested includes: (a) Any lot of tobacco...

  9. Dune vegetation fertilization by nesting sea turtles.

    PubMed

    Hannan, Laura B; Roth, James D; Ehrhart, Llewellyn M; Weishampel, John F

    2007-04-01

    Sea turtle nesting presents a potential pathway to subsidize nutrient-poor dune ecosystems, which provide the nesting habitat for sea turtles. To assess whether this positive feedback between dune plants and turtle nests exists, we measured N concentration and delta15N values in dune soils, leaves from a common dune plant (sea oats [Uniola paniculata]), and addled eggs of loggerhead (Caretta caretta) and green turtles (Chelonia mydas) across a nesting gradient (200-1050 nests/km) along a 40.5-km stretch of beach in east central Florida, USA. The delta15N levels were higher in loggerhead than green turtle eggs, denoting the higher trophic level of loggerhead turtles. Soil N concentration and delta15N values were both positively correlated to turtle nest density. Sea oat leaf tissue delta15N was also positively correlated to nest density, indicating an increased use of augmented marine-based nutrient sources. Foliar N concentration was correlated with delta15N, suggesting that increased nutrient availability from this biogenic vector may enhance the vigor of dune vegetation, promoting dune stabilization and preserving sea turtle nesting habitat.

  10. 7 CFR 29.6027 - Nested.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... INSPECTION Standards Definitions § 29.6027 Nested. Any tobacco which has been loaded, packed, or arranged to conceal foreign matter or tobacco of inferior grade, quality, or condition. Nested includes any lot...

  11. Teaching Ecological Interactions with Mud Dauber Nests.

    ERIC Educational Resources Information Center

    Matthews, Robert W.

    1997-01-01

    Describes the use of mud dauber wasp nests in laboratory activities in ecology and behavior and life science classes. Provides students with an opportunity to develop and practice basic skills including dissection, identification, observation, measurement, and communication. Discusses the life of mud daubers, obtaining and storing nests,…

  12. The evolution of eggshell cuticle in relation to nesting ecology.

    PubMed

    D'Alba, Liliana; Maia, Rafael; Hauber, Mark E; Shawkey, Matthew D

    2016-08-17

    Avian eggs are at risk of microbial infection prior to and during incubation. A large number of defence mechanisms have evolved in response to the severe costs imposed by these infections. The eggshell's cuticle is an important component of antimicrobial defence, and its role in preventing contamination by microorganisms in domestic chickens is well known. Nanometer-scale cuticular spheres that reduce microbial attachment and penetration have recently been identified on eggs of several wild avian species. However, whether these spheres have evolved specifically for antimicrobial defence is unknown. Here, we use comparative data on eggshell cuticular structure and nesting ecology to test the hypothesis that birds nesting in habitats with higher risk of infection (e.g. wetter and warmer) are more likely to evolve cuticular nanospheres on their eggshells than those nesting in less risky habitats. We found that nanostructuring, present in 54 of 296 analysed species, is the ancestral condition of avian eggshells and has been retained more often in taxa that nest in humid infection-prone environments, suggesting that they serve critical roles in antimicrobial egg defence. PMID:27488648

  13. Urine Nested Polymerase Chain Reaction in Neonatal Septicemia.

    PubMed

    Das, B K; Suri, Shipra; Nath, Gopal; Prasad, Rajniti

    2015-08-01

    This cross-sectional study was done to evaluate diagnostic efficacy of urine nested polymerase chain reaction (PCR) using broad-range 16SrDNA PCR-based amplification, followed by restriction analysis and sequencing in neonatal septicemia. The study included 50 babies; 48% had vaginal delivery, 46% were preterm, 20% had a history of prolonged rupture of membranes and 56% were low birth weight (≤2500 g). Clinical presentations were lethargy (96%), respiratory distress (80%) and bleeding diathesis (16%). Absolute neutrophil count <1800/mm(3) was observed in 60%, and positive C-reactive protein in 46%. Thirty neonates had positive blood culture, and Klebsiella pneumoniae (22%) was the predominant organism. Nested urine PCR was positive in 38 (76%) and detected bacterial DNA in 8 neonates with negative blood cultures. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy of nested PCR were 100, 60, 78.9, 100 and 84%, respectively, compared with blood culture. Nested PCR can detect most bacteria in single assay and identify unusual and unexpected causal agents.

  14. Evaluation of new primers for CSF1PO.

    PubMed

    Yoshida, K; Sekiguchi, K; Kasai, K; Sato, H; Seta, S; Sensabaugh, G F

    1997-01-01

    We describe new primers for the detection of the STR polymorphism at the CSF1PO locus. These primers have been designed to produce shorter amplicons (150-182 bp) than the primers in standard use (295-327 bp). The reliability of the new primers for CSF1PO typing has been demonstrated by testing on known samples and by sequence analysis. These primers are superior to the original primers with regard to electrophoretic resolution and utility for typing of severely degraded DNA.

  15. Evaluation of 16S rRNA Gene Primer Pairs for Monitoring Microbial Community Structures Showed High Reproducibility within and Low Comparability between Datasets Generated with Multiple Archaeal and Bacterial Primer Pairs

    PubMed Central

    Fischer, Martin A.; Güllert, Simon; Neulinger, Sven C.; Streit, Wolfgang R.; Schmitz, Ruth A.

    2016-01-01

    The application of next-generation sequencing technology in microbial community analysis increased our knowledge and understanding of the complexity and diversity of a variety of ecosystems. In contrast to Bacteria, the archaeal domain was often not particularly addressed in the analysis of microbial communities. Consequently, established primers specifically amplifying the archaeal 16S ribosomal gene region are scarce compared to the variety of primers targeting bacterial sequences. In this study, we aimed to validate archaeal primers suitable for high throughput next generation sequencing. Three archaeal 16S primer pairs as well as two bacterial and one general microbial 16S primer pairs were comprehensively tested by in-silico evaluation and performing an experimental analysis of a complex microbial community of a biogas reactor. The results obtained clearly demonstrate that comparability of community profiles established using different primer pairs is difficult. 16S rRNA gene data derived from a shotgun metagenome of the same reactor sample added an additional perspective on the community structure. Furthermore, in-silico evaluation of primers, especially those for amplification of archaeal 16S rRNA gene regions, does not necessarily reflect the results obtained in experimental approaches. In the latter, archaeal primer pair ArchV34 showed the highest similarity to the archaeal community structure compared to observed by the metagenomic approach and thus appears to be the appropriate for analyzing archaeal communities in biogas reactors. However, a disadvantage of this primer pair was its low specificity for the archaeal domain in the experimental application leading to high amounts of bacterial sequences within the dataset. Overall our results indicate a rather limited comparability between community structures investigated and determined using different primer pairs as well as between metagenome and 16S rRNA gene amplicon based community structure analysis

  16. Evaluation of 16S rRNA Gene Primer Pairs for Monitoring Microbial Community Structures Showed High Reproducibility within and Low Comparability between Datasets Generated with Multiple Archaeal and Bacterial Primer Pairs.

    PubMed

    Fischer, Martin A; Güllert, Simon; Neulinger, Sven C; Streit, Wolfgang R; Schmitz, Ruth A

    2016-01-01

    The application of next-generation sequencing technology in microbial community analysis increased our knowledge and understanding of the complexity and diversity of a variety of ecosystems. In contrast to Bacteria, the archaeal domain was often not particularly addressed in the analysis of microbial communities. Consequently, established primers specifically amplifying the archaeal 16S ribosomal gene region are scarce compared to the variety of primers targeting bacterial sequences. In this study, we aimed to validate archaeal primers suitable for high throughput next generation sequencing. Three archaeal 16S primer pairs as well as two bacterial and one general microbial 16S primer pairs were comprehensively tested by in-silico evaluation and performing an experimental analysis of a complex microbial community of a biogas reactor. The results obtained clearly demonstrate that comparability of community profiles established using different primer pairs is difficult. 16S rRNA gene data derived from a shotgun metagenome of the same reactor sample added an additional perspective on the community structure. Furthermore, in-silico evaluation of primers, especially those for amplification of archaeal 16S rRNA gene regions, does not necessarily reflect the results obtained in experimental approaches. In the latter, archaeal primer pair ArchV34 showed the highest similarity to the archaeal community structure compared to observed by the metagenomic approach and thus appears to be the appropriate for analyzing archaeal communities in biogas reactors. However, a disadvantage of this primer pair was its low specificity for the archaeal domain in the experimental application leading to high amounts of bacterial sequences within the dataset. Overall our results indicate a rather limited comparability between community structures investigated and determined using different primer pairs as well as between metagenome and 16S rRNA gene amplicon based community structure analysis

  17. Evaluation of 16S rRNA Gene Primer Pairs for Monitoring Microbial Community Structures Showed High Reproducibility within and Low Comparability between Datasets Generated with Multiple Archaeal and Bacterial Primer Pairs

    PubMed Central

    Fischer, Martin A.; Güllert, Simon; Neulinger, Sven C.; Streit, Wolfgang R.; Schmitz, Ruth A.

    2016-01-01

    The application of next-generation sequencing technology in microbial community analysis increased our knowledge and understanding of the complexity and diversity of a variety of ecosystems. In contrast to Bacteria, the archaeal domain was often not particularly addressed in the analysis of microbial communities. Consequently, established primers specifically amplifying the archaeal 16S ribosomal gene region are scarce compared to the variety of primers targeting bacterial sequences. In this study, we aimed to validate archaeal primers suitable for high throughput next generation sequencing. Three archaeal 16S primer pairs as well as two bacterial and one general microbial 16S primer pairs were comprehensively tested by in-silico evaluation and performing an experimental analysis of a complex microbial community of a biogas reactor. The results obtained clearly demonstrate that comparability of community profiles established using different primer pairs is difficult. 16S rRNA gene data derived from a shotgun metagenome of the same reactor sample added an additional perspective on the community structure. Furthermore, in-silico evaluation of primers, especially those for amplification of archaeal 16S rRNA gene regions, does not necessarily reflect the results obtained in experimental approaches. In the latter, archaeal primer pair ArchV34 showed the highest similarity to the archaeal community structure compared to observed by the metagenomic approach and thus appears to be the appropriate for analyzing archaeal communities in biogas reactors. However, a disadvantage of this primer pair was its low specificity for the archaeal domain in the experimental application leading to high amounts of bacterial sequences within the dataset. Overall our results indicate a rather limited comparability between community structures investigated and determined using different primer pairs as well as between metagenome and 16S rRNA gene amplicon based community structure analysis

  18. Evaluation of 16S rRNA Gene Primer Pairs for Monitoring Microbial Community Structures Showed High Reproducibility within and Low Comparability between Datasets Generated with Multiple Archaeal and Bacterial Primer Pairs.

    PubMed

    Fischer, Martin A; Güllert, Simon; Neulinger, Sven C; Streit, Wolfgang R; Schmitz, Ruth A

    2016-01-01

    The application of next-generation sequencing technology in microbial community analysis increased our knowledge and understanding of the complexity and diversity of a variety of ecosystems. In contrast to Bacteria, the archaeal domain was often not particularly addressed in the analysis of microbial communities. Consequently, established primers specifically amplifying the archaeal 16S ribosomal gene region are scarce compared to the variety of primers targeting bacterial sequences. In this study, we aimed to validate archaeal primers suitable for high throughput next generation sequencing. Three archaeal 16S primer pairs as well as two bacterial and one general microbial 16S primer pairs were comprehensively tested by in-silico evaluation and performing an experimental analysis of a complex microbial community of a biogas reactor. The results obtained clearly demonstrate that comparability of community profiles established using different primer pairs is difficult. 16S rRNA gene data derived from a shotgun metagenome of the same reactor sample added an additional perspective on the community structure. Furthermore, in-silico evaluation of primers, especially those for amplification of archaeal 16S rRNA gene regions, does not necessarily reflect the results obtained in experimental approaches. In the latter, archaeal primer pair ArchV34 showed the highest similarity to the archaeal community structure compared to observed by the metagenomic approach and thus appears to be the appropriate for analyzing archaeal communities in biogas reactors.