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Sample records for duplex allele-specific pcr

  1. A population-wide applicable HLA-DQ2 and DQ8 genotyping using DNA from dried blood spots and duplex allele-specific qPCR amplification.

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    Aguayo-Patrón, Sandra; Beltrán-Sauceda, Lizbeth; Calderón de la Barca, Ana María

    2016-11-01

    Genotyping of HLA-DQ2 and DQ8 haplotypes is important for diagnosis or for screening of early risk detection of celiac disease or type 1 diabetes. Usually, venous blood DNA extraction and expensive and time consuming amplification are used, that hinder population-wide studies. We assayed a friendly HLA-DQ2 and DQ8 genotyping procedure using a combination of DNA from dried blood spot (DBS) and duplex allele-specific qPCR amplification using SYBR Green. DNA was extracted using home-made buffers and compared to an extraction commercial kit. Duplex reactions by qPCR were designed using each Tm allele amplicon for reference samples (positive HLA-DQ2 or DQ8) with allele-specific primers. DBS samples from 558 children (7.99 ± 2.47 y) were collected. The DNA final yield obtained by the home-made extractive procedure was higher than from the commercial kit (1.11 ± 0.56 vs 0.23 ± 0.14 μg), while the quality was similar for both DNA samples. There was concordance in the amplification profiles for DNA samples obtained with both methods. All of four alleles from DQ2 and DQ8 haplotypes were accurately identified in duplex reactions. By using DBS samples and DNA extraction home-made procedure, the costs were reduced by 60%. The whole procedure is cost-effective for HLA-DQ2 and DQ8 genotyping.

  2. Simultaneous genotyping of single-nucleotide polymorphisms in alcoholism-related genes using duplex and triplex allele-specific PCR with two-step thermal cycles.

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    Shirasu, Naoto; Kuroki, Masahide

    2014-01-01

    We developed a time- and cost-effective multiplex allele-specific polymerase chain reaction (AS-PCR) method based on the two-step PCR thermal cycles for genotyping single-nucleotide polymorphisms in three alcoholism-related genes: alcohol dehydrogenase 1B, aldehyde dehydrogenase 2 and μ-opioid receptor. Applying MightyAmp(®) DNA polymerase with optimized AS-primers and PCR conditions enabled us to achieve effective and selective amplification of the target alleles from alkaline lysates of a human hair root, and simultaneously to determine the genotypes within less than 1.5 h using minimal lab equipment.

  3. [Allele-specific PCR and its application in forensic science].

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    Nie, Yan-chai; Wang, Bin; Zhao, Zi-qin; Zhou, Huai-gu

    2014-08-01

    Allele-specific polymerase chain reaction (AS-PCR) is a technique based on allele-specific primers, which can be used to analyze single nucleotide polymorphism (SNP) effectively including the transition, transversion and insertion/deletion polymorphism and has been exploited in the study of diseases research, molecular diagnosis, and forensic biological evidence. The article systematically reviews the principle, the detection methods, improvement of AS-PCR, and its research updates in the fields of autosome, Y chromosome and mitochondrial SNP, as well as its application in forensic science.

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

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    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.

  5. Epidemiological survey of Theileria parasite infection of cattle in Northeast China by allele-specific PCR.

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    Yu, Longzheng; Zhang, Shoufa; Liang, Wanfeng; Jin, Chunmei; Jia, Lijun; Luo, Yuzi; Li, Yan; Cao, Shinuo; Yamagishi, Junya; Nishikawa, Yoshifumi; Kawano, Suguru; Fujisaki, Kozo; Xuan, Xuenan

    2011-11-01

    An epidemiological survey on a Theileria parasite infection of cattle in Northeast China was carried out using allele-specific PCR and DNA sequence analysis of the major piroplasm surface protein (MPSP) gene. The results showed that 14 of 104 blood samples were positive for Theileria by PCR. Among the positive cases, co-infection with various combinations of C- and I-type parasites was detected in 12 samples; no B- and Thai-type parasites were detected by allele-specific PCR. Phylogenetic analysis based on the MPSP gene sequences revealed that Theileria parasites with the MPSP types 1, 2, and 4 were distributed in Northeast China.

  6. Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping.

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    Lee, Han B; Schwab, Tanya L; Koleilat, Alaa; Ata, Hirotaka; Daby, Camden L; Cervera, Roberto Lopez; McNulty, Melissa S; Bostwick, Hannah S; Clark, Karl J

    2016-06-01

    Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98-100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score

  7. Rapid ABO genotyping by high-speed droplet allele-specific PCR using crude samples.

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    Taira, Chiaki; Matsuda, Kazuyuki; Takeichi, Naoya; Furukawa, Satomi; Sugano, Mitsutoshi; Uehara, Takeshi; Okumura, Nobuo; Honda, Takayuki

    2017-03-13

    ABO genotyping has common tools for personal identification of forensic and transplantation field. We developed a new method based on a droplet allele-specific PCR (droplet-AS-PCR) that enabled rapid PCR amplification. We attempted rapid ABO genotyping using crude DNA isolated from dried blood and buccal cells. We designed allele-specific primers for three SNPs (at nucleotides 261, 526, and 803) in exons 6 and 7 of the ABO gene. We pretreated dried blood and buccal cells with proteinase K, and obtained crude DNAs without DNA purification. Droplet-AS-PCR allowed specific amplification of the SNPs at the three loci using crude DNA, with results similar to those for DNA extracted from fresh peripheral blood. The sensitivity of the methods was 5%-10%. The genotyping of extracted DNA and crude DNA were completed within 8 and 9 minutes, respectively. The genotypes determined by the droplet-AS-PCR method were always consistent with those obtained by direct sequencing. The droplet-AS-PCR method enabled rapid and specific amplification of three SNPs of the ABO gene from crude DNA treated with proteinase K. ABO genotyping by the droplet-AS-PCR has the potential to be applied to various fields including a forensic medicine and transplantation medical care. © 2017 Wiley Periodicals, Inc.

  8. Allele-specific PCR detection of sweet cherry self-incompatibility (S) alleles S1 to S16 using consensus and allele-specific primers.

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    Sonneveld, T; Tobutt, K R; Robbins, T P

    2003-10-01

    PCR-based identification of all 13 known self-incompatibility (S) alleles of sweet cherry is reported. Two pairs of consensus primers were designed from our previously published cDNA sequences of S(1) to S(6) S-RNases, the stylar components of self-incompatibility, to reveal length variation of the first and the second introns. With the exception of the first intron of S(13), these also amplified S(7) to S(14) and an allele previously referred to as S(x), which we now label S(16). The genomic PCR products were cloned and sequenced. The partial sequence of S(11) matched that of S(7) and the alleles were shown to have the same functional specificity. Allele-specific primers were designed for S(7) to S(16), so that allele-specific primers are now available for all 13 S alleles of cherry (S(8), S(11) and S(15) are duplicates). These can be used to distinguish between S alleles with introns of similar size and to confirm genotypes determined with consensus primers. The reliability of the PCR with allele-specific primers was improved by the inclusion of an internal control. The use of the consensus and allele-specific primers was demonstrated by resolving conflicting genotypes that have been published recently and by determining genotypes of 18 new cherry cultivars. Two new groups are proposed, Group XXIII (S(3) S(16)), comprising 'Rodmersham Seedling' and 'Strawberry Heart', and Group XXIV (S(6) S(12)), comprising 'Aida' and 'Flamentiner'. Four new self-compatibility genotypes, S(3) S(3)', S(4)' S(6), S(4)' S(9) and S(4)' S(13), were found. The potential use of the consensus primers to reveal incompatibility alleles in other cherry species is also demonstrated.

  9. A molecular method for S-allele identification in apple based on allele-specific PCR.

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    Janssens, G A; Goderis, I J; Broekaert, W F; Broothaerts, W

    1995-09-01

    cDNA sequences corresponding to two self-incompatibility alleles (S-alleles) of the apple cv 'Golden Delicious' have previously been described, and now we report the identification of three additional S-allele cDNAs of apple, one of which was isolated from a pistil cDNA library of cv 'Idared' and two of which were obtained by reverse transcription-PCR (RT-PCR) on pistil RNA of cv 'Queen's Cox'. A comparison of the deduced amino acid sequences of these five S-allele cDNAs revealed an average homology of 69%. Based on the nucleotide sequences of these S-allele cDNAs, we developed a molecular technique for the diagnostic identification of the five different S-alleles in apple cultivars. The method used consists of allele-specific PCR amplification of genomic DNA followed by digestion of the amplification product with an allele-specific restriction endonuclease. Analysis of a number of apple cultivars with known S-phenotype consistently showed coincidence of phenotypic and direct molecular data of the S-allele constitution of the cultivars. It is concluded that the S-allele identification approach reported here provides a rapid and useful method to determine the S-genotype of apple cultivars.

  10. Dideoxy single allele-specific PCR - DSASP new method to discrimination allelic

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    Eleonidas Moura Lima

    2015-06-01

    Full Text Available Gastric cancer (GC is a multifactorial disease with a high mortality rate in Brazil and worldwide. This work aimed to evaluate single nucleotide polymorphisms (SNP rs1695, in the Glutathione S-Transferase Pi (GSTP1 gene in GC samples by comparative analysis Specific PCR - ASP and Dideoxy Single Allele-Specific PCR - DSASP methods. The DSASP is the proposed new method for allelic discrimination. This work analyzed 60 GC samples, 26 diffuse and 34 intestinal types. The SNP rs1695 of the GSTP1 gene was significantly associated with GC analyzed by DSASP method (χ2 = 9.7, P 0.05. These results suggest that the SNP rs1695 of the GSTP1 gene was a risk factor associated with gastric carcinogens is and the DSASP method was a new successfully low-cost strategy to study allelic discrimination.

  11. Optimized Multiplex Detection of 7 KRAS Mutations by Taqman Allele-Specific qPCR

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    Orue, Andrea; Rieber, Manuel

    2016-01-01

    Establishing the KRAS mutational status of tumor samples is essential to manage patients with colorectal or lung cancer, since these mutations preclude treatment with monoclonal anti-epidermal growth factor receptor (EGFR) antibodies. We report an inexpensive, rapid multiplex allele-specific qPCR method detecting the 7 most clinically relevant KRAS somatic mutations with concomitant amplification of non-mutated KRAS in tumor cells and tissues from CRC patients. Positive samples evidenced in the multiplex assay were further subjected to individual allele-specific analysis, to define the specific mutation. Reference human cancer DNA harbouring either G12A, G12C, G12D, G12R, G12S, G12V and G13D confirmed assay specificity with ≤1% sensitivity of mutant alleles. KRAS multiplex mutation analysis usefulness was also demonstrated with formalin-fixed paraffin embedded (FFPE) from CRC biopsies. Conclusion. Co-amplification of non-mutated DNA avoided false negatives from degraded samples. Moreover, this cost effective assay is compatible with mutation detection by DNA sequencing in FFPE tissues, but with a greater sensitivity when mutant DNA concentrations are limiting. PMID:27632281

  12. A single tube modified allele-specific-PCR for rapid detection of erythromycin-resistant Mycoplasma pneumoniae in Beijing

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    LI Shao-li; SUN Hong-mei; ZHAO Han-qing; CAO Ling; YUAN Yi; FENG Yan-ling; XUE Guan-hua

    2012-01-01

    Background Mycoplasma pneumoniae (M.pneumoniae) is one of the common pathogens causing atypical pneumonia.In recent years,resistance to macrolides has become more common,especially in China.Previous studies have confirmed that the mutation at position 2063 in domain V of the 23S rRNA is the most prevalent,followed by the mutation at position 2064.Reported molecular detection methods for the identification of these mutations include direct sequencing,restriction fragment length polymorphism analysis,real-time polymerase chain reaction (PCR) with high-resolution melt analysis,and nested PCR-linked with capillary electrophoresis,etc.The most commonly used method for monitoring resistance-conferring mutations in M.pneumoniae is direct DNA sequencing of PCR or nested PCR products.However,these methods are time-consuming,labor-intensive or need expensive equipments.Therefore the development of rapid and sensitive methods is very important for monitoring the resistance globally.Methods In this study,we reported a fast and cost-effective method for detecting 2063 and/or 2064 macrolide resistant mutations from specimens using a modified allele-specific PCR analysis,and all results were compared with the sequencing data.We also analyzed the clinical courses of these samples to confirm the modified allele-specific PCR results.Results Among 97 M.pneumoniae specimens,88 were found to possess mutations by this method,and all modified allele-specific PCR analysis results were consistent with the sequencing data.The data of the clinical courses of these 97cases showed that they suffered from severe pneumonia.Erythromycin showed better efficacy on cases from which no macrolide resistance mutation was found on their specimens.However,in some cases from which mutations were detected,erythromycin monotherapy had poor efficacy,and on these patients severe symptoms improved only when azithromycin was added to the treatment.Conclusions The drug-resistant M.pneumoniae is very common in

  13. Detection of the MYD88 mutation by the combination of the allele-specific PCR and quenching probe methods.

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    Nogami, S; Kawaguchi-Ihara, N; Shiratori, E; Ohtaka, M; Itoh, M; Tohda, S

    2017-04-01

    The MYD88 missense mutation c.794T>C, p.Leu265Pro, is found in patients with Waldenstörm's macroglobulinemia and lymphoma. Direct sequencing, allele-specific PCR (AS-PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), and high-resolution melting analysis (HRM) are currently used to detect the mutation; however, they are either time-consuming or have low detection sensitivity. Here, we developed a novel highly sensitive and rapid detection method based on the quenching probe (QP) technique and AS-PCR. A lymphoma cell line heterozygous for the MYD88 mutation, two wild-type cell lines, and two samples from Waldenstörm's macroglobulinemia patients were analyzed by AS-PCR, PCR-RFLP, HRM, and QP, and their detection sensitivity was examined using the mixtures of the mutant and wild-type DNA. For mutation-carrying heterozygous samples, the QP method produced W-shaped melting profiles presenting curves derived from the wild-type and mutant alleles. The QP analysis was performed in 2 h and demonstrated the detection limit of 5%, which was similar to that of the other methods. However, the combination of AS-PCR and QP (AS-QP) improved the sensitivity to 0.62% of the mutant allele. The AS-QP analysis is rapid and minimally improves detection sensitivity compared to the AS-PCR. © 2016 John Wiley & Sons Ltd.

  14. Rapid identification of capybara (Hydrochaeris hydrochaeris through allele-specific PCR

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    Flávio Henrique-Silva

    2005-07-01

    Full Text Available The capybara is the largest rodent in the world and is widely distributed throughout Central and South America.  It is an animal of economic interest due to the pleasant flavor of its meat and higher protein content in comparison  to beef and pork meat.  The hide, hair and fat also have economic advantages. Thus,  as an animal with such high economic potential, it is the target of hunters, even though  hunting capybara is prohibited by law in Brazil.   Due to their  similarities,  capybara meat  is easily confused with  pork  meat.   This  occurs  upon  the apprehension of the  meat  from hunters, as well as in some restaurants that serve capybara meat that was slaughtered clandestinely. In both cases, when the meat is confiscated, those responsible for the crimes claim it is pork meat,  hindering  the enforcement of the law. A practical  course was ministered  to undergraduate biology students enrolled in the elective course Introduction to Genetic  Engineering  at Federal  University  of Sao Carlos (UFSCar, Sao Paulo  State, Brazil.  The  objective  of the  course was to establish  and  apply  a Polymerase  Chain  Reaction  (PCR assay to identify capybara meat and discriminate it in relation  to other types of meat,  including pork. Primers  were designed based  on 12S rRNA,  transthyretin and  growth  hormone  receptor  genes.  The primers generated  capybara specific fragments  of approximately 220, 290 and 330 bp for transthyretin,12S rRNA  and  growth  hormone  receptor,  respectively.   The  duplexes  developed  in the  present work can be used effectively to discriminate capybara meat  from other  animals,  contributing to combating predatory capybara hunting. The results were extensively discussed and the students have contributed to written a paper  to be submitted to a publication.

  15. Allele Specific Locked Nucleic Acid Quantitative PCR (ASLNAqPCR): An Accurate and Cost-Effective Assay to Diagnose and Quantify KRAS and BRAF Mutation

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    Morandi, Luca; de Biase, Dario; Visani, Michela; Cesari, Valentina; De Maglio, Giovanna; Pizzolitto, Stefano; Pession, Annalisa; Tallini, Giovanni

    2012-01-01

    The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes. PMID:22558339

  16. Assignment of SNP allelic configuration in polyploids using competitive allele-specific PCR: application to citrus triploid progeny

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    Cuenca, José; Aleza, Pablo; Navarro, Luis; Ollitrault, Patrick

    2013-01-01

    Background Polyploidy is a major component of eukaryote evolution. Estimation of allele copy numbers for molecular markers has long been considered a challenge for polyploid species, while this process is essential for most genetic research. With the increasing availability and whole-genome coverage of single nucleotide polymorphism (SNP) markers, it is essential to implement a versatile SNP genotyping method to assign allelic configuration efficiently in polyploids. Scope This work evaluates the usefulness of the KASPar method, based on competitive allele-specific PCR, for the assignment of SNP allelic configuration. Citrus was chosen as a model because of its economic importance, the ongoing worldwide polyploidy manipulation projects for cultivar and rootstock breeding, and the increasing availability of SNP markers. Conclusions Fifteen SNP markers were successfully designed that produced clear allele signals that were in agreement with previous genotyping results at the diploid level. The analysis of DNA mixes between two haploid lines (Clementine and pummelo) at 13 different ratios revealed a very high correlation (average = 0·9796; s.d. = 0·0094) between the allele ratio and two parameters [θ angle = tan−1 (y/x) and y′ = y/(x + y)] derived from the two normalized allele signals (x and y) provided by KASPar. Separated cluster analysis and analysis of variance (ANOVA) from mixed DNA simulating triploid and tetraploid hybrids provided 99·71 % correct allelic configuration. Moreover, triploid populations arising from 2n gametes and interploid crosses were easily genotyped and provided useful genetic information. This work demonstrates that the KASPar SNP genotyping technique is an efficient way to assign heterozygous allelic configurations within polyploid populations. This method is accurate, simple and cost-effective. Moreover, it may be useful for quantitative studies, such as relative allele-specific expression analysis and bulk segregant analysis

  17. Allele-specific PCR for detecting the deafness-associated mitochondrial 12S rRNA mutations.

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    Ding, Yu; Xia, Bo-Hou; Liu, Qi; Li, Mei-Ya; Huang, Shui-Xian; Zhuo, Guang-Chao

    2016-10-10

    Mutations in mitochondrial 12S rRNA (MT-RNR1) are the important causes of sensorineural hearing loss. Of these mutations, the homoplasmic m.1555A>G or m.1494C>T mutation in the highly conserved A-site of MT-RNR1 gene has been found to be associated with both aminoglycoside-induced and non-syndromic hearing loss in many families worldwide. Since the m.1555A>G and m.1494C>T mutations are sensitive to ototoxic drugs, therefore, screening for the presence of these mutations is important for early diagnosis and prevention of deafness. For this purpose, we recently developed a novel allele-specific PCR (AS-PCR) which is able to simultaneously detect these mutations. To assess its accuracy, in this study, we employed this method to screen the frequency of m.1555A>G and m.1494C>T mutations in 200 deafness patients and 120 healthy subjects. Consequently, four m.1555A>G and four m.1494C>T mutations were identified; among these, only one patient with the m.1494C>T mutation had an obvious family history of hearing loss. Strikingly, clinical evaluation showed that this family exhibited a high penetrance of hearing loss. In particular, the penetrances of hearing loss were 80% with the aminoglycoside included and 20% when excluded. PCR-Sanger sequencing of the mitochondrial genomes confirmed the presence of the m.1494C>T mutation and identified a set of polymorphisms belonging to mitochondrial haplogroup A. However, the lack of functional variants in mitochondrial and nuclear modified genes (GJB2 and TRMU) in this family indicated that mitochondrial haplogroup and nuclear genes may not play important roles in the phenotypic expression of the m.1494C>T mutation. Thus, other modification factors, such as environmental factor, aminoglycosides or epigenetic modification may have contributed to the high penetrance of hearing loss in this family. Taken together, our data showed that this assay is an effective approach that could be used for detection the deafness-associated MT-RNR1

  18. WASP: a Web-based Allele-Specific PCR assay designing tool for detecting SNPs and mutations

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    Assawamakin Anunchai

    2007-08-01

    Full Text Available Abstract Background Allele-specific (AS Polymerase Chain Reaction is a convenient and inexpensive method for genotyping Single Nucleotide Polymorphisms (SNPs and mutations. It is applied in many recent studies including population genetics, molecular genetics and pharmacogenomics. Using known AS primer design tools to create primers leads to cumbersome process to inexperience users since information about SNP/mutation must be acquired from public databases prior to the design. Furthermore, most of these tools do not offer the mismatch enhancement to designed primers. The available web applications do not provide user-friendly graphical input interface and intuitive visualization of their primer results. Results This work presents a web-based AS primer design application called WASP. This tool can efficiently design AS primers for human SNPs as well as mutations. To assist scientists with collecting necessary information about target polymorphisms, this tool provides a local SNP database containing over 10 million SNPs of various populations from public domain databases, namely NCBI dbSNP, HapMap and JSNP respectively. This database is tightly integrated with the tool so that users can perform the design for existing SNPs without going off the site. To guarantee specificity of AS primers, the proposed system incorporates a primer specificity enhancement technique widely used in experiment protocol. In particular, WASP makes use of different destabilizing effects by introducing one deliberate 'mismatch' at the penultimate (second to last of the 3'-end base of AS primers to improve the resulting AS primers. Furthermore, WASP offers graphical user interface through scalable vector graphic (SVG draw that allow users to select SNPs and graphically visualize designed primers and their conditions. Conclusion WASP offers a tool for designing AS primers for both SNPs and mutations. By integrating the database for known SNPs (using gene ID or rs number

  19. Detection of EGFR Mutations by TaqMan Mutation Detection Assays Powered by Competitive Allele-Specific TaqMan PCR Technology

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    Cristin Roma

    2013-01-01

    Full Text Available Epidermal growth factor receptor (EGFR mutations in non-small-cell lung cancer (NSCLC are predictive of response to treatment with tyrosine kinase inhibitors. Competitive Allele-Specific TaqMan PCR (castPCR is a highly sensitive and specific technology. EGFR mutations were assessed by TaqMan Mutation Detection Assays (TMDA based on castPCR technology in 64 tumor samples: a training set of 30 NSCLC and 6 colorectal carcinoma (CRC samples and a validation set of 28 NSCLC cases. The sensitivity and specificity of this method were compared with routine diagnostic techniques including direct sequencing and the EGFR Therascreen RGQ kit. Analysis of the training set allowed the identification of the threshold value for data analysis (0.2; the maximum cycle threshold (Ct=37; and the cut-off ΔCt value (7 for the EGFR TMDA. By using these parameters, castPCR technology identified both training and validation set EGFR mutations with similar frequency as compared with the Therascreen kit. Sequencing detected rare mutations that are not identified by either castPCR or Therascreen, but in samples with low tumor cell content it failed to detect common mutations that were revealed by real-time PCR based methods. In conclusion, our data suggest that castPCR is highly sensitive and specific to detect EGFR mutations in NSCLC clinical samples.

  20. Allele-specific real-time PCR testing for minor HIV-1 drug resistance mutations: assay preparation and application to reveal dynamic of mutations in vivo

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    GUO Dong-xing; LI Jing-yun; LI Han-ping; LI Lin; ZHUANG Dao-min; JIAO Li-yan; WANG Zheng; BAO Zuo-yi; LIU Si-yang; LIU Yong-jian

    2010-01-01

    Background It is very important for the clinical management to test for minor HIV-1 resistance mutations accurately and sensitively. The conventional genotypic assays of HIV drug resistance detection based on sequencing can only discriminate the mutations which present in more than 20%-30%. The aim of this study was to evaluate allele-specific real-time PCR (ASPCR) to detect the resistance-related mutations located at positions 103, 184 and 215.Methods We developed the allele-specific PCR assay, using the most common drug resistance mutations in Chinese AIDS patients, K103N, M184V/I, T215F/Y as a model system. The standards were constructed by cloning the wild-type and mutant DNA fragments into the T-vector. We designed specific primers to discriminate mutant templates in the real-time PCR using SYBR green as a fluorescence reporter. And then we evaluated the ASPCR assay and tested 140clinical samples using this method.Results The sensitivities of ASPCR assay were 0.04% for K103N, 0.30% for M1841, 0.40% for M184V, 0.03% for T215F and 0.02% for T215Y. The intra-assay and inter-assay coefficients of variation were less than 0.42. One hundred and forty plasma samples were tested by ASPCR and dynamic resistance curves of ten patients were obtained.Conclusions Drug resistance emerged half a year after the start of antiretroviral therapy. The mutation of T215Yemerged 1 to 1.5 years after starting treatment and then increased rapidly. The ASPCR assay we developed was a sensitive, accurate and rapid method to detect the minor HIV-1 variants and it can provide earlier and more drug-resistance information for HIV research and AIDS antiretroviral therapy.

  1. Detection of EGFR mutations in plasma and biopsies from non-small cell lung cancer patients by allele-specific PCR assays

    DEFF Research Database (Denmark)

    Weber, Britta; Meldgaard, Peter; Hager, Henrik

    2014-01-01

    BACKGROUND: Lung cancer patients with mutations in the epidermal growth factor receptor (EGFR) are primary candidates for EGFR-targeted therapy. Reliable analyses of such mutations have previously been possible only in tumour tissue. Here, we demonstrate that mutations can be detected in plasma...... samples with allele-specific PCR assays. METHODS: Pairs of the diagnostic biopsy and plasma obtained just prior to start of erlotinib treatment were collected from 199 patients with adenocarcinoma of non-small-cell lung cancer. DNA from both sample types was isolated and examined for the presence...... identified in 24/199 (12%) plasma samples and 28/196 (14%) biopsy samples, and 17/196 (9%) matched pairs contained the same mutation. Six EGFR mutations were present only in plasma samples but not in the biopsy samples. The overall concordance of the EGFR gene mutations detected in plasma and biopsy tissue...

  2. Concordance between allele-specific PCR and ultra-deep pyrosequencing for the detection of HIV-1 non-nucleoside reverse transcriptase inhibitor resistance mutations

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    Hunt, Gillian M; Morris, Lynn; Moorthy, Anitha; Coovadia, Ashraf; Abrams, Elaine J; Strehlau, Renate; Kuhn, Louise; Persaud, Deborah

    2014-01-01

    Recent advances in genotyping technologies have allowed for detection of HIV-1 drug resistance mutations present at low levels. The presence and percentage of Y181C and K103N drug-resistant variants in the blood of 105 subtype C HIV-infected infants who failed single-dose nevirapine prophylaxis for HIV transmission were compared using two highly sensitive genotyping methods, allele-specific PCR (AS-PCR) and ultra-deep pyrosequencing. Significant correlations in detection between both methods were found for both Y181C (correlation coefficients of 0.94 [95% CI 0.91-0.96]) and K103N (0.89 [95% CI 0.84 – 0.92]) mutations. The majority of discordant specimens (3/5 Y181C and 8/11 K103N) had wild-type variants when population sequencing was used, but mutant variants were detectable at very low levels (≤5%) with either assay. This difference is most likely due to stochastic variations in the appearance of mutant variants. Overall, both AS-PCR and ultra-deep pyrosequencing methods have proven to be sensitive and accurate, and may confidently be used where feasible. PMID:25034127

  3. Development of duplex-PCR for identification of Aeromonas species

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    Carina Lucena Mendes-Marques

    2013-10-01

    Full Text Available Introduction The number of reports of intestinal infections caused by Aeromonas spp. has increased significantly in recent years. In most clinical laboratories, identification of these bacteria is carried out by general phenotypic tests that sometimes do not accurately differentiate Aeromonas and Vibrio. Methods A duplex-polymerase chain reaction (PCR was developed directed to 2 targets identifying Aeromonas spp. pathogenic to humans. Results The duplex-PCR results were reproducible and specific for Aeromonas spp. pathogenic to humans. Conclusions This method will allow differentiation between Vibrio and Aeromonas spp. in patients with in cholera-like symptoms and can also be used in water quality monitoring.

  4. Development of duplex-PCR for identification of Aeromonas species

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    Carina Lucena Mendes-Marques

    2013-05-01

    Full Text Available Introduction The number of reports of intestinal infections caused by Aeromonas spp. has increased significantly in recent years. In most clinical laboratories, identification of these bacteria is carried out by general phenotypic tests that sometimes do not accurately differentiate Aeromonas and Vibrio. Methods A duplex-polymerase chain reaction (PCR was developed directed to 2 targets identifying Aeromonas spp. pathogenic to humans. Results The duplex-PCR results were reproducible and specific for Aeromonas spp. pathogenic to humans. Conclusions This method will allow differentiation between Vibrio and Aeromonas spp. in patients with in cholera-like symptoms and can also be used in water quality monitoring.

  5. Disagreement in genotyping results of drug resistance alleles of the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) gene by allele-specific PCR (ASPCR) assays and Sanger sequencing.

    Science.gov (United States)

    Sharma, Divya; Lather, Manila; Dykes, Cherry L; Dang, Amita S; Adak, Tridibes; Singh, Om P

    2016-01-01

    The rapid spread of antimalarial drug resistance in Plasmodium falciparum over the past few decades has necessitated intensive monitoring of such resistance for an effective malaria control strategy. P. falciparum dihydropteroate synthase (Pfdhps) and P. falciparum dihydrofolate reductase (Pfdhfr) genes act as molecular markers for resistance against the antimalarial drugs sulphadoxine and pyrimethamine, respectively. Resistance to pyrimethamine which is used as a partner drug in artemisinin combination therapy (ACT) is associated with several mutations in the Pfdhfr gene, namely A16V, N51I, C59R, S108N/T and I164L. Therefore, routine monitoring of Pfdhfr-drug-resistant alleles in a population may help in effective drug resistance management. Allele-specific PCR (ASPCR) is one of the commonly used methods for molecular genotyping of these alleles. In this study, we genotyped 55 samples of P. falciparum for allele discrimination at four codons of Pfdhfr (N51, C59, S108 and I164) by ASPCR using published methods and by Sanger's DNA sequencing method. We found that the ASPCR identified a significantly higher number of mutant alleles as compared to the DNA sequencing method. Such discrepancies arise due to the non-specificity of some of the allele-specific primer sets and due to the lack of sensitivity of Sanger's DNA sequencing method to detect minor alleles present in multiple clone infections. This study reveals the need of a highly specific and sensitive method for genotyping and detecting minor drug-resistant alleles present in multiple clonal infections.

  6. Development and validation of a bi-directional allele-specific PCR tool for differentiation in nurseries of dura, tenera and pisifera oil palms

    Directory of Open Access Journals (Sweden)

    Paola A. Reyes

    2015-04-01

    Full Text Available Oil palm (Elaeis guineensis Jacq. fruits are classified by shell thickness into three types: dura, pisifera, and tenera, the last one being the product of a dura × pisifera cross. The palm oil industry relies on the use of high-yield tenera plant material for production; however, it is usually generated with female infertile pisifera, so early identification of this trait is very important to oil production and breeding programs. Recently, the mapping and sequencing of the SHELL gene, which is responsible for endocarp formation in oil palms, made it possible to identify two mutations (type SNP, single nucleotide polymorphism that affect its function and that are useful to developing molecular markers for predicting shell thickness. The aim of this study was to standardize PCR-based methodologies in order to detect the SNP observed in codon 30 and validate it under our E. guineensis biological collections. We achieved the differentiation of SHELL alleles with both allele specific PCR and CAPS with the restriction enzyme HindIII in homozygous and heterozygous plants that contained the described mutation, and the prediction was correlated with the phenotype observed in oil palm fruits. These methodologies facilitated the discrimination of plants by fruit type in nursery and pre-nursery stages 24 months before production started, thereby reducing the time and area used in oil palm breeding programs.

  7. Minority drug-resistant HIV-1 variants in treatment naive East-African and Caucasian patients detected by allele-specific real-time PCR.

    Directory of Open Access Journals (Sweden)

    Halime Ekici

    Full Text Available To assess the presence of two major non-nucleoside reverse transcriptase inhibitors (NNRTI drug resistance mutations (DRMs, Y181C and K103N, in minor viral quasispecies of treatment naïve HIV-1 infected East-African and Swedish patients by allele-specific polymerase chain reaction (AS-PCR.Treatment naïve adults (n=191 with three epidemiological backgrounds were included: 92 Ethiopians living in Ethiopia; 55 East-Africans who had migrated to Sweden; and 44 Caucasians living in Sweden. The pol gene was analysed by standard population sequencing and by AS-PCR for the detection of Y181C and K103N.The Y181C was detected in the minority quasispecies of six Ethiopians (6.5%, in two Caucasians (4.5%, and in one East-African (1.8%. The K103N was detected in one East- African (1.8%, by both methods. The proportion of mutants ranged from 0.25% to 17.5%. Additional DRMs were found in all three treatment naïve patient groups by population sequencing.Major NNRTI mutations can be found by AS-PCR in minor quasispecies of treatment naïve HIV-1 infected Ethiopians living in Ethiopia, in East-African and Caucasian patients living in Sweden in whom population sequencing reveal wild-type virus only. Surveys with standard sequencing are likely to underestimate transmitted drug resistance and the presence of resistant minor quasispecies in treatment naïve patients should be topic for future large scale studies.

  8. A Multiplex Allele Specific Polymerase Chain Reaction (MAS-PCR) for the Detection of Factor V Leiden and Prothrombin G20210A.

    Science.gov (United States)

    Bagheri, Morteza; Rad, Isa Abdi

    2011-01-01

    In order to determine the frequencies of factor V Leiden and prothrombin G20210A point mutations in the Iranian population with Azeri Turkish origin. 120 unrelated individuals from general population randomly selected and were examined for factor V Leiden and prothrombin G20210A mutations using a multiplex allele specific polymerase chain reaction (MAS-PCR) assayOutcomes: The frequency of prothrombin G20210A mutation was 2.08%, which means 5 chromosomes out of 240 chromosomes had prothrombin G20210A mutation. The distribution of prothrombin 20210 GG, GA, AA genotypes and prothrombin 20210A allele were 37(92.5%), 3(7.5%), 0(0%) and 3(3.75%) in males and 78(97.5%), 2(2.5%), 0(0%) and 2(1.25%) in females, respectively. Factor V Leiden was not found in our tested group (zero chromosomes out of 240 chromosomes). Analysis of the observed frequencies in the studied groups indicates that there is no statistically significant difference between females and males, regarding prothrombin G20210A mutation (p value>0.05). This is the first study in its own kind in this population and implies that the frequency of Factor V Leiden G1691A (R506Q, FV-Leiden) allele is extremely low but the prothrombin G20210A mutation is more frequent in the tested group.

  9. A suitable duplex PCR for ovine embryo sex and genotype of PrnP gene determination for MOET-based selection programmes.

    Science.gov (United States)

    Dervishi, E; Sánchez, P; Alabart, J L; Cocero, M J; Folch, J; Calvo, J H

    2011-12-01

    The objective of this study was to test the suitability of a duplex PCR assay for sex and scrapie resistance genotype determination in fresh embryos. Duplex PCR amplified a repetitive and specific fragment of Y chromosome, used for sex diagnosis, and a PrnP fragment. PrnP codons 134 and 156, and codon 171 were genotyped by restriction fragment length polymorphisms and allele-specific PCR, respectively, after re-amplification of PrnP fragment. The specificity of the method was first assessed by testing 359 blood samples from Rasa Aragonesa sheep breed (161 males and 198 females). No amplification failures and total agreement between genotypic and phenotypic sex were found. In the same way, PrnP genotype determination by duplex PCR assay was in agreement with the PrnP animal's genotype established by sequencing. Finally, 73 samples of 1-10 cells from compact morulae were aspirated through the zona pellucida and genotyped for sex and PrnP. The efficiency was 96% when three or more cells were sampled. These results confirm that the duplex PCR assay reported in this work can be used for rapid sex determination in ovine embryos, with a high efficiency and accuracy (96%) when three or more cells are sampled, allowing sexed fresh embryos of known PrnP genotype to be transferred in multiple ovulation and embryo transfer programmes.

  10. Sex determination in 6 bovid species by duplex PCR.

    Science.gov (United States)

    Prashant; Gour, Digpal S; Dubey, Prem P; Jain, Anubhav; Gupta, Subhash C; Joshi, Balwinder K; Kumar, Dinesh

    2008-01-01

    Sex determination in domestic animals is of potential value to livestock breeding programs. The aim of this study was to develop a simple and accurate PCR-based sex determination protocol, which can be applicable to 6 major domesticated species of the family Bovidae, viz. Bos frontalis, B. grunniens, B. indicus, Bubalus bubalis, Capra hircus, and Ovis aries. In silico analysis was done to identify conserved DNA sequence in the HMG box region of the sex-determining region of the Y-chromosome (SRY gene) across the bovids. Duplex PCR assay, including the SRY gene and the GAPDH housekeeping gene, was optimized by using genomic DNA extracted from blood samples of known sex. It was possible to identify the sex of animals by amplifying both gender-specific (SRY) and autosomal (GAPDH) genes simultaneously in the duplex reaction, with the male yielding two bands and the female one band. The protocol was subjected to a blind test that showed a 100 percent specificity and accuracy, thus it can be used in sex determination in livestock breeding programs.

  11. Detection of Hepatitis B Virus DNA by Duplex Scorpion Primer-based PCR Assay

    Institute of Scientific and Technical Information of China (English)

    KONG De-Ming孔德明; SHEN Han-Xi沈含熙; MI Huai-Feng宓怀风

    2004-01-01

    The application of a new fiuorogenic probe-based PCR assay (PCR duplex scorpion primer assay) to the detection of Hepatitis B virus (HBV) DNA in human sera was described. Duplex scorpion primer is a modified variant of duplex Amplifluor, and the incorporation of a PCR stopper between probe and primer sequences improve the detection specificity and sensitivity. Combined with PCR amplification, this probe can give unambiguous positive results for the reactions initiated with more than 20 HBV molecules. In addition, the particular unimolecular probing mechanism of this probe makes the use of short target-specific probe sequence possible, which will render this probe applicable in some specific systems.

  12. DETEKSI MUTASI V1016G PADA GEN VOLTAGE-GATED SODIUM CHANNEL PADA POPULASI Aedes aegypti (DIPTERA: CULICIDAE DI KABUPATEN KLATEN, JAWA TENGAH DENGAN METODE ALLELE-SPECIFIC PCR

    Directory of Open Access Journals (Sweden)

    Dyah Widiastuti

    2015-10-01

    Full Text Available AbstrakMeluasnya kejadian resistensi pada vektor virus Dengue di Jawa Tengah memerlukan strategi pengelolaan resistensi insektisida secara efektif. Oleh karena itu, informasi mengenai mutasi gen pada posisi 1016 di domain II segmen ke­6 gen VGSC pada nyamuk Aedes aegypti yang menyebabkan perubahan asam amino valin (V menjadi glisin (G akan dapat memperkuat penelitian operasional mengenai strategi pemilihan insektisida dalam program­pengendalian­vektor­Dengue.­Penelitian­ini­menggunakan­uji­Allele-Specific­Polymerase­Chain­Reaction(AS­PCR yang dapat mendeteksi mutasi V1016G. Sampel penelitian ini adalah 22 ekor nyamuk Aedes aegypti dari Kabupaten Klaten yang berumur 2­5 hari. Hasil penelitian menunjukkan bahwa 22,7% nyamuk belum mengalami mutasi (V/V, 59,1% nyamuk mengalami mutasi heterozigot (V/G dan 18,2% nyamuk mengalami mutasi homozigot (G/G. Hal ini menunjukkan indikasi terjadinya resistensi populasi nyamuk Ae.aegypti terhadap insektisida sintetik piretroid yang disebabkan oleh mekanisme knockdown resistance.Kata Kunci:­Aedes­aegypti,­mutasi­V1016G,­Allele-Specific­PCR,­VGSCAbstractInsecticides resistance has spread rapidly among dengue vectors from Central Java, and require an effective insecticide resistance management strategies.one of the resistance mechanism in Aedes aegypti may arise through knockdown resistance or kdr which consists of single point mutation within the genes that are targeted by insecticide compounds. Mutation at position 1016 in domain II, segment 6 of the Voltage Gated Sodium Channel gene in Ae. aegypti leads to a valine to glycine substitution (V1016G is associated with resistance to the type II pyrethroid. The result of this study will help us to strengthen basic and operational research on the­development­of­strategies­for­Dengue­vector­control­in­Indonesia.­This­study­utilized­an­allele-specificPolymerase Chain Reaction (AS­PCR assay that could be used to detect the V1016G

  13. Determination of cis/trans phase of variations in the MC1R gene with allele-specific PCR and single base extension

    DEFF Research Database (Denmark)

    Mengel-From, Jonas; Børsting, Claus; Sanchez, Juan J

    2008-01-01

    The MC1R gene encodes a protein with key regulatory functions in the melanin synthesis. A multiplex PCR and a multiplex single base extension protocol were established for genotyping six exonic MC1R variations highly penetrant for red hair (R), four exonic MC1R variations weakly penetrant for red...

  14. 454 next generation-sequencing outperforms allele-specific PCR, Sanger sequencing, and pyrosequencing for routine KRAS mutation analysis of formalin-fixed, paraffin-embedded samples.

    Science.gov (United States)

    Altimari, Annalisa; de Biase, Dario; De Maglio, Giovanna; Gruppioni, Elisa; Capizzi, Elisa; Degiovanni, Alessio; D'Errico, Antonia; Pession, Annalisa; Pizzolitto, Stefano; Fiorentino, Michelangelo; Tallini, Giovanni

    2013-01-01

    Detection of KRAS mutations in archival pathology samples is critical for therapeutic appropriateness of anti-EGFR monoclonal antibodies in colorectal cancer. We compared the sensitivity, specificity, and accuracy of Sanger sequencing, ARMS-Scorpion (TheraScreen®) real-time polymerase chain reaction (PCR), pyrosequencing, chip array hybridization, and 454 next-generation sequencing to assess KRAS codon 12 and 13 mutations in 60 nonconsecutive selected cases of colorectal cancer. Twenty of the 60 cases were detected as wild-type KRAS by all methods with 100% specificity. Among the 40 mutated cases, 13 were discrepant with at least one method. The sensitivity was 85%, 90%, 93%, and 92%, and the accuracy was 90%, 93%, 95%, and 95% for Sanger sequencing, TheraScreen real-time PCR, pyrosequencing, and chip array hybridization, respectively. The main limitation of Sanger sequencing was its low analytical sensitivity, whereas TheraScreen real-time PCR, pyrosequencing, and chip array hybridization showed higher sensitivity but suffered from the limitations of predesigned assays. Concordance between the methods was k = 0.79 for Sanger sequencing and k > 0.85 for the other techniques. Tumor cell enrichment correlated significantly with the abundance of KRAS-mutated deoxyribonucleic acid (DNA), evaluated as ΔCt for TheraScreen real-time PCR (P = 0.03), percentage of mutation for pyrosequencing (P = 0.001), ratio for chip array hybridization (P = 0.003), and percentage of mutation for 454 next-generation sequencing (P = 0.004). Also, 454 next-generation sequencing showed the best cross correlation for quantification of mutation abundance compared with all the other methods (P < 0.001). Our comparison showed the superiority of next-generation sequencing over the other techniques in terms of sensitivity and specificity. Next-generation sequencing will replace Sanger sequencing as the reference technique for diagnostic detection of KRAS mutation in archival tumor tissues.

  15. Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay

    Science.gov (United States)

    Wang, Zengguo; Zhang, Xiujuan; Zhao, Xiaomin; Du, Qian; Chang, Lingling; Tong, Dewen

    2015-01-01

    Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility. PMID:26544710

  16. Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay.

    Directory of Open Access Journals (Sweden)

    Yong Huang

    Full Text Available Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus and PCV2 (DNA virus from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29% and TGEV (11.7% preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility.

  17. Genotyping of the 19-bp insertion/deletion polymorphism in the 5' flank of beta-hydroxylase gene by dissociation analysis of allele-specific PCR products

    DEFF Research Database (Denmark)

    Rasmussen, Henrik Berg; Werge, Thomas

    2005-01-01

    The 19-bp insertion/deletion polymorphism in the 5' flank of the dopamine beta-hydroxylase (DBH) gene has been associated with psychiatric disorders. We have developed a simple, reliable and inexpensive closed-tube assay for genotyping of this polymorphism based upon T(m) determination of amplified...... and a conventional approach based upon agarose gel electrophoresis of amplified fragments revealed complete concordance between the two procedures. The insights obtained in this study may be utilized to develop assays based upon dissociation analysis of PCR products for genotyping of other insertion...

  18. Allele-specific PCR genotyping of the HSP70 gene polymorphism discriminating the green and red color variants sea cucumber (Apostichopus japonicus)

    Institute of Scientific and Technical Information of China (English)

    Jung-Ha Kang; Ki Hwan Yu; Jung-Youn Park; Chul-Min An; Je-Cheon Jun; Sang-Jun Lee

    2011-01-01

    Color variation is a well-known feature of sea cucumbers (Apostichopus japonicus),which are classified into three groups based on their colors of red,green and black.It is also one of the most important traits related to how they taste,and it thereby affects their market price.Attempts were made to identify single-nucleotide polymorphisms (SNPs) and to analyze differences associated with SNP genotypes between green and red color variants using HSP70 as the target gene.The HSP70 gene,which is found universally in organisms from bacteria to humans,is one of the most evolutionarily conserved genes and the most widely studied biomarker of stress response.DNA fragments of 1074 bp covering a partial sequence of the sea cucumber HSP70 gene,were amplified from both red and green variants,and subsequently analyzed for the presence of SNPs.Twenty-seven polymorphic sites in total,including heterozygous sites,were observed.Of these,six sites were found to be significantly different SNP genotypes between green and red variants.Furthermore,PCR with an internal primer designed to include an allelespecific SNP at the 3' end (site 443) showed differentiation between the two variants,100% and 4.2% amplification in green and red variants,respectively.The validated SNPs may serve as informative genetic markers that can be used to distinguish variants at the early developmental stage,prior to color differentiation.

  19. Duplex PCR for detection of Salmonella and Shigella spp in cockle samples.

    Science.gov (United States)

    Senachai, Pachara; Chomvarin, Chariya; Wongboot, Warawan; Boonyanugomol, Wongwarut; Tangkanakul, Waraluk

    2013-09-01

    Salmonella and Shigella spp are important causative agents of foodborne diseases. A sensitive, specific and rapid method is essential for detection of these pathogens. In this study, a duplex PCR method was developed for simultaneous detection of Salmonella and Shigella spp in cockle samples and compared with the traditional culture method. Enrichment broths for Salmonella spp recovery were also compared. Sensitivity of the duplex PCR for simultaneous detection of Salmonella and Shigella spp from pure culture was 10(3) CFU/ml (40 CFU/PCR reaction), and that of sterile cockle samples spiked with these two pathogens was 1 CFU/10 g of cockle tissue after 9 hours enrichment [3 hours in buffered peptone water (BPW), followed by 6 hours in Rappaport Vasiliadis (RV) broth or tetrathionate (TT) broth for Salmonella spp and 6 hours enrichment in Shigella broth (SB) for Shigella spp]. There was no significant difference in detection sensitivity between enrichment in RV and TT broths. Salmonella spp detected in cockles in Khon Kaen, Thailand by duplex PCR and culture method was 17% and 13%, respectively but Shigella spp was not detected. The duplex PCR technique developed for simultaneous detection of Salmonella and Shigella spp in cockle samples was highly sensitive, specific and rapid and could serve as a suitable method for food safety assessment.

  20. A duplex PCR for rapid and simultaneous detection of Brucella spp. in human blood samples.

    Science.gov (United States)

    Mirnejad, Reza; Mohamadi, Mozafar; Piranfar, Vahbeh; Mortazavi, Seied Mojtaba; Kachuei, Reza

    2013-06-01

    To design a duplex PCR for rapid and simultaneous detection of Brucella species. in human blood samples. Fifty-two peripheral bloods samples were collected from suspicious patients with brucellosis. Following DNA extraction, PCR assay were performed, using three primers that could simultaneously identify and differentiate three major species of pathogenic Brucella in humans and animals. Of the 52 peripheral bloods samples tested, 25 sample (48%) showed positive reactions in PCR. Twelve samples were positive for Brucella abortus 39 (B. abortus 39) (23%), 13 for Brucella melitensis 39 (B. melitensis 39) (25%) and 0 for Brucella ovis 39 (B. ovis 39) (0%). This work demonstrates that in case where specific primers were utilized, duplex PCR has proved to be a simple, fast, and relatively inexpensive method for simultaneous detection of important species of Brucella in clinical samples. Copyright © 2013 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

  1. A duplex PCR for the rapid and simultaneous detection of Brucella spp. in human blood samples

    Institute of Scientific and Technical Information of China (English)

    Reza Mirnejad; Mozafar mohamadi; Vahbeh Piranfar; Seied Mojtaba Mortazavi; Reza Kachuei

    2013-01-01

    Objective: To design a duplex PCR for rapid and simultaneous detection of Brucella species. in human blood samples. Methods: Fifty-two peripheral bloods samples were collected from suspicious patients with brucellosis. Following DNA extraction, PCR assay were performed, using three primers that could simultaneously identify and differentiate three major species of pathogenic Brucella in humans and animals. Results: Of the 52 peripheral bloods samples tested, 25 sample (48%) showed positive reactions in PCR. Twelve samples were positive for Brucella abortus (B. abortus) (23%), 13 for Brucella melitensis (B. melitensis) (25%) and 0 for Brucella ovis (B. ovis) (0%). Conclusions: This work de=monstrates that in case where specific primers were utilized, duplex PCR has proved to be a simple, fast, and relatively inexpensive method for simultaneous detection of important species of Brucella in clinical samples.

  2. 全血AS-PCR方法检测Wilson病ATP7B基因四个突变%Whole blood allele-specific PCR, a simple method to detect four ATP7B gene mutations in Wilson disease

    Institute of Scientific and Technical Information of China (English)

    孙玮; 管俊杰; 王进; 秦正红

    2014-01-01

    Objective To establish a simple method to detect four ATP7B gene mutations in Wilson disease using allele-specific PCR (AS-PCR) with whole blood polymerase chain reaction.Methods Four allele-specific PCR primers specific for the mutations(G2333T,C2850T,G2855A,G2975T) were designed,and PCR was optimized to screen the whole blood samples.The amplified gene products with mutation were separated with agarose gel electrophoresis to detect the pattern of point mutation and allele types.Exons 8,12 and 13 of the ATP7B gene were amplified with PCR,and the amplification products were sequenced to confirm the mutation.Results The detection of four ATP7B gene mutations by AS-PCR with whole blood was accomplished with 100% accuracy.In the 27 healthy subjects,the mutation rate of G2855A was 51.8%.No mutation was detected for G2333T,C2850T and G2975T.Among the 22 patients,11 were mutated for G2333T,C2850T or G2975T.The mutation rate was therefore 50%.Conclusion Our experiment has established an AS-PCR based method for detecting four ATP7B gene mutations using whole blood samples,which has provided a simple and effective means for the early diagnosis of Wilson disease.This method is rapid,convenient,accurate and economical for detecting point mutations of the ATP7B gene.%目的 研究肝豆状核变性ATP7B基因高频突变位点,探索全血等位基因特异性-PCR(allelespecific PCR,AS-PCR)技术在该基因4个常见突变检测中的应用.方法 针对ATP7B基因4个突变位点(G2333T、C2850T、G2855A、G2975T)设计等位基因特异性引物,应用高保真酶对人抗凝全血样本进行聚合酶链反应,扩增产物经琼脂糖凝胶电泳以判断待检样本有无基因突变及其等位基因型.PCR扩增人基因组A TP7B基因第8、12、13外显子,扩增产物直接进行基因序列测定.结果 全血AS-PCR法检测ATP7B基因4个基因突变,各位点检测结果与基因序列测定完全相符.27份健康对照血样分型结果G2333T、C2850T

  3. A duplex real-time RT-PCR assay for profiling inhibitors of four dengue serotypes.

    Science.gov (United States)

    Gong, Edwin Yunhao; Smets, Alexandra; Verheyen, Nick; Clynhens, Marleen; Gustin, Emmanuel; Lory, Pedro; Kraus, Guenter

    2013-01-01

    We have developed a duplex real-time RT-PCR assay for profiling antiviral inhibitors of four dengue virus (DENV) serotypes. In this assay, the primers and the probe for amplifying DENV were designed in the conserved regions of the genome after aligned more than 300 nucleotide sequences of four dengue serotypes deposited in the GeneBank. To discriminate the antiviral activity from the cytotoxicity of compounds, a housekeeping gene of the Vero cells, β-actin, was used to design the primers and the probe for the second set of PCR as an internal control, which is used to normalize the RNA levels of dengue-specific PCR due to the cellular toxicity of test compounds. For compound profiling, the duplex PCR is performed using LightCycler(®) in a single tube to simultaneously amplify both the dengue target gene and the Vero cell housekeeping gene from the compound-treated Vero cell lysates. This assay was validated against a panel of reference compounds. The results show that the universal primers and probe in this duplex RT-PCR assay can efficiently amplify all four dengue serotypes and that the PCR efficiency for both the dengue target gene and the Vero cells β-actin gene is 100%.

  4. Use of a Combined Duplex PCR/Dot Blot Assay for more sensitive genetic characterization

    Directory of Open Access Journals (Sweden)

    Erin Curry

    2008-01-01

    Full Text Available A reliable and sensitive method of genetic analysis is necessary to detect multiple specific nucleic acid sequences from samples containing limited template. The most widely utilized method of specific gene detection, polymerase chain reaction (PCR, imparts inconsistent results when assessing samples with restricted template, especially in a multiplex reaction when copies of target genes are unequal. This study aimed to compare two methods of PCR product analysis, fluorescent detection following agarose gel electrophoresis or dot blot hybridization with chemiluminescent evaluation, in the detection of a single copy gene (SRY and a multicopy gene (β-actin. Bovine embryo sex determination was employed to exploit the limited DNA template available and the target genes of unequal copies. Primers were used either independently or together in a duplex reaction with purified bovine genomic DNA or DNA isolated from embryos. When used independently, SRY and β-actin products were detected on a gel at the equivalent of 4-cell or 1-cell of DNA, respectively; however, the duplex reaction produced visible SRY bands at the 256 cell DNA equivalent and β-actin products at the 64 cell DNA equivalent. Upon blotting and hybridization of the duplex PCR reaction, product was visible at the 1–4 cell DNA equivalent. Duplex PCR was also conducted on 186 bovine embryos and product was subjected to gel electrophoresis or dot-blot hybridization in duplicate. Using PCR alone, sex determination was not possible for 22.6% of the samples. Using PCR combined with dot blot hybridization, 100.0% of the samples exhibited either both the male specific and β-actin products or the β-actin signal alone, indicating that the reaction worked in all samples. This study demonstrated that PCR amplification followed by dot blot hybridization provided more conclusive results in the evaluation of samples with low DNA concentrations and target genes of unequal copies.

  5. Development of quantitative duplex real-time PCR method for screening analysis of genetically modified maize.

    Science.gov (United States)

    Oguchi, Taichi; Onishi, Mari; Minegishi, Yasutaka; Kurosawa, Yasunori; Kasahara, Masaki; Akiyama, Hiroshi; Teshima, Reiko; Futo, Satoshi; Furui, Satoshi; Hino, Akihiro; Kitta, Kazumi

    2009-06-01

    A duplex real-time PCR method was developed for quantitative screening analysis of GM maize. The duplex real-time PCR simultaneously detected two GM-specific segments, namely the cauliflower mosaic virus (CaMV) 35S promoter (P35S) segment and an event-specific segment for GA21 maize which does not contain P35S. Calibration was performed with a plasmid calibrant specially designed for the duplex PCR. The result of an in-house evaluation suggested that the analytical precision of the developed method was almost equivalent to those of simplex real-time PCR methods, which have been adopted as ISO standard methods for the analysis of GMOs in foodstuffs and have also been employed for the analysis of GMOs in Japan. In addition, this method will reduce both the cost and time requirement of routine GMO analysis by half. The high analytical performance demonstrated in the current study would be useful for the quantitative screening analysis of GM maize. We believe the developed method will be useful for practical screening analysis of GM maize, although interlaboratory collaborative studies should be conducted to confirm this.

  6. Comparison of 454 Ultra-Deep Sequencing and Allele-Specific Real-Time PCR with Regard to the Detection of Emerging Drug-Resistant Minor HIV-1 Variants after Antiretroviral Prophylaxis for Vertical Transmission.

    Directory of Open Access Journals (Sweden)

    Andrea Hauser

    Full Text Available Pregnant HIV-infected women were screened for the development of HIV-1 drug resistance after implementation of a triple-antiretroviral transmission prophylaxis as recommended by the WHO in 2006. The study offered the opportunity to compare amplicon-based 454 ultra-deep sequencing (UDS and allele-specific real-time PCR (ASPCR for the detection of drug-resistant minor variants in the HIV-1 reverse transcriptase (RT.Plasma samples from 34 Tanzanian women were previously analysed by ASPCR for key resistance mutations in the viral RT selected by AZT, 3TC, and NVP (K70R, K103N, Y181C, M184V, T215Y/F. In this study, the RT region of the same samples was investigated by amplicon-based UDS for resistance mutations using the 454 GS FLX System.Drug-resistant HIV-variants were identified in 69% (20/29 of women by UDS and in 45% (13/29 by ASPCR. The absolute number of resistance mutations identified by UDS was twice that identified by ASPCR (45 vs 24. By UDS 14 of 24 ASPCR-detected resistance mutations were identified at the same position. The overall concordance between UDS and ASPCR was 61.0% (25/41. The proportions of variants quantified by UDS were approximately 2-3 times lower than by ASPCR. Amplicon generation from samples with viral loads below 20,000 copies/ml failed more frequently by UDS compared to ASPCR (limit of detection = 650 copies/ml, resulting in missing or insufficient sequence coverage.Both methods can provide useful information about drug-resistant minor HIV-1 variants. ASPCR has a higher sensitivity than UDS, but is restricted to single resistance mutations. In contrast, UDS is limited by its requirement for high viral loads to achieve sufficient sequence coverage, but the sequence information reveals the complete resistance patterns within the genomic region analysed. Improvements to the UDS limit of detection are in progress, and UDS could then facilitate monitoring of drug-resistant minor variants in the HIV-1 quasispecies.

  7. Detection and quantification of beef and pork materials in meat products by duplex droplet digital PCR.

    Science.gov (United States)

    Cai, Yicun; He, Yuping; Lv, Rong; Chen, Hongchao; Wang, Qiang; Pan, Liangwen

    2017-01-01

    Meat products often consist of meat from multiple animal species, and inaccurate food product adulteration and mislabeling can negatively affect consumers. Therefore, a cost-effective and reliable method for identification and quantification of animal species in meat products is required. In this study, we developed a duplex droplet digital PCR (dddPCR) detection and quantification system to simultaneously identify and quantify the source of meat in samples containing a mixture of beef (Bos taurus) and pork (Sus scrofa) in a single digital PCR reaction tube. Mixed meat samples of known composition were used to test the accuracy and applicability of this method. The limit of detection (LOD) and the limit of quantification (LOQ) of this detection and quantification system were also identified. We conclude that our dddPCR detection and quantification system is suitable for quality control and routine analyses of meat products.

  8. A simple Duplex-PCR to evaluate the DNA quality of anthropological and forensic samples prior short tandem repeat typing.

    Science.gov (United States)

    von Wurmb-Schwark, Nicole; Schwark, Thorsten; Harbeck, Michaela; Oehmichen, Manfred

    2004-04-01

    Typing of DNA from ancient or otherwise highly degraded material, e.g. formalin fixed tissues, can be difficult, time consuming and costly. Very often, genetic typing is not possible at all. We present an inexpensive and easy to use Duplex-PCR that amplifies a 164 bp fragment specific for nuclear DNA together with a 260 bp mitochondrial DNA fragment and that can be employed as a pretest prior to short tandem repeat (STR) typing. All together, we analyzed DNA from 20 ancient bones, 20 formalin fixed tissues and 20 other forensic samples in different concentrations. Each sample that failed in the presented Duplex-amplification was also negative for STR typing, while samples that showed strong and clear signals in the Duplex-PCR led to reproducible genetic profiles using the multiplex kits AmpFLSTR Identifiler and Powerplex ES. The Duplex-PCR worked as a reliable indicator of DNA quality in the sample.

  9. Sex determination in goat by amplification of the HMG box using duplex PCR.

    Science.gov (United States)

    Shi, Lei; Yue, Wenbin; Ren, Youshe; Lei, Fulin; Zhao, Junxing

    2008-05-01

    The objective of this study was to obtain a fast, accurate and reliable method of determining the sex of goat embryos prior to implantation through amplification of the high-motility-group (HMG) box of the sex-determining region of the Y chromosome (SRY) gene of the goats. Goat specific primers were designed for duplex polymerase chain reaction (PCR). As an internal control gene, the goat beta-action gene sequence was simultaneously amplified together with the HMG box of goat SRY gene. Males showed both 1 SRY band and 1 beta-action band, but only 1 beta-action band was present in the agarose gel electrophoresis of females. The result indicated that the goat HMG-box sequence motif of SRY was male specific. Afterward, the optimized PCR procedure was applied in 30 embryo biopsies and the biopsied embryos were transferred into 30 recipient female goats. The sex of the 13 kids proved anatomically corresponded to the sex determined by PCR (100% accuracy). Thus, this study showed that this duplex PCR method can be applied to sex the goat pre-implantation embryos and to manipulate the sex ratio of offspring in goat breeding programs.

  10. Allele-Specific DNA Methylation Detection by Pyrosequencing®

    DEFF Research Database (Denmark)

    Sommer Kristensen, Lasse; Johansen, Jens Vilstrup; Grønbæk, Kirsten

    2015-01-01

    DNA methylation is an epigenetic modification that plays important roles in healthy as well as diseased cells, by influencing the transcription of genes. In spite the fact that human somatic cells are diploid, most of the currently available methods for the study of DNA methylation do not provide......-effective protocol for allele-specific DNA methylation detection based on Pyrosequencing(®) of methylation-specific PCR (MSP) products including a single nucleotide polymorphism (SNP) within the amplicon....

  11. Detection of chicken contamination in beef meatball using duplex-PCR Cyt b gene

    Science.gov (United States)

    Sari, E. P.; Kartikasari, L. R.; Cahyadi, M.

    2017-04-01

    Beef is one of expensive animal protein sources compared to other meats, on the other hand, chicken is cheap animal protein source. Mixing of chicken into beef meatball is possibly performed to decrease production cost. The aim of this study was to detect chicken contamination in beef meatball using Cytochrome b (Cyt b) gene by duplex-PCR. Sample was designed and prepared as follows, 100% of chicken meatball, 100% of beef meatball and serial level of chicken contaminations in beef meatball (1, 5, 10 and 25%, respectively). Isolation of DNA genome from meatball was according to the guideline of gSYNCTM DNA Extraction Kit for animal tissue. The PCR reaction was carried out using KAPA2G Fast Multiplex Mix. This study found that the DNA genome was succesfully extracted. Moreover, chicken contamination in beef meatball was indicated by the presence of 227 bp DNA band on 2% of agarose gels. Current study revealed that duplex-PCR using Cyt b gene as a genetic marker was able to detect chicken contamination in beef meatball until 1% of chicken meat in the sample. It can be effectively used to identify contamination and also authenticate species origin in animal products to protect consumer from undesirable contents in the food.

  12. A new general model for predicting melting thermodynamics of complementary and mismatched B-form duplexes containing locked nucleic acids: application to probe design for digital PCR detection of somatic mutations.

    Science.gov (United States)

    Hughesman, Curtis; Fakhfakh, Kareem; Bidshahri, Roza; Lund, H Louise; Haynes, Charles

    2015-02-17

    quantitative PCR or dPCR assay. This potential is demonstrated by using the model to design allele-specific probes that completely discriminate and quantify clinically relevant mutant alleles (BRAF V600E and KIT D816V) in a dPCR assay.

  13. A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids

    Directory of Open Access Journals (Sweden)

    Wilson Zoe A

    2008-06-01

    Full Text Available Abstract Background Real-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule. Results We describe a real-time PCR technique employing attached universal duplex probes (AUDP, which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time PCR methods. AUDP involves one set of universal duplex probes in which the 5' end of the fluorescent probe (FP and a complementary quenching probe (QP lie in close proximity so that fluorescence can be quenched. The PCR primer pair with attached universal template (UT and the FP are identical to the UT sequence. We have shown that the AUDP technique can be used for detecting multiple target DNA sequences in both simplex and duplex real-time PCR assays for gene expression analysis, genotype identification, and genetically modified organism (GMO quantification with comparable sensitivity, reproducibility, and repeatability with other real-time PCR methods. Conclusion The results from GMO quantification, gene expression analysis, genotype identification, and GMO quantification using AUDP real-time PCR assays indicate that the AUDP real-time PCR technique has been successfully applied in nucleic acids analysis, and the developed AUDP real-time PCR technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost.

  14. Simultaneous detection of bovine and porcine DNA in pharmaceutical gelatin capsules by duplex PCR assay for Halal authentication.

    Science.gov (United States)

    Nikzad, Jafar; Shahhosseini, Soraya; Tabarzad, Maryam; Nafissi-Varcheh, Nastaran; Torshabi, Maryam

    2017-02-14

    In the pharmaceutical industry, hard- and soft-shelled capsules are typically made from gelatin, commonly derived from bovine and porcine sources. To ensure that pharmaceutical products comply with halal regulations in Muslim countries (no porcine products allowed), development of a valid, reliable, quick, and most importantly, cost-effective tests are of utmost importance. We developed a species-specific duplex polymerase chain reaction (PCR) assay targeting 149 bp porcine and 271 bp bovine mitochondrial DNA (mtDNA) to simultaneously detect both porcine and bovine DNA (in one reaction at the same time) in gelatin. Some additional simplex PCR tests (targeting 126 bp bovine and 212 bp porcine mtDNA) and real-time PCR using a commercially available kit (for identification of porcine DNA) were used to verify the selectivity and sensitivity of our duplex PCR. After optimization of DNA extraction and PCR methods, hard/soft pharmaceutical gelatin capsules (containing drug) were tested for the presence of porcine and/or bovine DNA. Duplex PCR detected the presence of as little as 0.1% porcine DNA, which was more accurate than the commercially available kit. Of all gelatin capsules tested (n = 24), 50% contained porcine DNA (pure porcine gelatin alone or in combination with bovine gelatin). Duplex PCR presents an easy-to-follow, quick, low-cost and reliable method to simultaneously detect porcine and bovine DNAs (>100 bp) in minute amounts in highly processed gelatin-containing pharmaceutical products (with a 0.1% sensitivity for porcine DNA) which may be used for halal authentication. Simultaneous detection of porcine and bovine DNA in gelatin capsules by duplex PCR.

  15. Development of duplex PCR for simultaneous detection of Theileria spp. and Anaplasma spp. in sheep and goats.

    Science.gov (United States)

    Cui, Yanyan; Zhang, Yan; Jian, Fuchun; Zhang, Longxian; Wang, Rongjun; Cao, Shuxuan; Wang, Xiaoxing; Yan, Yaqun; Ning, Changshen

    2017-05-01

    Theileria spp. and Anaplasma spp., which are important tick-borne pathogens (TBPs), impact the health of humans and animals in tropical and subtropical areas. Theileria and Anaplasma co-infections are common in sheep and goats. Following alignment of the relevant DNA sequences, two primer sets were designed to specifically target the Theileria spp. 18S rRNA and Anaplasma spp. 16S rRNA gene sequences. Genomic DNA from the two genera was serially diluted tenfold for testing the sensitivities of detection of the primer sets. The specificities of the primer sets were confirmed when DNA from Anaplasma and Theileria (positive controls), other related hematoparasites (negative controls) and ddH2O were used as templates. Fifty field samples were also used to evaluate the utility of single PCR and duplex PCR assays, and the detection results were compared with those of the PCR methods previously published. An optimized duplex PCR assay was established from the two primer sets based on the relevant genes from the two TBPs, and this assay generated products of 298-bp (Theileria spp.) and 139-bp (Anaplasma spp.). The detection limit of the assay was 29.4 × 10(-3) ng per μl, and there was no cross-reaction with the DNA from other hematoparasites. The results showed that the newly developed duplex PCR assay had an efficiency of detection (P > 0.05) similar to other published PCR methods. In this study, a duplex PCR assay was developed that can simultaneously identify Theileria spp. and Anaplasma spp. in sheep and goats. This duplex PCR is a potentially valuable assay for epidemiological studies of TBPs in that it can detect cases of mixed infections of the pathogens. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. A duplex real-time PCR assay for the quantitative detection of Naegleria fowleri in water samples.

    Science.gov (United States)

    Behets, Jonas; Declerck, Priscilla; Delaedt, Yasmine; Verelst, Lieve; Ollevier, Frans

    2007-01-01

    A fast and accurate duplex real-time PCR (qPCR) was developed to detect and quantify the human pathogenic amoeba Naegleria fowleri in water samples. In this study, primers and probe based on the Mp2Cl5 gene were designed to amplify and quantify N. fowleri DNA in a single duplex reaction. The qPCR detection limit (DL) corresponds to the minimum DNA quantity showing significant fluorescence with at least 90% of the positive controls in a duplex reaction. Using fluorescent Taqman technology the qPCR was found to be 100% specific for N. fowleri with a DL of 3 N. fowleri cell equivalents and a PCR efficiency of 99%. The quantification limit (QL) was 16 N. fowleri cell equivalents (corresponded with 320 N. fowleri cell equivalents l(-1) water sample) in a duplex qPCR reaction and corresponds to the lowest DNA quantity amplifiable with a coefficient of variation less than 25%. To detect inhibition an exogenous internal positive control (IPC) was included in each PCR reaction preventing false negative results. Comparison of qPCR and most probable number (MPN) culture results confirms that the developed qPCR is well suited for rapid and quantitative detection of this human pathogen in real water samples. Nevertheless 'low contamination levels' of water samples (fowleri cells l(-1)) still require culture method analyses. When other thermophilic Naegleria are very dominant, the MPN culture method could result in an underestimation in the real number of N. fowleri and some caution is necessary to interpret the data. The N. fowleri qPCR could be a useful tool to study further competitive phenomena between thermophilic Naegleria strains.

  17. A duplex PCR assay for the detection of Ralstonia solanacearum phylotype II strains in Musa spp.

    Directory of Open Access Journals (Sweden)

    Gilles Cellier

    Full Text Available Banana wilt outbreaks that are attributable to Moko disease-causing strains of the pathogen Ralstonia solanacearum (Rs remain a social and economic burden for both multinational corporations and subsistence farmers. All known Moko strains belong to the phylotype II lineage, which has been previously recognized for its broad genetic basis. Moko strains are paraphyletic and are distributed among seven related but distinct phylogenetic clusters (sequevars that are potentially major threats to Musaceae, Solanaceae, and ornamental crops in many countries. Although clustered within the Moko IIB-4 sequevar, strains of the epidemiologically variant IIB-4NPB do not cause wilt on Cavendish or plantain bananas; instead, they establish a latent infection in the vascular tissues of plantains and demonstrate an expanded host range and high aggressiveness toward Solanaceae and Cucurbitaceae. Although most molecular diagnostic methods focus on strains that wilt Solanaceae (particularly potato, no relevant protocol has been described that universally detects strains of the Musaceae-infecting Rs phylotype II. Thus, a duplex PCR assay targeting Moko and IIB-4NPB variant strains was developed, and its performance was assessed using an extensive collection of 111 strains representing the known diversity of Rs Moko-related strains and IIB-4NPB variant strains along with certain related strains and families. The proposed diagnostic protocol demonstrated both high accuracy (inclusivity and exclusivity and high repeatability, detected targets on either pure culture or spiked plant extracts. Although they did not belong to the Moko clusters described at the time of the study, recently discovered banana-infecting strains from Brazil were also detected. According to our comprehensive evaluation, this duplex PCR assay appears suitable for both research and diagnostic laboratories and provides reliable detection of phylotype II Rs strains that infect Musaceae.

  18. Duplex real-time PCR assay for rapid detection of ampicillin-resistant Enterococcus faecium.

    Science.gov (United States)

    Mohn, Stein Christian; Ulvik, Arve; Jureen, Roland; Willems, Rob J L; Top, Janetta; Leavis, Helen; Harthug, Stig; Langeland, Nina

    2004-02-01

    Rapid and accurate identification of carriers of resistant microorganisms is an important aspect of efficient infection control in hospitals. Traditional identification methods of antibiotic-resistant bacteria usually take at least 3 to 4 days after sampling. A duplex real-time PCR assay was developed for rapid detection of ampicillin-resistant Enterococcus faecium (ARE). Primers and probes that are used in this assay specifically detected the D-Ala-D-Ala ligase gene of E. faecium and the modified penicillin-binding protein 5 gene (pbp5) carrying the Glu-to-Val substitution at position 629 (Val-629) in a set of 129 tested E. faecium strains with known pbp5 sequence. Presence of the Val-629 in the strain set from 11 different countries was highly correlated with ampicillin resistance. In a screening of hospitalized patients, the real-time PCR assay yielded a sensitivity and a specificity for the detection of ARE colonization of 95% and 100%, respectively. The results were obtained 4 h after samples were harvested from overnight broth of rectal swab samples, identifying both species and the resistance marker mutation in pbp5. This novel assay reliably identifies ARE 2 to 3 days more quickly than traditional culture methods, thereby increasing laboratory throughput, making it useful for rectal screening of ARE. The assay demonstrates the advantages of real-time PCR for detection of nosocomial pathogens.

  19. Duplex real-time PCR for rapid simultaneous detection of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans in Amphibian samples.

    Science.gov (United States)

    Blooi, M; Pasmans, F; Longcore, J E; Spitzen-van der Sluijs, A; Vercammen, F; Martel, A

    2013-12-01

    Chytridiomycosis is a lethal fungal disease contributing to declines and extinctions of amphibian species worldwide. The currently used molecular screening tests for chytridiomycosis fail to detect the recently described species Batrachochytrium salamandrivorans. In this study, we present a duplex real-time PCR that allows the simultaneous detection of B. salamandrivorans and Batrachochytrium dendrobatidis. With B. dendrobatidis- and B. salamandrivorans-specific primers and probes, detection of the two pathogens in amphibian samples is possible, with a detection limit of 0.1 genomic equivalent of zoospores of both pathogens per PCR. The developed real-time PCR shows high degrees of specificity and sensitivity, high linear correlations (r(2) > 0.995), and high amplification efficiencies (>94%) for B. dendrobatidis and B. salamandrivorans. In conclusion, the described duplex real-time PCR can be used to detect DNA of B. dendrobatidis and B. salamandrivorans with highly reproducible and reliable results.

  20. Duplex real-time PCR for detection and quantification of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV) in Penaeus monodon.

    Science.gov (United States)

    Tang, Kathy F J; Lightner, Donald V

    2011-02-22

    We describe a duplex real-time PCR assay using TaqMan probes for the simultaneous detection of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV). Both MBV and HPV are shrimp enteric viruses that infect intestinal and hepatopancreatic epithelial cells. Both viruses can cause significant mortalities and depressed growth in infected larval, postlarval, and early juvenile stages of shrimp, and thus present a risk to commercial aquaculture. In this duplex assay, we combined 2 single real-time PCRs, amplifying MBV and HPV, in a one-tube PCR reaction. The 2 viruses were distinguished by specific fluorescent labels at the 5' end of TaqMan probes: the MBV probe was labeled with dichlorodimethoxyfluorescein (JOE), and the HPV probe was labeled with 6-carboxyfluorescein (FAM). The duplex real-time PCR assay was performed in a multi-channel real-time PCR detection system, and MBV and HPV amplification signals were separately detected by the JOE and FAM channels. This duplex assay was validated to be specific to the target viruses and found to have a detection limit of single copies for each virus. The dynamic range was found to be from 1 to 1 x 10(8) copies per reaction. This assay was further applied to quantify MBV and HPV in samples of infected Penaeus monodon collected from Malaysia, Indonesia, and Thailand. The specificity and sensitivity of this duplex real-time PCR assay offer a valuable tool for routine diagnosis and quantification of MBV and HPV from both wild and farmed shrimp stocks.

  1. Standardization and validation of simple PCR, duplex PCR and RAPD in comparison to blood smear examination for diagnosing bovine tropical theileriosis.

    Science.gov (United States)

    Sudan, Vikrant; Shanker, Daya; Jaiswal, Amit; Singh, Amit; Pandey, Vijay

    2017-03-01

    Bovine Tropical Theileriosis (BTT) is an important vector-borne protozoan disease that imposing serious constraints on the health and productivity of domestic cattle. It is matter of common fact that following recovery from primary infection, cattle become persistent carriers and act as reservoirs of infection thereby, playing a critical role in disease epidemiology. The present study describes the comparative diagnostic efficiency of simplex PCR, duplex PCR and RAPD assays for detection of Theileria annulata in cattle. An optimized simple PCR and duplex PCR assay were established using TAMS F/R as primer sets encoding for 721 bp amplicon alongside a RAPD with arbitrary primer coding for 963 bp product of T. annulata. The simple PCR and duplex PCR detected pathogen with almost same level of sensitivity, irrespective of whether its DNA was amplified in isolation or together with DNA of another pathogen without nonspecific amplifications. RAPD failed to give comparable results and suffered from limitations of sensitivity as well as specificity. The developed assays may be seen as a good tool for epidemiological studies aiming at assessing the burden of chronic infections and improving control of the associated diseases in endemic regions. Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  2. Rv1458c: a new diagnostic marker for identification of Mycobacterium tuberculosis complex in a novel duplex PCR assay.

    Science.gov (United States)

    Shrivastava, Kamal; Garima, Kushal; Narang, Anshika; Bhattacharyya, Kausik; Vishnoi, Ekta; Singh, Roshan Kumar; Chaudhry, Anil; Prasad, Rajendra; Bose, Mridula; Varma-Basil, Mandira

    2017-03-01

    We explored the efficiency of Rv1458c, the gene encoding a putative ABC drug transporter specific for the Mycobacterium tuberculosis complex (MTBC), as a diagnostic marker. A 190 bp region of Rv1458c and a 300 bp region of hsp65 were targeted in a novel duplex PCR assay and the results were compared with those for PCR restriction analysis(PRA) using the restriction enzymes NruI and BamHI. Species identification of a subset of the isolates (n=50) was confirmed by sequencing. Clinical isolates of M. tuberculosis (n=426) obtained from clinically suspected patients of pulmonary tuberculosis and mycobacterial (n=13) and non-mycobacterial (n=8) reference strains were included in the study. The duplex PCR assay correctly identified 320/426 isolates as MTBC and 106/426 isolates as non-tuberculous mycobacteria(NTM). The test was 100 % specific and sensitive when compared with NruI/BamHI PCR restriction analysis and highlighted the use of Rv1458c as a diagnostic marker for MTBC. The duplex PCR assay could be developed for use as a screening test to identify MTBC in clinical specimens in peripheral laboratories with limited resources.

  3. Duplex real-time PCR assay for rapid identification of Staphylococcus aureus isolates from dairy cow milk.

    Science.gov (United States)

    Pilla, Rachel; Snel, Gustavo G M; Malvisi, Michela; Piccinini, Renata

    2013-05-01

    Staphylococcus aureus isolates from dairy cow mastitis are not always consistent with the characteristic morphology described, and molecular investigation is often needed. The aim of the study was to develop a duplex real-time PCR assay for rapid identification of Staph. aureus isolates, targeting both nuc and Sa442. Overall, 140 isolates collected from dairy cow mastitis in 90 different herds, were tested. All strains had been identified using morphological and biochemical characteristics. DNA from each strain was amplified in real-time PCR assay, to detect nuc or Sa442. Thereafter, a duplex real-time PCR assay was performed, and specificity of the amplified products was assessed by high resolution melting curve analysis. Out of 124 Staph. aureus isolates, 33 did not show the typical morphology or enzymic activity; in 118 strains, the two melt-curve peaks consistent with nuc and Sa442 were revealed, while 2 isolates showed only the peak consistent with Sa442. Four isolates bacteriologically identified as Staph. aureus, were PCR-negative and were further identified as Staph. pseudintermedius by sequencing. Staph. pseudintermedius and coagulase-negative staphylococci did not carry nuc or Sa442. The results showed the correct identification of all isolates, comprehending also coagulase-or nuc-negative Staph. aureus, while other coagulase-positive Staphylococci were correctly identified as non-Staph. aureus. Both sensitivity and specificity were 100%. High resolution melting analysis allowed easy detection of unspecific products. Finally, the duplex real-time PCR was applied directly to 40 milk samples, to detect infected mammary quarters. The assay confirmed the results of bacteriological analysis, on Staph. aureus-positive or-negative samples. Therefore, the proposed duplex real-time PCR could be used in laboratory routine as a cost-effective and powerful tool for high-throughput identification of atypical Staph. aureus isolates causing dairy cow mastitis. Also, it

  4. Identification of pork contamination in meatball using genetic marker mitochondrial DNA cytochrome b gene by duplex-PCR

    Science.gov (United States)

    Novianty, E.; Kartikasari, L. R.; Lee, J. H.; Cahyadi, M.

    2017-04-01

    Meat based food products have a big opportunity to mix and adulterated with other meats. Muslim communities are prohibited to consume pork-containing product or other pig derivatives in food. Therefore, the high sensitivity, fast, cheap and accurate approach is needed to detect pig contamination in raw meat and meat-processed product such as meatball. The aim of this study was to identify pork contamination in meatball using genetic marker of mitochondrial DNA cytochrome b gene by duplex-PCR. Samples were prepared and designed by following the proportions 0, 1, 5, 10, 25% of pork in meatballs, respectively. The DNA genome was extracted from meatballs and polymerase chain reaction (PCR) was performed using species specific primer to isolate mt-DNA cytochrome b gene. The results showed that the DNA genome was successfully isolated from pork, beef, and contaminated meatballs. Furthermore, 2% agarose gels was able to visualize of duplex-PCR to identify pork contamination in meatballs up to very small proportion (1%). It can be concluded that duplex-PCR of mt-DNA cytochrome b gene was very sensitive to identify pork contamination in meatball with the presence of specific 398 bp DNA band.

  5. Use of site-directed mutagenesis of allele-specific PCR primers to identify the GSTM1 A, GSTM1 B, GSTM1 A,B and GSTM1 null polymorphisms at the glutathione S-transferase, GSTM1 locus.

    OpenAIRE

    Fryer, A A; Zhao, L.; Alldersea, J; Pearson, W R; Strange, R C

    1993-01-01

    We describe the identification of the GSTM1 null, GSTM1 A, GSTM1 B and GSTM1 A,B polymorphisms at the glutathione S-transferase GSTM1 locus using a single-step PCR method. Target DNA was amplified using primers to intron 6 and exon 7 with site-directed mutagenesis being used to introduce a restriction site in DNA amplified from GSTM1 *A, thereby allowing differentiation of this allele and GSTM1 *B. The accuracy of this approach in identifying the GSTM1 A, GSTM1 B, GSTM1 A,B and GSTM1 null pol...

  6. Application of three duplex real-time PCR assays for simultaneous detection of human seasonal and avian influenza viruses.

    Science.gov (United States)

    Stefańska, Ilona; Dzieciatkowski, Tomasz; Brydak, Lidia B; Romanowska, Magdalena

    2013-08-01

    This study was performed to develop real-time PCR (qPCR) for detection of human seasonal and avian influenza viruses in duplex format. First duplex qPCR detects haemagglutinin (HA) gene of influenza virus A(H1N1)pdm09 and HA gene of influenza virus A(H3N2), the second reaction detects neuraminidase (NA) gene of influenza virus A(H3N2) and NA gene of influenza virus A(H1N1)pdm09 and A(H5N1), and the third reaction detects HA gene of influenza A(H5N1) and nonstructural protein gene of influenza B virus. Primers and probes were designed using multiple alignments of target gene sequences of different reference strains. Assays were optimised for identical thermocycling conditions. Their specificity was confirmed by conventional PCR and monoplex qPCR with nucleic acids isolated from different influenza viruses and other respiratory pathogens. Plasmid constructs with a fragment of specific gene were used to assess sensitivity of the assay. The limit of detection ranged from 27 to 96 cDNA copies/reaction. Clinical specimens (n = 107) have been tested using new assays, immunofluorescence and monoplex qRT-PCR. It has been shown that developed assays have been capable of rapid and accurate simultaneous detection and differentiation of influenza viruses. They are more sensitive than immunofluorescence and at least as sensitive as monoplex qRT-PCR.

  7. A single-tube duplex and multiplex PCR for simultaneous detection of four cassava mosaic begomovirus species in cassava plants.

    Science.gov (United States)

    Aloyce, R C; Tairo, F; Sseruwagi, P; Rey, M E C; Ndunguru, J

    2013-04-01

    A single-tube duplex and multiplex PCR was developed for the simultaneous detection of African cassava mosaic virus (ACMV), East African cassava mosaic Cameroon virus (EACMCV), East African cassava mosaic Malawi virus (EACMMV) and East African cassava mosaic Zanzibar virus (EACMZV), four cassava mosaic begomoviruses (CMBs) affecting cassava in sub-Saharan Africa. Co-occurrence of the CMBs in cassava synergistically enhances disease symptoms and complicates their detection and diagnostics. Four primer pairs were designed to target DNA-A component sequences of cassava begomoviruses in a single tube PCR amplification using DNA extracted from dry-stored cassava leaves. Duplex and multiplex PCR enabled the simultaneous detection and differentiation of the four CMBs, namely ACMV (940bp), EACMCV (435bp), EACMMV (504bp) and EACMZV (260bp) in single and mixed infections, and sequencing results confirmed virus identities according to the respective published sequences of begomovirus species. In addition, we report here a modified Dellapotra et al. (1983) protocol, which was used to extract DNA from dry and fresh cassava leaves with comparable results. Using the duplex and multiplex techniques, time was saved and amount of reagents used were reduced, which translated into reduced cost of the diagnostics. This tool can be used by cassava breeders screening for disease resistance; scientists doing virus diagnostic studies; phytosanitary officers checking movement of diseased planting materials, and seed certification and multipliers for virus indexing.

  8. Cloned plasmid DNA fragments as calibrators for controlling GMOs: different real-time duplex quantitative PCR methods.

    Science.gov (United States)

    Taverniers, Isabel; Van Bockstaele, Erik; De Loose, Marc

    2004-03-01

    Analytical real-time PCR technology is a powerful tool for implementation of the GMO labeling regulations enforced in the EU. The quality of analytical measurement data obtained by quantitative real-time PCR depends on the correct use of calibrator and reference materials (RMs). For GMO methods of analysis, the choice of appropriate RMs is currently under debate. So far, genomic DNA solutions from certified reference materials (CRMs) are most often used as calibrators for GMO quantification by means of real-time PCR. However, due to some intrinsic features of these CRMs, errors may be expected in the estimations of DNA sequence quantities. In this paper, two new real-time PCR methods are presented for Roundup Ready soybean, in which two types of plasmid DNA fragments are used as calibrators. Single-target plasmids (STPs) diluted in a background of genomic DNA were used in the first method. Multiple-target plasmids (MTPs) containing both sequences in one molecule were used as calibrators for the second method. Both methods simultaneously detect a promoter 35S sequence as GMO-specific target and a lectin gene sequence as endogenous reference target in a duplex PCR. For the estimation of relative GMO percentages both "delta C(T)" and "standard curve" approaches are tested. Delta C(T) methods are based on direct comparison of measured C(T) values of both the GMO-specific target and the endogenous target. Standard curve methods measure absolute amounts of target copies or haploid genome equivalents. A duplex delta C(T) method with STP calibrators performed at least as well as a similar method with genomic DNA calibrators from commercial CRMs. Besides this, high quality results were obtained with a standard curve method using MTP calibrators. This paper demonstrates that plasmid DNA molecules containing either one or multiple target sequences form perfect alternative calibrators for GMO quantification and are especially suitable for duplex PCR reactions.

  9. Detection and prevalence of pathogenic Yersinia enterocolitica in refrigerated and frozen dairy products by duplex PCR and dot hybridization targeting the virF and ail genes.

    Science.gov (United States)

    Ye, Y W; Ling, N; Han, Y J; Wu, Q P

    2014-11-01

    Pathogenic Yersinia enterocolitica is involved in yersiniosis through expression of chromosome-borne or plasmid-borne virulence factors. Yersinia enterocolitica is a cold-tolerant pathogen frequently isolated from refrigerated or frozen foods. However, little attention has been focused on the prevalence of pathogenic Y. enterocolitica in refrigerated or frozen dairy samples in China. In this study, we developed a new duplex PCR targeting the plasmid-borne virF gene and chromosome-borne ail gene for detection of pathogenic Y. enterocolitica isolates. We established a detection limit for the duplex PCR of 6.5 × 10(2)cfu/mL in artificially contaminated dairy samples. In addition, the duplex PCR could detect directly 4.5 to 5.7 cfu of Y. enterocolitica in 5 mL of brain heart infusion broth after 6 h of enrichment at 28 °C. A newly developed dot hybridization assay further confirmed specificity of the duplex PCR for detection of virulent Y. enterocolitica. Furthermore, 13 Y. enterocolitica and 5 pathogenic strains, from 88 commercial frozen or refrigerated dairy products, were detected successfully by the China National Standard method (GB/T4789.8-2008) and the duplex PCR, respectively. Finally, biotypes and serotypes of pathogenic Y. enterocolitica strains were further characterized. The duplex PCR developed here is reliable for large-scale screening, routine monitoring, and risk assessment of pathogenic Y. enterocolitica in refrigerated or frozen dairy products.

  10. A Novel Pretreatment-Free Duplex Chamber Digital PCR Detection System for the Absolute Quantitation of GMO Samples

    Science.gov (United States)

    Zhu, Pengyu; Wang, Chenguang; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

    2016-01-01

    Digital polymerase chain reaction (PCR) has developed rapidly since it was first reported in the 1990s. However, pretreatments are often required during preparation for digital PCR, which can increase operation error. The single-plex amplification of both the target and reference genes may cause uncertainties due to the different reaction volumes and the matrix effect. In the current study, a quantitative detection system based on the pretreatment-free duplex chamber digital PCR was developed. The dynamic range, limit of quantitation (LOQ), sensitivity and specificity were evaluated taking the GA21 event as the experimental object. Moreover, to determine the factors that may influence the stability of the duplex system, we evaluated whether the pretreatments, the primary and secondary structures of the probes and the SNP effect influence the detection. The results showed that the LOQ was 0.5% and the sensitivity was 0.1%. We also found that genome digestion and single nucleotide polymorphism (SNP) sites affect the detection results, whereas the unspecific hybridization within different probes had little side effect. This indicated that the detection system was suited for both chamber-based and droplet-based digital PCR. In conclusion, we have provided a simple and flexible way of achieving absolute quantitation for genetically modified organism (GMO) genome samples using commercial digital PCR detection systems. PMID:26999129

  11. A Novel Pretreatment-Free Duplex Chamber Digital PCR Detection System for the Absolute Quantitation of GMO Samples

    Directory of Open Access Journals (Sweden)

    Pengyu Zhu

    2016-03-01

    Full Text Available Digital polymerase chain reaction (PCR has developed rapidly since it was first reported in the 1990s. However, pretreatments are often required during preparation for digital PCR, which can increase operation error. The single-plex amplification of both the target and reference genes may cause uncertainties due to the different reaction volumes and the matrix effect. In the current study, a quantitative detection system based on the pretreatment-free duplex chamber digital PCR was developed. The dynamic range, limit of quantitation (LOQ, sensitivity and specificity were evaluated taking the GA21 event as the experimental object. Moreover, to determine the factors that may influence the stability of the duplex system, we evaluated whether the pretreatments, the primary and secondary structures of the probes and the SNP effect influence the detection. The results showed that the LOQ was 0.5% and the sensitivity was 0.1%. We also found that genome digestion and single nucleotide polymorphism (SNP sites affect the detection results, whereas the unspecific hybridization within different probes had little side effect. This indicated that the detection system was suited for both chamber-based and droplet-based digital PCR. In conclusion, we have provided a simple and flexible way of achieving absolute quantitation for genetically modified organism (GMO genome samples using commercial digital PCR detection systems.

  12. A Novel Pretreatment-Free Duplex Chamber Digital PCR Detection System for the Absolute Quantitation of GMO Samples.

    Science.gov (United States)

    Zhu, Pengyu; Wang, Chenguang; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

    2016-03-18

    Digital polymerase chain reaction (PCR) has developed rapidly since it was first reported in the 1990s. However, pretreatments are often required during preparation for digital PCR, which can increase operation error. The single-plex amplification of both the target and reference genes may cause uncertainties due to the different reaction volumes and the matrix effect. In the current study, a quantitative detection system based on the pretreatment-free duplex chamber digital PCR was developed. The dynamic range, limit of quantitation (LOQ), sensitivity and specificity were evaluated taking the GA21 event as the experimental object. Moreover, to determine the factors that may influence the stability of the duplex system, we evaluated whether the pretreatments, the primary and secondary structures of the probes and the SNP effect influence the detection. The results showed that the LOQ was 0.5% and the sensitivity was 0.1%. We also found that genome digestion and single nucleotide polymorphism (SNP) sites affect the detection results, whereas the unspecific hybridization within different probes had little side effect. This indicated that the detection system was suited for both chamber-based and droplet-based digital PCR. In conclusion, we have provided a simple and flexible way of achieving absolute quantitation for genetically modified organism (GMO) genome samples using commercial digital PCR detection systems.

  13. Allelic-specific expression in relation to Bombyx mori resistance to Bt toxin.

    Science.gov (United States)

    Chen, Yazhou; Li, Muwang; Islam, Iftakher; You, Lang; Wang, Yueqiang; Li, Zhiqian; Ling, Lin; Zeng, Baosheng; Xu, Jun; Huang, Yongping; Tan, Anjiang

    2014-11-01

    Understanding the mechanism of Bt resistance is one of the key elements of the effective application of Bt in pest control. The lepidopteran model insect, the silkworm, demonstrates qualities that make it an ideal species to use in achieving this understanding. We screened 45 strains of silkworm (Bombyx mori) using a Cry1Ab toxin variant. The sensitivity levels of the strains varied over a wide range. A resistant strain (P50) and a phylogenetically related susceptible strain (Dazao) were selected to profile the expressions of 12 Bt resistance-related genes. The SNPs in these genes were detected based on EST analysis and were validated by allelic-specific PCR. A comparison of allelic-specific expression between P50 and Dazao showed that the transcript levels of heterozygous genes containing two alleles rather than an imbalanced allelic expression contribute more to the resistance of P50 against Bt. The responses of the allelic-specific expression to Bt in hybrid larvae were then investigated. The results showed that the gene expression pattern of an ATP-binding cassette transporter C2 (ABCC2) and an aminopeptidase N (APN3), changed in an allelic-specific manner, with the increase of the resistant allele expression correlated with larval survival. The results suggest that a trans-regulatory mechanism in ABCC2 and APN3 allelic-specific expression is involved in the insect's response to the Bt toxin. The potential role of allelic-specific gene regulation in insect resistance to Bt toxins is discussed.

  14. High-speed droplet-allele-specific polymerase chain reaction for genotyping of single nucleotide polymorphisms.

    Science.gov (United States)

    Matsuda, Kazuyuki; Honda, Takayuki

    2015-01-01

    Single nucleotide alternations such as single nucleotide polymorphisms (SNPs) or single nucleotide mutations are useful genetic markers for molecular diagnosis, prognosis, drug response, and predisposition to diseases. Rapid identification of SNPs or mutations is clinically important, especially for determining drug responses and selection of molecular-targeted therapy. Here, we describe a rapid genotyping assay based on the allele-specific polymerase chain reaction (AS-PCR) by using our droplet-PCR machine (droplet-AS-PCR).

  15. Allele specific expression and methylation in the bumblebee, Bombus terrestris

    Directory of Open Access Journals (Sweden)

    Zoë Lonsdale

    2017-09-01

    Full Text Available The social hymenoptera are emerging as models for epigenetics. DNA methylation, the addition of a methyl group, is a common epigenetic marker. In mammals and flowering plants methylation affects allele specific expression. There is contradictory evidence for the role of methylation on allele specific expression in social insects. The aim of this paper is to investigate allele specific expression and monoallelic methylation in the bumblebee, Bombus terrestris. We found nineteen genes that were both monoallelically methylated and monoallelically expressed in a single bee. Fourteen of these genes express the hypermethylated allele, while the other five express the hypomethylated allele. We also searched for allele specific expression in twenty-nine published RNA-seq libraries. We found 555 loci with allele-specific expression. We discuss our results with reference to the functional role of methylation in gene expression in insects and in the as yet unquantified role of genetic cis effects in insect allele specific methylation and expression.

  16. Development of a duplex real-time RT-PCR for the simultaneous detection and differentiation of Theiler's murine encephalomyelitis virus and rat theilovirus.

    Science.gov (United States)

    Yuan, Wen; Wang, Jing; Xu, Fengjiao; Huang, Bihong; Lian, Yuexiao; Rao, Dan; Yin, Xueqin; Wu, Miaoli; Zhu, Yujun; Zhang, Yu; Huang, Ren; Guo, Pengju

    2016-10-01

    Theiler's murine encephalomyelitis virus (TMEV) and rat theilovirus (RTV), the member of the genus Cardiovirus, are widespread in laboratory mice and rats, and are potential contaminants of biological materials. Cardioviruses infection may cause serious complications in biomedical research. To improve the efficiency of routine screening for Cardioviruses infection, a duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for simultaneous detection and differentiation of TMEV and RTV. The duplex assay was specific for reference strains of TMEV and RTV, and no cross-reaction was found with seven other rodent viruses. The limits of detection of both TMEV and RTV were 4×10(1) copies RNA/reaction. Reproducibility was estimated using standard dilutions, with coefficients of variation PCR and conventional RT-PCR. For 439 clinical samples,95 samples were positive for TMEV and 72 samples were positive for RTV using duplex real-time RT-PCR approach, whereas only 77 samples were positive for TMEV and 66 samples were positive for RTV when conventional RT-PCR was applied. Mixed infections were found in 20 samples when analyzed by conventional RT-PCR whereas 30 samples were found to be mixed infection when duplex real-time RT-PCR was applied. This duplex assay provides a useful tool for routine health monitoring and screening of contaminated biological materials of these two viruses.

  17. Development of a one-step duplex RT-qPCR for the quantification of phocine distemper virus.

    Science.gov (United States)

    Bogomolni, Andrea L; Frasca, Salvatore; Matassa, Keith A; Nielsen, Ole; Rogers, Kara; De Guise, Sylvain

    2015-04-01

    Worldwide, stranded marine mammals and the network personnel who respond to marine mammal mortality have provided much of the information regarding marine morbillivirus infections. An assay to determine the amount of virus present in tissue samples would be useful to assist in routine surveying of animal health and for monitoring large-scale die-off events. False negatives from poor-quality samples prevent determination of the true extent of infection, while only small amounts of tissue samples or archived RNA may be available at the time of collection for future retrospective analysis. We developed a one-step duplex real-time reverse transcriptase-quantitative-PCR assay (RT-qPCR) based on Taqman probe technology to quantify phocine distemper virus (PDV) isolated from an outbreak in harbor (Phoca vitulina concolor) and gray seals (Halichoerus grypus) along the northeast US coast in 2006. The glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) gene was selected to assess RNA quality. This duplex assay is specific for PDV and sensitive through a range of 10(0) to 10(9) copies ds-plasmid DNA. For the GAPDH target, the reaction in duplex amplified 10(0) to 10(9) copies of ds-plasmid DNA and was detectable in multiple seal species. This assay reduced the likelihood of false negative results due to degradation of tissues and well-to-well variability while providing sensitive and specific detection of PDV, which would be applicable in molecular epidemiologic studies and pathogen detection in field and laboratory investigations involving a variety of seal species.

  18. Development of allele-specific PCR for detection of herbicide-resistance in Monochoria korsakowii Regel et Maack%采用ASPCR技术检测抗药性雨久花ALS突变基因碱基种类的探讨

    Institute of Scientific and Technical Information of China (English)

    吴明根; 金万赫; 许勇男

    2011-01-01

    等位基因特异性扩增(Allele-specific PCR简称:ASPCR)技术是一项快速、简便、低成本的检测等位基因单核苷酸多态性的新方法.作者针对雨久花感抗磺酰脲类生态型ALS基因编码区第592位碱基密码(ALS氨基酸第197位)特征,采用在引物3'末端设有胸腺嘧啶核苷酸(感性)或鸟嘌呤(抗性)核苷酸的2种引物,对延边稻田发生的雨久花感抗磺酰脲类生态型ALS基因编码区第592位进行了ASPCR.检测结果显示,当以引物3'末端设有胸腺嘧啶引物时,感性类型出现ASPCR扩增带,而抗性类型无带出现,反之抗性类型有带而感性类型无带,检测到延边稻田发生的抗磺酰脲类雨久花生态型的ALS 197位点的脯氨酸被组氨酸替代,证明了该方法的有效性和可靠性.%Allele-specific PCR is a rapid, simple, and low-cost method for single-nucleotide polymorphism identification. Using this technique, a single-nucleotide polymorphism primer marker of thymine(susceptible) or guanine(resistant) at the 3'end on 592 point of ALS(acetolactate synthetase) gene encoding site (197 point of ALS amino acid) was designed to detect the herbicide-resistant type of Monochoria korsakowii Regel et Maack. in Yanbian region. The result shows that susceptibility primer marker can be used to detect the susceptibility biotype;otherwise can be detected resistant biotype; the cause of resistance to sulfonylurea herbicide type can be induced by substitution of Pro197 to histidine. It proved that the technique was efficient and feasible to detect ALS gene of herbicide-resistant type.

  19. Duplex Real-Time RT-PCR Assays for the Detection and Typing of Epizootic Haemorrhagic Disease Virus

    Science.gov (United States)

    Viarouge, Cyril; Breard, Emmanuel; Zientara, Stephan; Vitour, Damien; Sailleau, Corinne

    2015-01-01

    Epizootic haemorrhagic disease virus (EHDV) may cause severe clinical episodes in some species of deer and sometimes in cattle. Laboratory diagnosis provides a basis for the design and timely implementation of disease control measures. There are seven distinct EHDV serotypes, VP2 coding segment 2 being the target for serotype specificity. This paper reports the development and validation of eight duplex real-time RT-PCR assays to simultaneously amplify the EHDV target (S9 for the pan-EHDV real-time RT-PCR assay and S2 for the serotyping assays) and endogenous control gene Beta-actin. Analytical and diagnostic sensitivity and specificity, inter- and intra-assay variation and efficiency were evaluated for each assay. All were shown to be highly specific and sensitive. PMID:26161784

  20. RNA-FISH to analyze allele-specific expression.

    Science.gov (United States)

    Braidotti, G

    2001-01-01

    One of the difficulties associated with the analysis of imprinted gene expression is the need to distinguish RNA synthesis occurring at the maternal vs the paternally inherited copy of the gene. Most of the techniques used to examine allele-specific expression exploit naturally occurring polymorphisms and measure steady-state levels of RNA isolated from a pool of cells. Hence, a restriction fragment length polymorphism (RFLP) an be exploited in a heterozygote, by a reverse transcriptase polymerase chain reaction (RT-PCR)- based procedure, to analyze maternal vs paternal gene expression. The human IGF2R gene was analyzed in this way. Smrzka et al. (1) were thus able to show that the IGF2R gene possesses a hemimethylated, intronic CpG island analogous to the mouse imprinting box. However, IGF2R mRNA was detected that possessed the RFLP from both the maternal and paternal alleles in all but one of the 70 lymphoblastoid samples. (The one monoallelic sample reactivated its paternal allele with continued cell culturing.) It was concluded that monoallelic expression of the human gene is a polymorphic trait occurring in a small minority of all tested samples (reviewed in refs. 2,3). Although this is a sound conclusion, the question remains: Is the human IGF2R gene imprinted?

  1. Simultaneous detection of Fusarium culmorum and F. graminearum in plant material by duplex PCR with melting curve analysis

    Directory of Open Access Journals (Sweden)

    Karlovsky Petr

    2006-01-01

    Full Text Available Abstract Background Fusarium head blight (FHB is a disease of cereal crops, which has a severe impact on wheat and barley production worldwide. Apart from reducing the yield and impairing grain quality, FHB leads to contamination of grain with toxic secondary metabolites (mycotoxins, which pose a health risk to humans and livestock. The Fusarium species primarily involved in FHB are F. graminearum and F. culmorum. A key prerequisite for a reduction in the incidence of FHB is an understanding of its epidemiology. Results We describe a duplex-PCR-based method for the simultaneous detection of F. culmorum and F. graminearum in plant material. Species-specific PCR products are identified by melting curve analysis performed in a real-time thermocycler in the presence of the fluorescent dye SYBR Green I. In contrast to multiplex real-time PCR assays, the method does not use doubly labeled hybridization probes. Conclusion PCR with product differentiation by melting curve analysis offers a cost-effective means of qualitative analysis for the presence of F. culmorum and F. graminearum in plant material. This method is particularly suitable for epidemiological studies involving a large number of samples.

  2. Comparison of CYP2D6 genotyping by allele-specific PCR with DXT phe-notype and gene chip testing%CYP2D6 PCR基因型与DXT表型和基因芯片检测的比较

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    目的:为了评价CYP2D6的基因型和表型的联系以及基因芯片在CYP2D6多基因分析中的应用.方法:242健康志愿者,口服dextromethorphan后收集尿液测定其代谢率,收集20ml血提取DNA,并通过基因特异性PCR和/(或)基因芯片分析CYP2D6*2--*11,*17和多拷贝CYP2D6基因,其中5个基因(*3、*4、*6、*7和*9)用PCR和CYD450基因芯片同时分析.结果:CYP2D6基因型比表型更富有信息和更能反映CYP2D6酶的表达.CYP2D6*3、*4、*6、*7和*9的基因检测在CYP450基因芯片和基因特异性PCR中显示高度的一致性.结论:基因芯片在检测基因多位点的多基因中是一个有发展前途和可靠的方法.%To evaluate association of genotype and phenotype of CYP2D6 and the application of oligonucleotide microarray hybridization genetic testing in CYP2D6 multiple alleles analyses. METHODS: Two hundred forty-two healthy volunteers were recruited, and a 60 mg oral dose of dextromethorphan (DXT) was administered to each for assessment of the DXT metabolic ratio [ MR]. A 20 ml blood sample was also collected for DNA isolation and testing. CYP2D6 alleles * 2-*11; * 17 and multiple CYP2D6 gene copies were tested by allele-specific PCR and/or the affymetrix CYP450 gene chip assay. Five of the CYP2D6 alleles ( * 3, * 4, *6, * 7, and * 9) were evaluated by both PCR and the CYP450 gene chip assay. RESULTS: The CYP2D6genotype was more informative and reflective in CYP2D6 enzyme expression than a phenotype. Genetic tests for the CYP2D6 * 3, * 4, * 6, * 7 and * 9 alleles showed a high degree of concordance between the CYP450 gene chip and AS-PCR methods. CONCLUSION: Oligonucleotide microarray hybridization is a promising and reliable approach for detecting multiple alleles at gene loci.

  3. Development and Evaluation of a Single-Step Duplex PCR for Simultaneous Detection of Fasciola hepatica and Fasciola gigantica (Family Fasciolidae, Class Trematoda, Phylum Platyhelminthes)

    Science.gov (United States)

    Nguyen, Khue Thi; Nguyen, Nga Thi Bich; Doan, Huong Thi Thanh; Le, Xuyen Thi Kim; Hoang, Chau Thi Minh; De, Nguyen Van

    2012-01-01

    A single-step multiplex PCR (here referred to as a duplex PCR) has been developed for simultaneous detection and diagnosis of Fasciola hepatica and F. gigantica. These species overlap in distribution in many countries of North and East Africa and Central and Southeast Asia and are similar in egg morphology, making identification from fecal samples difficult. Based on a comparative alignment of mitochondrial DNA (mtDNA) spanning the region of cox1-trnT-rrnL, two species-specific forward primers were designed, FHF (for F. hepatica) and FGF (for F. gigantica), and a single reverse primer, FHGR (common for both species). Conventional PCR followed by sequencing was applied using species-specific primer pairs to verify the specificity of primers and the identity of Fasciola DNA templates. Duplex PCR (using three primers) was used for testing with the DNA extracted from adult worms, miracidia, and eggs, producing amplicons of 1,031 bp for F. hepatica and 615 bp for F. gigantica. The duplex PCR failed to amplify from DNA of other common liver and intestinal trematodes, including two opisthorchiids, three heterophyids, an echinostomid, another fasciolid, and a taeniid cestode. The sensitivity assay showed that the duplex PCR limit of detection for each Fasciola species was between 0.012 ng and 0.006 ng DNA. Evaluation using DNA templates from 32 Fasciola samples (28 adults and 4 eggs) and from 25 field-collected stools of ruminants and humans revealed specific bands of the correct size and the presence of Fasciola species. This novel mtDNA duplex PCR is a sensitive and fast tool for accurate identification of Fasciola species in areas of distributional and zonal overlap. PMID:22692744

  4. Development and evaluation of a single-step duplex PCR for simultaneous detection of Fasciola hepatica and Fasciola gigantica (family Fasciolidae, class Trematoda, phylum Platyhelminthes).

    Science.gov (United States)

    Le, Thanh Hoa; Nguyen, Khue Thi; Nguyen, Nga Thi Bich; Doan, Huong Thi Thanh; Le, Xuyen Thi Kim; Hoang, Chau Thi Minh; De, Nguyen Van

    2012-08-01

    A single-step multiplex PCR (here referred to as a duplex PCR) has been developed for simultaneous detection and diagnosis of Fasciola hepatica and F. gigantica. These species overlap in distribution in many countries of North and East Africa and Central and Southeast Asia and are similar in egg morphology, making identification from fecal samples difficult. Based on a comparative alignment of mitochondrial DNA (mtDNA) spanning the region of cox1-trnT-rrnL, two species-specific forward primers were designed, FHF (for F. hepatica) and FGF (for F. gigantica), and a single reverse primer, FHGR (common for both species). Conventional PCR followed by sequencing was applied using species-specific primer pairs to verify the specificity of primers and the identity of Fasciola DNA templates. Duplex PCR (using three primers) was used for testing with the DNA extracted from adult worms, miracidia, and eggs, producing amplicons of 1,031 bp for F. hepatica and 615 bp for F. gigantica. The duplex PCR failed to amplify from DNA of other common liver and intestinal trematodes, including two opisthorchiids, three heterophyids, an echinostomid, another fasciolid, and a taeniid cestode. The sensitivity assay showed that the duplex PCR limit of detection for each Fasciola species was between 0.012 ng and 0.006 ng DNA. Evaluation using DNA templates from 32 Fasciola samples (28 adults and 4 eggs) and from 25 field-collected stools of ruminants and humans revealed specific bands of the correct size and the presence of Fasciola species. This novel mtDNA duplex PCR is a sensitive and fast tool for accurate identification of Fasciola species in areas of distributional and zonal overlap.

  5. Evaluation of two singleplex reverse transcription-Insulated isothermal PCR tests and a duplex real-time RT-PCR test for the detection of porcine epidemic diarrhea virus and porcine deltacoronavirus.

    Science.gov (United States)

    Zhang, Jianqiang; Tsai, Yun-Long; Lee, Pei-Yu Alison; Chen, Qi; Zhang, Yan; Chiang, Cheng-Jen; Shen, Yu-Han; Li, Fu-Chun; Chang, Hsiao-Fen Grace; Gauger, Phillip C; Harmon, Karen M; Wang, Hwa-Tang Thomas

    2016-08-01

    Recent outbreaks of porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) in multiple countries have caused significant economic losses and remain a serious challenge to the swine industry. Rapid diagnosis is critical for the implementation of efficient control strategies before and during PEDV and PDCoV outbreaks. Insulated isothermal PCR (iiPCR) on the portable POCKIT™ device is user friendly for on-site pathogen detection. In the present study, a singleplex PEDV RT-iiPCR, a singleplex PDCoV RT-iiPCR, and a duplex PEDV/PDCoV real-time RT-PCR (rRT-PCR) commercial reagents targeting the M gene were compared to an N gene-based PEDV rRT-PCR and an M gene-based PDCoV rRT-PCR that were previously published and used as reference PCRs. All PCR assays were highly specific and did not cross react with other porcine enteric pathogens. Analytical sensitivities of the PEDV RT-iiPCR, PDCoV RT-iiPCR and duplex PEDV/PDCoV rRT-PCR were determined using in vitro transcribed RNA as well as viral RNA extracted from ten-fold serial dilutions of PEDV and PDCoV cell culture isolates. Performance of each PCR assay was further evaluated using 170 clinical samples (86 fecal swabs, 24 feces, 19 intestines, and 41 oral fluids). Compared to the reference PEDV rRT-PCR, the sensitivity, specificity and accuracy of the PEDV RT-iiPCR were 97.73%, 98.78%, and 98.24%, respectively, and those of the duplex PEDV/PDCoV rRT-PCR were 98.86%, 96.34%, and 97.65%, respectively. Compared to the reference PDCoV rRT-PCR, the sensitivity, specificity and accuracy of the PDCoV RT-iiPCR were 100%, 100%, and 100%, respectively, and those of the PEDV/PDCoV duplex rRT-PCR were 96.34%, 100%, and 98.24%, respectively. Overall, all three new PCR assays were comparable to the reference rRT-PCRs for detection of PEDV and/or PDCoV. The PEDV and PDCoV RT-iiPCRs are potentially useful tools for on-site detection and the duplex PEDV/PDCoV rRT-PCR provides a convenient method to simultaneously detect

  6. Duplex PCR assay for the detection of avian adeno virus and chicken anemia virus prevalent in Pakistan

    Directory of Open Access Journals (Sweden)

    Iqbal Aqib

    2011-09-01

    Full Text Available Abstract Avian Adeno viruses and Chicken Anemia Viruses cause serious economic losses to the poultry industry of Pakistan each year. Timely and efficient diagnosis of the viruses is needed in order to practice prevention and control strategies. In the first part of this study, we investigated broilers, breeder and Layer stocks for morbidity and mortality rates due to AAV and CAV infections and any co-infections by examining signs and symptoms typical of their infestation or post mortem examination. In the second part of the study, we developed a duplex PCR assay for the detection of AAV and CAV which is capable to simultaneously detect both the viral types prevalent in Pakistan with high sensitivity and 100% specificity.

  7. Development and application of absolute quantitative detection by duplex chamber-based digital PCR of genetically modified maize events without pretreatment steps.

    Science.gov (United States)

    Zhu, Pengyu; Fu, Wei; Wang, Chenguang; Du, Zhixin; Huang, Kunlun; Zhu, Shuifang; Xu, Wentao

    2016-04-15

    The possibility of the absolute quantitation of GMO events by digital PCR was recently reported. However, most absolute quantitation methods based on the digital PCR required pretreatment steps. Meanwhile, singleplex detection could not meet the demand of the absolute quantitation of GMO events that is based on the ratio of foreign fragments and reference genes. Thus, to promote the absolute quantitative detection of different GMO events by digital PCR, we developed a quantitative detection method based on duplex digital PCR without pretreatment. Moreover, we tested 7 GMO events in our study to evaluate the fitness of our method. The optimized combination of foreign and reference primers, limit of quantitation (LOQ), limit of detection (LOD) and specificity were validated. The results showed that the LOQ of our method for different GMO events was 0.5%, while the LOD is 0.1%. Additionally, we found that duplex digital PCR could achieve the detection results with lower RSD compared with singleplex digital PCR. In summary, the duplex digital PCR detection system is a simple and stable way to achieve the absolute quantitation of different GMO events. Moreover, the LOQ and LOD indicated that this method is suitable for the daily detection and quantitation of GMO events. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Quantification of viable Escherichia coli O157:H7 in meat products by duplex real-time PCR assays.

    Science.gov (United States)

    Gordillo, Rubén; Rodríguez, Alicia; Werning, María L; Bermúdez, Elena; Rodríguez, Mar

    2014-02-01

    Rapid and specific detection of viable Escherichia coli O157:H7 cells in ready-to-eat (RTE) meat products, by duplex quantitative PCR (qPCR) procedures with mRNA and SYBR Green and TaqMan methodologies were developed. Specific primers and probes were designed based on the serotype of E. coli O157:H7, fliCh7 and rfbE genes. No cross-reactivity with other microorganisms was observed. The detection limit of the assays was 10(1) or 10(2)CFU/g for artificially contaminated meat products, and after a 4h enrichment period at 37 °C, the detection limit decreased to about 1 CFU/g. Time-to completion of the assay was approximately 8h. Thus, these qPCR methods offer a useful, rapid and efficient tool for screening viable E. coli O157:H7 in RTE meat products. This tool could also be proposed for monitoring these foodborne pathogens in HACCP programs.

  9. Quantitative detection of Borrelia burgdorferi sensu lato in erythema migrans skin lesions using internally controlled duplex real time PCR.

    Science.gov (United States)

    O'Rourke, Maria; Traweger, Andreas; Lusa, Lara; Stupica, Dasa; Maraspin, Vera; Barrett, P Noel; Strle, Franc; Livey, Ian

    2013-01-01

    B. burgdorferi sensu stricto, B. afzelii, B. garinii and B. bavariensis are the principal species which account for Lyme borreliosis (LB) globally. We have developed an internally controlled duplex quantitative real time PCR assay targeting the Borrelia 16S rRNA and the human RNAseP genes. This assay is well-suited for laboratory confirmation of suspected cases of LB and will be used to assess the efficacy of a vaccine against LB in clinical trials. The assay is highly specific, successfully detecting DNA extracted from 83 diverse B. burgdorferi sensu lato strains representing all major species causing LB, while 21 unrelated microbial species and human genomic DNA tested negative. The assay was highly reproducible and sensitive, with a lower limit of detection of 6 copies per PCR reaction. Together with culture, the assay was used to evaluate paired 3 mm skin biopsy samples taken from 121 patients presenting with solitary erythema migrans (EM) lesion. PCR testing identified more positive biopsy samples than culture (77.7% PCR positive versus 55.1% culture positive) and correctly identified all specimens scored as culture positive. OspA-based typing identified the majority of isolates as B. afzelii (96.8%) and the bacterial load was significantly higher in culture positive biopsies than in culture negative biopsies (Phistory of LB (P = 0.10). This is the first quantitative PCR study of human skin biopsies predominantly infected with B. afzelii and the first study to demonstrate a clear relationship between clinical symptoms in B. afzelii-infected patients and Borrelia burden.

  10. 用等位基因特异性PCR/限制性片段长度多态性策略构建原发性青光眼致病基因CYP1B1的单倍型%Construction of CYP1B1 gene haplotypes predisposing to primary congenital glaucoma through allele-specific PCR/restriction fragment length polymorphism analysis

    Institute of Scientific and Technical Information of China (English)

    张爱平; 李圣杰; 欧阳琦; 汤荔; 王晓蕾; 吉建; 曹文俊

    2015-01-01

    目的 建立等位基因特异性PCR/限制性片段长度多态性(allele-specific PCR/restriction fragment length polymorphism,AS-PCR/RFLP)法检测原发性先天性青光眼(primary congenital glaucoma,PCG)致病基因CYP1B1常见单核苷酸多态性(single nucleotide polymorphisms,SNPs)及其单倍型的方法.方法 收集20例原发性先天性青光眼患者和20名正常对照为研究对象,首先经测序筛查SNP位点后,再分别以PCR-RFLP和AS-PCR/RFLP策略构建CYP1B1基因rs10012(S1)和rs1056827(S2)及rs1056836(S3)和rs1056837(S4)位点的单倍型,并对这两种策略进行评价.结果 测序共发现4个SNP位点,为第2外显子rs10012 G/C(S1)及rs1056827 T/G (S2)、第3外显子rs1056836C/G(S3)及rs1056837T/C(S4).这些位点在PCG患者和正常对照中的分布呈现不同特点,同时存在rs10012 (S1)和rs1056827(S2)位点的PCG患者和正常对照分别为10例(50%)和5人(25%);同时存在rs1056836 (S3)和rs1056837(S4)位点的PCG患者和正常对照分别为5例(25%)和2人(10%);均未发现上述SNP位点单独存在.确定各位点的分布特点后,首先用PCR-RFLP策略构建rs10012(S1)和rs1056827(S2)位点单倍型,显示杂合突变型除出现目的条带外还存在底物条带,虽提示同时存在rs10012(S1)和rs1056827 (S2),但仍不能证实存在位点间的连锁;AS-PCR/RFLP构建的结果显示,AS-PCR扩增rs10012(S1)位点获得阳性结果的同时,针对rs1056827 (S2)位点的特异性RFLP分析也获得阳性结果.AS-PCR/RFLP对位点rs1056836(S3)和rs1056837 (S4)的分析获得了类似的结果.应用AS-PCR/RFLP成功构建了C-G[rs10012(S1)-rs1056827(S2)]和G-C[rs1056836 (S3)-rs1056837 (S4)]两种单倍型.结论 应用AS-PCR/RFLP策略成功构建PCG致病基因CYP1B1单倍型,该方法准确高效特异可用于构建遗传性疾病基因的单倍型.%Objective To develop an allele-specific PCR (AS-PCR)/restriction fragment length polymorphism (RFLP) assay for CYP1B1 gene haplotypes

  11. Optimized Pan-species and Speciation Duplex Real-time PCR Assays for Plasmodium Parasites Detection in Malaria Vectors

    Science.gov (United States)

    Sandeu, Maurice Marcel; Moussiliou, Azizath; Moiroux, Nicolas; Padonou, Gilles G.; Massougbodji, Achille; Corbel, Vincent; Tuikue Ndam, Nicaise

    2012-01-01

    Background An accurate method for detecting malaria parasites in the mosquito’s vector remains an essential component in the vector control. The Enzyme linked immunosorbent assay specific for circumsporozoite protein (ELISA-CSP) is the gold standard method for the detection of malaria parasites in the vector even if it presents some limitations. Here, we optimized multiplex real-time PCR assays to accurately detect minor populations in mixed infection with multiple Plasmodium species in the African malaria vectors Anopheles gambiae and Anopheles funestus. Methods Complementary TaqMan-based real-time PCR assays that detect Plasmodium species using specific primers and probes were first evaluated on artificial mixtures of different targets inserted in plasmid constructs. The assays were further validated in comparison with the ELISA-CSP on 200 field caught Anopheles gambiae and Anopheles funestus mosquitoes collected in two localities in southern Benin. Results The validation of the duplex real-time PCR assays on the plasmid mixtures demonstrated robust specificity and sensitivity for detecting distinct targets. Using a panel of mosquito specimen, the real-time PCR showed a relatively high sensitivity (88.6%) and specificity (98%), compared to ELISA-CSP as the referent standard. The agreement between both methods was “excellent” (κ = 0.8, PPlasmodium DNA between the two Anopheles species analyzed showed no significant difference (P = 0, 2). All infected mosquito samples contained Plasmodium falciparum DNA and mixed infections with P. malariae and/or P. ovale were observed in 18.6% and 13.6% of An. gambiae and An. funestus respectively. Plasmodium vivax was found in none of the mosquito samples analyzed. Conclusion This study presents an optimized method for detecting the four Plasmodium species in the African malaria vectors. The study highlights substantial discordance with traditional ELISA-CSP pointing out the utility of employing an accurate

  12. Identification of Orientia tsutsugamushi,spotted fever group and typhus group Rickettsia by duplex and nested PCR methods

    Institute of Scientific and Technical Information of China (English)

    M-C Luan; D-Z Yu; L Tang; L-J Zhang

    2008-01-01

    Objective:To identify members of genera of rickettsia and O.tsutsugamushi simultaneously.Methods:Rapid and duplex and nested PCR methods have been established by designing primers based on the conserved re-gions of heat shock protein GroEL gene.345 mouse viscera samples including liver,spleen and kidney,96 Xe-nopsylla cheopis and 32 chiggers collected from Hongta areas of Yuxi city,Yunnan province were tested by the new PCR methods.Results:The result of the study showed that the new PCR methods could identify most members of genera -Rickettsia and Orientia simultaneously with 100% specificity and its sensitivity could test one copy per microliter.The results of detection prevalence of rickettsioses in mouse,flea and mites DNA sam-ples showed that the total rickettsia infection rate in mouse was 34.78% (120 /345).The total infection rates in R.typhi,O.t Karp and R.sibirica of mouse samples were 28.12% (97 /345),19.71% (68 /345)and 0. 29% (1 /345)respectively.Co-infection rates in R.typhi and O.t Karp of mouse samples were 13.33% (46 /345).O.t Karp type has been the main epidemic strain in these areas.Conclusion:We concluded that this PCR method could be used to detect multi-genera rickettsia simultaneously.Molecular evidences provided in this and previous studies strongly support that Hongta areas of Yuxi city are a natural focus for typhus and scrub typhus with the common occurrence of their confection.

  13. Interlaboratory validation of quantitative duplex real-time PCR method for screening analysis of genetically modified maize.

    Science.gov (United States)

    Takabatake, Reona; Koiwa, Tomohiro; Kasahara, Masaki; Takashima, Kaori; Futo, Satoshi; Minegishi, Yasutaka; Akiyama, Hiroshi; Teshima, Reiko; Oguchi, Taichi; Mano, Junichi; Furui, Satoshi; Kitta, Kazumi

    2011-01-01

    To reduce the cost and time required to routinely perform the genetically modified organism (GMO) test, we developed a duplex quantitative real-time PCR method for a screening analysis simultaneously targeting an event-specific segment for GA21 and Cauliflower Mosaic Virus 35S promoter (P35S) segment [Oguchi et al., J. Food Hyg. Soc. Japan, 50, 117-125 (2009)]. To confirm the validity of the method, an interlaboratory collaborative study was conducted. In the collaborative study, conversion factors (Cfs), which are required to calculate the GMO amount (%), were first determined for two real-time PCR instruments, the ABI PRISM 7900HT and the ABI PRISM 7500. A blind test was then conducted. The limit of quantitation for both GA21 and P35S was estimated to be 0.5% or less. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSD(R)). The determined bias and RSD(R) were each less than 25%. We believe the developed method would be useful for the practical screening analysis of GM maize.

  14. Detection of invasive infection caused by Fusarium solani and non-Fusarium solani species using a duplex quantitative PCR-based assay in a murine model of fusariosis.

    Science.gov (United States)

    Bernal-Martínez, Leticia; Buitrago, Maria J; Castelli, Maria V; Rodríguez-Tudela, Juan L; Cuenca-Estrella, Manuel

    2012-04-01

    A duplex Real Time PCR (RT-PCR) assay for detecting DNA of members of the genus Fusarium has been developed and validated by using two mouse models of invasive infection. The duplex RT-PCR technique employed two specific molecular beacon probes targeting a highly conserved region of the fungal rDNA gene. This technique showed a detection limit of 10 fg DNA per μl of sample and a specificity of 100%. The sensitivity in a total of 48 samples from a murine model of Fusarium solani infection was 93.9% for lung tissues and 86.7% for serum samples. In comparison, the sensitivity in a total of 45 samples of a F. oxysporum murine model infection was 87% for lung tissues and 42.8% for serum samples. This molecular technique could be a reliable method for the quantification and the evaluation of the disease in animal models and for the clinical diagnosis of fusariosis.

  15. Duplex Real-Time PCR Method for the Differentiation of Cronobacter sakazakii and Cronobacter malonaticus.

    Science.gov (United States)

    Li, Xiaofang; Cui, Jinghua; DU, Xiaoli; Cui, Zhigang; Huang, Yibing; Kan, Biao

    2017-01-01

    Cronobacter sakazakii and Cronobacter malonaticus are the most common species of Cronobacter , so it is necessary to detect the two species as soon as possible in surveillance programs. We developed a real-time PCR method for identifying C. sakazakii and C. malonaticus from the genus Cronobacter . In this study, the two pairs of primers and probes were designed, targeting 16S rRNA and fusA, respectively. The specificity of the real-time PCR assay was validated with 112 strains of Cronobacter , including 56 C. sakazakii , 32 C. malonaticus , 16 Cronobacter dublinensis , 6 Cronobacter turicensis , and 2 Cronobacter muytjensii . The results showed that C. sakazakii and C. malonaticus were all correctly identified, consistent with the results of another method by analyzing the clustering of the fusA sequence. The detection limit for pure culture was 10(2) CFU/ml and 10(3) CFU/g for artificially contaminated rehydrated powdered infant formula. Therefore, the developed real-time PCR was a rapid, sensitive, and reliable method for the identification of C. sakazakii and C. malonaticus .

  16. DETECTION OF PORK CONTAMINATION IN FRESH AND COOKED BEEF USING GENETIC MARKER MITOCHONDRIAL-DNA CYTOCHROME B BY DUPLEX-PCR

    Directory of Open Access Journals (Sweden)

    A. Ni’mah

    2016-03-01

    Full Text Available By mixing with pork, beef adulteration is frequently found in the traditional  market that very disturbing Moeslem community in Indonesia. This study was conducted to detect pork contamination in fresh and cooked beef using genetic marker mitochondrial DNA cytochrome b (mt-DNA Cyt b by duplex-PCR. A total of twelve samples was used in this study consisting six fresh meat samples and six cooked meat samples, respectively. Those beef and pork were bought from animal slaughterhouse and a supermarket in Surakarta. Cooked samples were prepared by boiling the meats in hot water at 100oC for 30 minutes. We designed pork contamination in beef in the level of 0, 1, 5, 10, 25%, respectively. The DNA genome was extracted and polymerase chain reaction (PCR was performed using species specific primer to isolate mt-DNA Cyt b gene from the samples. The results showed that the DNA genome was successfully extracted from pork, beef, and contaminated meat samples. In addition, visualization of duplex-PCR on 1.5% agarose gel was able to detect pork contamination in both fresh and cooked beef up to very small proportion (1%. The existence of pork in beef was indicated with the presence of specific 398 bp DNA band. It can be concluded, duplex-PCR of mt-DNA Cyt b gene was very sensitive in detection of pork contamination in fresh and cooked beef.

  17. Single-cell duplex RT-LATE-PCR reveals Oct4 and Xist RNA gradients in 8-cell embryos

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    Hartung Odelya

    2007-12-01

    Full Text Available Abstract Background The formation of two distinctive cell lineages in preimplantation mouse embryos is characterized by differential gene expression. The cells of the inner cell mass are pluripotent and express high levels of Oct4 mRNA, which is down-regulated in the surrounding trophectoderm. In contrast, the trophectoderm of female embryos contains Xist mRNA, which is absent from cells of the inner mass. Prior to blastocyst formation, all blastomeres of female embryos still express both of these RNAs. We, thus, postulated that simultaneous quantification of Oct4 and Xist transcripts in individual blastomeres at the 8-cell stage could be informative as to their subsequent fate. Testing this hypothesis, however, presented numerous technical challenges. We overcame these difficulties by combining PurAmp, a single-tube method for RNA preparation and quantification, with LATE-PCR, an advanced form of asymmetric PCR. Results We constructed a duplex RT-LATE-PCR assay for real-time measurement of Oct4 and Xist templates and confirmed its specificity and quantitative accuracy with different methods. We then undertook analysis of sets of blastomeres isolated from embryos at the 8-cell stage. At this stage, all cells in the embryo are still pluripotent and morphologically equivalent. Our results demonstrate, however, that both Oct4 and Xist RNA levels vary in individual blastomeres comprising the same embryo, with some cells having particularly elevated levels of either transcript. Analysis of multiple embryos also shows that Xist and Oct4 expression levels are not correlated at the 8-cell stage, although transcription of both genes is up-regulated at this time in development. In addition, comparison of data from males and females allowed us to determine that the efficiency of the Oct4/Xist assay is unaffected by sex-related differences in gene expression. Conclusion This paper describes the first example of multiplex RT-LATE-PCR and its utility, when

  18. Quantitative detection of Borrelia burgdorferi sensu lato in erythema migrans skin lesions using internally controlled duplex real time PCR.

    Directory of Open Access Journals (Sweden)

    Maria O'Rourke

    Full Text Available B. burgdorferi sensu stricto, B. afzelii, B. garinii and B. bavariensis are the principal species which account for Lyme borreliosis (LB globally. We have developed an internally controlled duplex quantitative real time PCR assay targeting the Borrelia 16S rRNA and the human RNAseP genes. This assay is well-suited for laboratory confirmation of suspected cases of LB and will be used to assess the efficacy of a vaccine against LB in clinical trials. The assay is highly specific, successfully detecting DNA extracted from 83 diverse B. burgdorferi sensu lato strains representing all major species causing LB, while 21 unrelated microbial species and human genomic DNA tested negative. The assay was highly reproducible and sensitive, with a lower limit of detection of 6 copies per PCR reaction. Together with culture, the assay was used to evaluate paired 3 mm skin biopsy samples taken from 121 patients presenting with solitary erythema migrans (EM lesion. PCR testing identified more positive biopsy samples than culture (77.7% PCR positive versus 55.1% culture positive and correctly identified all specimens scored as culture positive. OspA-based typing identified the majority of isolates as B. afzelii (96.8% and the bacterial load was significantly higher in culture positive biopsies than in culture negative biopsies (P<0.001. The quantitative data also enabled relationships between Borrelia burden and patient symptoms to be evaluated. The bacterial load was significantly higher among patients with systemic symptoms than without (P = 0.02 and was significantly higher for biopsies retrieved from patients with EM lesions with central clearing (P<0.001. 16S copy numbers were moderately lower in samples from patients reporting a history of LB (P = 0.10. This is the first quantitative PCR study of human skin biopsies predominantly infected with B. afzelii and the first study to demonstrate a clear relationship between clinical symptoms in B. afzelii

  19. Diferenciação específica entre Taenia saginata e Taenia solium por ensaio de PCR e duplex-PCR

    Directory of Open Access Journals (Sweden)

    Jardim Eurione Antônio Garcia da Veiga

    2006-01-01

    Full Text Available Este estudo teve como objetivo a padronização de protocolos e a seleção de novos primers para a identificação espécie-específica de Taenia saginata e Taenia solium através da reação em cadeia da polimerase (PCR e duplex-PCR. Inicialmente, foram recuperadas seqüências depositadas no GenBank (acesso ndegrees AB020399 para T. saginata e ndegrees AB020395 para T. solium referentes ao gene da subunidade maior do ribossomo (LSU RNAr de tenídeos. A partir do alinhamento das seqüências, um primer genérico denominado TBR-3 (5'-ggcttgtttgaatggtttgacg- 3' foi selecionado de região conservada e, de diferentes regiões semi-conservadas, os primers específicos TBR-4 para T. saginata (5'-cgactcatgaagataaacaaggt-3' e TBR-5 (5'-cggtcgaacagaccataaatct-3' e TBR-6 (5'-gctactacacctaaattctaacc- 3' para T. solium. Os primers foram avaliados quanto à especificidade através da PCR empregando-se DNA total (DNAt de amostras de cisticercos e proglotes dos parasitos, previamente identificadas por critérios morfológicos. O par de primers TBR-3/TBR-4 permitiu a amplificação específica do fragmento esperado de 328 pb a partir do DNAt de T. saginata. Os pares TBR-3/TBR-5 e TBR-3/TBR-6 permitiram a amplificação, respectivamente, dos fragmentos específicos de 310pb e 286pb a partir do DNAt de T. solium. A identidade dos produtos de PCR foi comprovada comparando-se a seqüência dos amplicons obtidos às seqüências de referência do gene LSU RNAr registrado no GenBank (ndegrees AB020399 e ndegrees AB020395. As reações apresentaram sensibilidade para detecção de até 1fg do DNAt de T. solium e 0,2fg do DNAt de T. saginata. A combinação dos primers TBR-3/TBR-4 e TBR3/TBR-6 e o tamanho dos fragmentos gênicos obtidos permitiram o estabelecimento de ensaios de duplex-PCR, eficaz na detecção simultânea do DNA de T. saginata e T. solium em sistema único de reação. Os primers utilizados não geraram qualquer produto de amplificação cruzada

  20. Milk adulteration: Detection of bovine milk in bulk goat milk produced by smallholders in northeastern Brazil by a duplex PCR assay.

    Science.gov (United States)

    Rodrigues, N P A; Givisiez, P E N; Queiroga, R C R E; Azevedo, P S; Gebreyes, W A; Oliveira, C J B

    2012-05-01

    The aim of this study was to investigate the adulteration of goat milk produced by smallholders in semiarid northeastern Brazil with bovine milk as an adulterant. The study was requested by the association of smallholder producers in the region to investigate and to inhibit adulteration practices as a need to ensure the quality and safety of goat milk. A duplex PCR assay has been developed and standardized. Further validation was performed in 160 fresh bulk goat milk samples. The detection limit of the duplex PCR was 0.5% bovine milk in goat milk and the results indicated that 41.2% of the goat milk presented to market was positive for bovine milk. Making the test available to the association of producers, together with extension activities, have been applied to reduce adulteration in goat milk sold to small-scale dairy plants and to ensure the species origin for goat milk in the state of Paraíba.

  1. Allele-specific KRT1 expression is a complex trait.

    Directory of Open Access Journals (Sweden)

    Heng Tao

    2006-06-01

    Full Text Available The differential expression of alleles occurs commonly in humans and is likely an important genetic factor underlying heritable differences in phenotypic traits. Understanding the molecular basis of allelic expression differences is thus an important challenge. Although many genes have been shown to display differential allelic expression, this is the first study to examine in detail the cumulative effects of multiple cis-regulatory polymorphisms responsible for allele-specific expression differences. We have used a variety of experimental approaches to identify and characterize cis-regulatory polymorphisms responsible for the extreme allele-specific expression differences of keratin-1 (KRT1 in human white blood cells. The combined data from our analyses provide strong evidence that the KRT1 allelic expression differences result from the haplotypic combinations and interactions of five cis-regulatory single nucleotide polymorphisms (SNPs whose alleles differ in their affinity to bind transcription factors and modulate KRT1 promoter activity. Two of these cis-regulatory SNPs bind transcriptional activators with the alleles on the high-expressing KRT1 haplotype pattern having a higher affinity than the alleles on the low-expressing haplotype pattern. In contrast, the other three cis-regulatory SNPs bind transcriptional inhibitors with the alleles on the low-expressing haplotype pattern having a higher affinity than the alleles on the high-expressing haplotype pattern. Our study provides important new insights into the degree of complexity that the cis-regulatory sequences responsible for allele-specific transcriptional regulation have. These data suggest that allelic expression differences result from the cumulative contribution of multiple DNA sequence polymorphisms, with each having a small effect, and that allele-specific expression can thus be viewed as a complex trait.

  2. Duplex PCR for differentiation of the vaccine strain Brucella suis S2 and B. suis biovar 1 from other strains of Brucella spp.

    Science.gov (United States)

    Nan, Wenlong; Tan, Pengfei; Wang, Yong; Xu, Zouliang; Mao, Kairong; Peng, Daxin; Chen, Yiping

    2014-09-01

    Immunisation with attenuated Brucella spp. vaccines prevents brucellosis, but may also interfere with diagnosis. In this study, a duplex PCR was developed to distinguish Brucella suis vaccine strain S2 from field strains of B. suis biovar 1 and other Brucella spp. The PCR detected 60 fg genomic DNA of B. suis S2 or biovar 1 field strains and was able to distinguish B. suis S2 and wild-type strains of B. suis biovar 1 among 76 field isolates representing all the common species and biovars, as well as four vaccine strains, of Brucella.

  3. Improving the molecular diagnosis of Chlamydia psittaci and Chlamydia abortus infection with a species-specific duplex real-time PCR.

    Science.gov (United States)

    Opota, Onya; Jaton, Katia; Branley, James; Vanrompay, Daisy; Erard, Veronique; Borel, Nicole; Longbottom, David; Greub, Gilbert

    2015-10-01

    Chlamydia psittaci and Chlamydia abortus are closely related intracellular bacteria exhibiting different tissue tropism that may cause severe but distinct infection in humans. C. psittaci causes psittacosis, a respiratory zoonotic infection transmitted by birds. C. abortus is an abortigenic agent in small ruminants, which can also colonize the human placenta and lead to foetal death and miscarriage. Infections caused by C. psittaci and C. abortus are underestimated mainly due to diagnosis difficulties resulting from their strict intracellular growth. We developed a duplex real-time PCR to detect and distinguish these two bacteria in clinical samples. The first PCR (PCR1) targeted a sequence of the 16S-23S rRNA operon allowing the detection of both C. psittaci and C. abortus. The second PCR (PCR2) targeted the coding DNA sequence CPSIT_0607 unique to C. psittaci. The two PCRs showed 100 % detection for ≥ 10 DNA copies per reaction (1000 copies ml(- 1)). Using a set of 120 samples, including bacterial reference strains, clinical specimens and infected cell culture material, we monitored 100 % sensitivity and 100 % specificity for the detection of C. psittaci and C. abortus for PCR1. When PCR1 was positive, PCR2 could discriminate C. psittaci from C. abortus with a positive predictive value of 100 % and a negative predictive value of 88 %. In conclusion, this new duplex PCR represents a low-cost and time-saving method with high-throughput potential, expected to improve the routine diagnosis of psittacosis and pregnancy complication in large-scale screening programs and also during outbreaks.

  4. 空肠弯曲菌和沙门菌双重实时荧光定量 PCR 检测方法的建立%Duplex Real-time PCR for Simultaneous Detection of Campylobacter jejuni and Salmonella

    Institute of Scientific and Technical Information of China (English)

    高瑞娟; 张凯; 吕嘉敏; 黄永兴; 罗开健

    2014-01-01

    This study aimed to establish a rapid detection of Campylobacter jejuni and Salmonella by duplex real-time PCR.The primers and Taq man probes were designed based on the invA gene of Salmonella and hipO gene of Campylobacter jejuni ,using FAM,JOE and TAMRA fluorescently labeled specific probes aimed at the conserved genes of target pathogens,respectively.We compared Tm values to fix these primers and probes,and optimize the reaction conditions to establish the duplex real-time PCR approach for the detection of Campylobacter jejuni and Salmonella.The sensitivity of the duplex real-time PCR was 10 CFU/mL,230 CFU/mL for Campylobacterjejuni and Salmonella.The duplex real-time PCR showed good specificity which was validated by testing various bacteria isolates.In this study,a duplex PCR with high specificity and sensitivity was developed and it would provide useful information for the simultaneous detection of Campylobacter jejuni and Salmonella in food.%为建立一种空肠弯曲菌和沙门菌的双重实时荧光定量 PCR 检测方法,根据空肠弯曲菌的保守基因 hipO 和沙门菌的保守基因 invA 分别设计合成引物和 TaqMan 探针,分别使用 FAM、JOE 作为探针报告基团,TAMRA 作为探针淬灭基团。优化反应体系及条件,建立适用于检测食品中空肠弯曲菌和沙门菌的双重实时荧光定量 PCR 方法。结果显示,该检测方法特异性强,灵敏度高,空肠弯曲菌检测限可达10 CFU/mL,沙门菌检测限达230 CFU/mL。表明建立的双重实时荧光定量 PCR 可为实现食品中空肠弯曲菌和沙门菌的同时检测提供新方法。

  5. Development of a One-Step Duplex RT-PCR Method for the Simultaneous Detection of VP3/VP1 and VP1/P2B Regions of the Hepatitis A Virus.

    Science.gov (United States)

    Kim, Mi-Ju; Lee, Shin-Young; Kim, Hyun-Joong; Lee, Jeong Su; Joo, In Sun; Kwak, Hyo Sun; Kim, Hae-Yeong

    2016-08-28

    The simultaneous detection and accurate identification of hepatitis A virus (HAV) is critical in food safety and epidemiological studies to prevent the spread of HAV outbreaks. Towards this goal, a one-step duplex reverse-transcription (RT)-PCR method was developed targeting the VP1/P2B and VP3/VP1 regions of the HAV genome for the qualitative detection of HAV. An HAV RT-qPCR standard curve was produced for the quantification of HAV RNA. The detection limit of the duplex RT-PCR method was 2.8 × 10(1) copies of HAV. The PCR products enabled HAV genotyping analysis through DNA sequencing, which can be applied for epidemiological investigations. The ability of this duplex RT-PCR method to detect HAV was evaluated with HAV-spiked samples of fresh lettuce, frozen strawberries, and oysters. The limit of detection of the one-step duplex RT-PCR for each food model was 9.4 × 10(2) copies/20 g fresh lettuce, 9.7 × 10(3) copies/20 g frozen strawberries, and 4.1 × 10(3) copies/1.5 g oysters. Use of a one-step duplex RT-PCR method has advantages such as shorter time, decreased cost, and decreased labor owing to the single amplification reaction instead of four amplifications necessary for nested RT-PCR.

  6. A Novel Duplex Real-Time Reverse-Transcription PCR Assay for the Detection of Influenza A and the Novel Influenza A(H1N1 Strain

    Directory of Open Access Journals (Sweden)

    Theo P. Sloots

    2009-12-01

    Full Text Available Timely implementation of antiviral treatment and other public health based responses are dependent on accurate and rapid diagnosis of the novel pandemic influenza A(H1N1 strain. In this study we developed a duplex real-time PCR (RT-PCR (dFLU-TM assay for the simultaneous detection of a broad range of influenza A subtypes and specific detection of the novel H1N1 2009 pandemic strain. The assay was compared to the combined results of two previously described monoplex RT-PCR assays using 183 clinical samples and 10 seasonal influenza A isolates. Overall, the results showed that the dFLU-TM RT-PCR method is suitable for detection of influenza A, including the novel H1N1 pandemic strain, in clinical samples.

  7. Detection of A. phagocytophilum and E. chaffeensis in patient and mouse blood and ticks by a duplex real-time PCR assay.

    Directory of Open Access Journals (Sweden)

    Tuo Dong

    Full Text Available Human granulocytic anaplasmosis (HGA and human monocytic ehrlichiosis (HME are emerging, tick-borne, zoonotic infectious diseases caused by Anaplasma phagocytophilum and Ehrlichia chaffeensis, respectively. Early diagnosis is essential for rapid clinical treatment to avoid misdiagnosis and severe patient outcomes. Simple, sensitive and reliable diagnostic methods are urgently needed. In this study, we developed a duplex real-time PCR assay targeting the A. phagocytophilum ankA gene and the E. chaffeensis TRP120 gene, respectively. The lowest limit of detection of the duplex real-time PCR assay was 100 copies of the targeted A. phagocytophilum ankA gene and the E. chaffeensis TRP120 gene per reaction, and the specificity was 100%. Detection in blood DNA samples from the acute stage of illness for 22 HGA cases and 8 HME cases indicated that the duplex real-time PCR assay was more sensitive than the nested PCR assay. The infection of Citellusundulatus Pallas with A. phagocytophilum and E. chaffeensis was first confirmed in Xinjiang Province and the positive rate was 3.1% for A. phagocytophilum, 6.3% for E. chaffeensis and 3.1% for co-infection with both pathogens. The rates of A. phagocytophilum and E. chaffeensis infection of D. silvarum ticks collected from Shanxi Province were 8.2% and 14.8%, respectively, and the co-infection rate was 3.3%. The rates of A. phagocytophilum and E. chaffeensis infection in H. longicornis ticks collected from Shandong Province were 1.6% and 6.3%, respectively, and the co-infection rate was 1.6%.

  8. Detection of A. phagocytophilum and E. chaffeensis in patient and mouse blood and ticks by a duplex real-time PCR assay.

    Science.gov (United States)

    Dong, Tuo; Qu, Zhangyi; Zhang, Lijuan

    2013-01-01

    Human granulocytic anaplasmosis (HGA) and human monocytic ehrlichiosis (HME) are emerging, tick-borne, zoonotic infectious diseases caused by Anaplasma phagocytophilum and Ehrlichia chaffeensis, respectively. Early diagnosis is essential for rapid clinical treatment to avoid misdiagnosis and severe patient outcomes. Simple, sensitive and reliable diagnostic methods are urgently needed. In this study, we developed a duplex real-time PCR assay targeting the A. phagocytophilum ankA gene and the E. chaffeensis TRP120 gene, respectively. The lowest limit of detection of the duplex real-time PCR assay was 100 copies of the targeted A. phagocytophilum ankA gene and the E. chaffeensis TRP120 gene per reaction, and the specificity was 100%. Detection in blood DNA samples from the acute stage of illness for 22 HGA cases and 8 HME cases indicated that the duplex real-time PCR assay was more sensitive than the nested PCR assay. The infection of Citellusundulatus Pallas with A. phagocytophilum and E. chaffeensis was first confirmed in Xinjiang Province and the positive rate was 3.1% for A. phagocytophilum, 6.3% for E. chaffeensis and 3.1% for co-infection with both pathogens. The rates of A. phagocytophilum and E. chaffeensis infection of D. silvarum ticks collected from Shanxi Province were 8.2% and 14.8%, respectively, and the co-infection rate was 3.3%. The rates of A. phagocytophilum and E. chaffeensis infection in H. longicornis ticks collected from Shandong Province were 1.6% and 6.3%, respectively, and the co-infection rate was 1.6%.

  9. Duplex detection of the Mycobacterium tuberculosis complex and medically important non-tuberculosis mycobacteria by real-time PCR based on the rnpB gene.

    Science.gov (United States)

    Abdeldaim, Guma; Svensson, Erik; Blomberg, Jonas; Herrmann, Björn

    2016-11-01

    A duplex real-time PCR based on the rnpB gene was developed for Mycobacterium spp. The assay was specific for the Mycobacterium tuberculosis complex (MTB) and also detected all 19 tested species of non-tuberculous mycobacteria (NTM). The assay was evaluated on 404 clinical samples: 290 respiratory samples and 114 from tissue and other non-respiratory body sites. M. tuberculosis was detected by culture in 40 samples and in 30 samples by the assay. The MTB assay showed a sensitivity similar to Roche Cobas Amplicor MTB-PCR (Roche Molecular Systems, Pleasanton, CA, USA). There were only nine samples with non-tuberculous mycobacteria detected by culture. Six of them were detected by the PCR assay.

  10. Rapid detection of Opisthorchis viverrini and Strongyloides stercoralis in human fecal samples using a duplex real-time PCR and melting curve analysis.

    Science.gov (United States)

    Janwan, Penchom; Intapan, Pewpan M; Thanchomnang, Tongjit; Lulitanond, Viraphong; Anamnart, Witthaya; Maleewong, Wanchai

    2011-12-01

    Human opisthorchiasis caused by the liver fluke Opisthorchis viverrini is an endemic disease in Southeast Asian countries including the Lao People's Democratic Republic, Cambodia, Vietnam, and Thailand. Infection with the soil-transmitted roundworm Strongyloides stercoralis is an important problem worldwide. In some areas, both parasitic infections are reported as co-infections. A duplex real-time fluorescence resonance energy transfer (FRET) PCR merged with melting curve analysis was developed for the rapid detection of O. viverrini and S. stercoralis in human fecal samples. Duplex real-time FRET PCR is based on fluorescence melting curve analysis of a hybrid of amplicons generated from two genera of DNA elements: the 162 bp pOV-A6 DNA sequence specific to O. viverrini and the 244 bp 18S rRNA sequence specific to S. stercoralis, and two pairs of specific fluorophore-labeled probes. Both O. viverrini and S. stercoralis can be differentially detected in infected human fecal samples by this process through their different fluorescence channels and melting temperatures. Detection limit of the method was as little as two O. viverrini eggs and four S. stercoralis larvae in 100 mg of fecal sample. The assay could distinguish the DNA of both parasites from the DNA of negative fecal samples and fecal samples with other parasite materials, as well as from the DNA of human leukocytes and other control parasites. The technique showed 100% sensitivity and specificity. The introduced duplex real-time FRET PCR can reduce labor time and reagent costs and is not prone to carry over contamination. The method is important for simultaneous detection especially in areas where both parasites overlap incidence and is useful as the screening tool in the returning travelers and immigrants to industrialized countries where number of samples in the diagnostic units will become increasing.

  11. Duplex real-time PCR method for the detection of sesame (Sesamum indicum) and flaxseed (Linum usitatissimum) DNA in processed food products.

    Science.gov (United States)

    López-Calleja, Inés María; de la Cruz, Silvia; Martín, Rosario; González, Isabel; García, Teresa

    2015-01-01

    The development of a duplex real-time polymerase chain reaction (PCR) method allowing the simultaneous detection of sesame and flaxseed DNA in commercial food products is described. This duplex real-time PCR technique is based in the design of sesame- and flaxseed-specific primers based on the ITS1 region and two TaqMan fluorescent probes. The method was positive for sesame and flaxseed, and showed no cross-reactivity for all other heterologous plant and animal species tested. Sesame and flaxseed could be detected in a series of model samples with defined raw and heat-treated sesame in flaxseed, and flaxseed in sesame, respectively, with detection limits of 1.3 mg kg(-1) for sesame and 1.4 mg kg(-1) for flaxseed. The applicability of the assay for determining sesame and flaxseed in different food matrices was investigated by analysing a total of 238 commercial foodstuffs. This PCR method is useful for highly selective and sensitive detection of traces of sesame and flaxseed in commercial food products.

  12. Allele-specific amplification in cancer revealed by SNP array analysis.

    Directory of Open Access Journals (Sweden)

    Thomas LaFramboise

    2005-11-01

    Full Text Available Amplification, deletion, and loss of heterozygosity of genomic DNA are hallmarks of cancer. In recent years a variety of studies have emerged measuring total chromosomal copy number at increasingly high resolution. Similarly, loss-of-heterozygosity events have been finely mapped using high-throughput genotyping technologies. We have developed a probe-level allele-specific quantitation procedure that extracts both copy number and allelotype information from single nucleotide polymorphism (SNP array data to arrive at allele-specific copy number across the genome. Our approach applies an expectation-maximization algorithm to a model derived from a novel classification of SNP array probes. This method is the first to our knowledge that is able to (a determine the generalized genotype of aberrant samples at each SNP site (e.g., CCCCT at an amplified site, and (b infer the copy number of each parental chromosome across the genome. With this method, we are able to determine not just where amplifications and deletions occur, but also the haplotype of the region being amplified or deleted. The merit of our model and general approach is demonstrated by very precise genotyping of normal samples, and our allele-specific copy number inferences are validated using PCR experiments. Applying our method to a collection of lung cancer samples, we are able to conclude that amplification is essentially monoallelic, as would be expected under the mechanisms currently believed responsible for gene amplification. This suggests that a specific parental chromosome may be targeted for amplification, whether because of germ line or somatic variation. An R software package containing the methods described in this paper is freely available at http://genome.dfci.harvard.edu/~tlaframb/PLASQ.

  13. QuASAR: quantitative allele-specific analysis of reads.

    Science.gov (United States)

    Harvey, Chris T; Moyerbrailean, Gregory A; Davis, Gordon O; Wen, Xiaoquan; Luca, Francesca; Pique-Regi, Roger

    2015-04-15

    Expression quantitative trait loci (eQTL) studies have discovered thousands of genetic variants that regulate gene expression, enabling a better understanding of the functional role of non-coding sequences. However, eQTL studies are costly, requiring large sample sizes and genome-wide genotyping of each sample. In contrast, analysis of allele-specific expression (ASE) is becoming a popular approach to detect the effect of genetic variation on gene expression, even within a single individual. This is typically achieved by counting the number of RNA-seq reads matching each allele at heterozygous sites and testing the null hypothesis of a 1:1 allelic ratio. In principle, when genotype information is not readily available, it could be inferred from the RNA-seq reads directly. However, there are currently no existing methods that jointly infer genotypes and conduct ASE inference, while considering uncertainty in the genotype calls. We present QuASAR, quantitative allele-specific analysis of reads, a novel statistical learning method for jointly detecting heterozygous genotypes and inferring ASE. The proposed ASE inference step takes into consideration the uncertainty in the genotype calls, while including parameters that model base-call errors in sequencing and allelic over-dispersion. We validated our method with experimental data for which high-quality genotypes are available. Results for an additional dataset with multiple replicates at different sequencing depths demonstrate that QuASAR is a powerful tool for ASE analysis when genotypes are not available. http://github.com/piquelab/QuASAR. fluca@wayne.edu or rpique@wayne.edu Supplementary Material is available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  14. Duplex real-time reverse transcriptase PCR to determine cytokine mRNA expression in a hamster model of New World cutaneous leishmaniasis

    Directory of Open Access Journals (Sweden)

    Osorio Yaneth

    2010-06-01

    Full Text Available Abstract Background The Syrian hamster, Mesocricetus auratus, has distinct immunological features and is uniquely susceptible to intracellular pathogens. Studies in hamsters are limited by the relative unavailability of tools to conduct immunological studies. To address this limitation we developed duplex real-time reverse transcriptase (RT PCR assays for the relative quantification of the mRNAs of hamster cytokines, chemokines, and related immune response molecules. Results Real-time RT-PCR primers and probes were synthesized for analysis of interleukin (IL-4, IFN-γ, TNF-α, IL-10, IL-12p40, TGF-β, IL-13, IL-21, chemokine ligand (CCL 22, CCL17, Chemokine (C-C motif receptor 4 and FoxP3 expression. Standard curves and validation experiments were performed for each real-time RT-PCR assay, allowing us to use the comparative Ct (2-ΔΔCt method to calculate changes in gene expression. Application of the real-time RT PCR assays to a biological model was demonstrated by comparing mRNA expression in skin and lymph node tissues between uninfected and Leishmania panamensis infected hamsters. Conclusions The duplex real-time RT PCR assays provide a powerful approach for the quantification of cytokine transcription in hamsters, and their application to a model of cutaneous leishmaniasis suggests that a balanced type 1 and type 2 cytokine response contributes to the chronic, nonprogressive course of disease. These new molecular tools will further facilitate investigation into the mechanisms of disease in the hamster, not only for models of leishmaniasis, but also for other viral, bacterial, fungal, and parasitic infections.

  15. Duplex real-time reverse transcriptase PCR to determine cytokine mRNA expression in a hamster model of New World cutaneous leishmaniasis

    Science.gov (United States)

    2010-01-01

    Background The Syrian hamster, Mesocricetus auratus, has distinct immunological features and is uniquely susceptible to intracellular pathogens. Studies in hamsters are limited by the relative unavailability of tools to conduct immunological studies. To address this limitation we developed duplex real-time reverse transcriptase (RT) PCR assays for the relative quantification of the mRNAs of hamster cytokines, chemokines, and related immune response molecules. Results Real-time RT-PCR primers and probes were synthesized for analysis of interleukin (IL)-4, IFN-γ, TNF-α, IL-10, IL-12p40, TGF-β, IL-13, IL-21, chemokine ligand (CCL) 22, CCL17, Chemokine (C-C motif) receptor 4 and FoxP3 expression. Standard curves and validation experiments were performed for each real-time RT-PCR assay, allowing us to use the comparative Ct (2-ΔΔCt) method to calculate changes in gene expression. Application of the real-time RT PCR assays to a biological model was demonstrated by comparing mRNA expression in skin and lymph node tissues between uninfected and Leishmania panamensis infected hamsters. Conclusions The duplex real-time RT PCR assays provide a powerful approach for the quantification of cytokine transcription in hamsters, and their application to a model of cutaneous leishmaniasis suggests that a balanced type 1 and type 2 cytokine response contributes to the chronic, nonprogressive course of disease. These new molecular tools will further facilitate investigation into the mechanisms of disease in the hamster, not only for models of leishmaniasis, but also for other viral, bacterial, fungal, and parasitic infections. PMID:20569429

  16. Survey of Cherry necrotic rusty mottle virus and Cherry green ring mottle virus incidence in Korea by Duplex RT-PCR

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    Seung-Yeol Lee

    2014-12-01

    Full Text Available The incidence of Cherry necrotic rusty mottle virus (CNRMV and Cherry green ring mottle virus (CGRMV have recently been occurred in Korea, posing a problem for sweet cherry cultivation. Since infected trees have symptomless leaves or ring-like spots on the pericarp, it is difficult to identify a viral infection. In this study, the incidence of CNRMV and CGRMV in sweet cherry in Gyeongbuk province was surveyed using a newly developed duplex reverse transcriptase polymerase chain reaction (RT-PCR method that can detect both viruses in a single reaction. CNRMV and CGRMV co-infection rates were 29.6%, 53.6%, and 17.6%, respectively, in samples collected from three different sites (Daegu, Gyeongju and Gyeongsan in Gyeongbuk province during 2012 and 2013. This duplex RT-PCR method offers a simple, rapid, and effective way of identifying CNRMV and CGRMV simultaneously in sweet cherry trees, which can aid in the management of viral infections that could undermine yield.

  17. Combination of 768-well microplate array diagonal gel electrophoresis with duplex PCR of X and Y chromosome markers for quality control of epidemiological DNA banks.

    Science.gov (United States)

    Huang, Shuwen; Chen, Xiao-he; Day, Ian N M

    2006-08-01

    Large DNA banks for human epidemiological studies have become an increasingly important research tool. The power of genotype-phenotype studies is dependent both on the quality of phenotyping and of genotyping and of correct linking of phenotypes to genotypes. Samples must be tracked through numerous steps between subject or patient and post-genotypic data. Only one phenotype, sex, has a perfect and binary correlation with genotype. In mixed sex studies, it may be advantageous for purposes of quality control to keep sexes mixed during the steps from acquisition to DNA bank, in order to be able to check later for sample swaps. We have designed a duplex PCR combining an amplicon from MAOA marking the X chromosome and an amplicon from DDX3Y marking the Y chromosome. We combined this with a simple economical palmtop sized 768-well microplate compatible electrophoresis system developed in-house for examination of duplex PCR products. We applied this quality control test in the validation of two DNA banks.

  18. Ultrafast real-time PCR with integrated melting curve analysis and duplex capacities using a low-cost polymer lab-on-a-chip system

    Science.gov (United States)

    Gransee, Rainer; Schneider, Tristan; Elyorgun, Deniz; Strobach, Xenia; Schunck, Tobias; Gatscha, Theresia; Winkler, Christian; Höth, Julian

    2015-05-01

    Nucleic amplification using quantitative polymeric chain reaction (qPCR) has become the gold standard of molecular testing. These systems offer both amplification and simultaneous fluorescence detection. An ultrafast microfluidic module (allowing 30 PCR cycles in 6 minutes) based on the oscillating fluid plug concept was previously developed [1,2] allowing the amplification of native genomic deoxyribonucleic acid (DNA) molecules. This abstract presents the actual status of the advanced system. The upgraded system generates high quality qPCR amplification plots and additional sensitive melting point analysis comparable to data obtained from commercial real-time cyclers. These features provide the user with all information needed to analyze PCR products. The system uses light emitting diodes (LED) for illumination and a low cost Charge-coupled Device (CCD) camera for optical detection. Image data processing allows the automated process control of the overall system components. The system enables the performance of rapid and robust nucleic acid amplifications together with the integration of real time measurement technology. This allows the amplification and simultaneous quantification of the targeted pathogens. The integration of duplex amplification performance allows the incorporation of the necessary controls into the device to validate the PCR performance. This demonstrator can be run either as fully autonomously working device or as OEM part of a sample-to-answer platform.

  19. Comparison between two standardized cultural methods and 24 hour duplex SYBR green real-time PCR assay for Salmonella detectionin meat samples.

    Science.gov (United States)

    Delibato, Elisabetta; Fiore, Alfonsina; Anniballi, Fabrizio; Auricchio, Bruna; Filetici, Emma; Orefice, Leucio; Losio, Marina Nadia; De Medici, Dario

    2011-07-01

    Food-borne diseases caused by Salmonella represent a worldwide public health problem. Salmonella must be absent in an established amount depending on the kind of the product and usually cultural methods have to be applied to evaluate the compliance of the products. ISO 6579:2002 in Europe and FSIS MLG 4.04.:2008 in the USA have usually been employed to detect Salmonella in meat, poultry and egg products. A Real Time PCR method using probes has recently been validated against the NMKL (Nordic Committee on Food Analysis) standard method. This method has been modified using the less expensive Sybr Green Real Time PCR approach and applied directly in the 18 hours preenrichment broth for the purpose of detecting Salmonella in meat products in less than 24 hours. The purpose of this study was to: - compare the effectiveness of ISO and FSIS cultural methods; - develop a new 24 hour duplex Sybr Green Real Time PCR-melting curve analysis; - evaluate the performance of Salmonella, Standard Method, Rapid Method, SYBR Green Real Time PCR. The equivalence between ISO and FSIS methods was demonstrated and the use of SYBR Green Real Time PCR as a screening tool for negative results seems appealing especially to evaluate compliance with the HACCP systems.

  20. Duplex quantitative real-time PCR assay for the detection and discrimination of the eggs of Toxocara canis and Toxocara cati (Nematoda, Ascaridoidea in soil and fecal samples

    Directory of Open Access Journals (Sweden)

    Durant Jean-Francois

    2012-12-01

    Full Text Available Abstract Background Toxocarosis is a zoonotic disease caused by Toxocara canis (T. canis and/or Toxocara cati (T. cati, two worldwide distributed roundworms which are parasites of canids and felids, respectively. Infections of humans occur through ingestion of embryonated eggs of T. canis or T. cati, when playing with soils contaminated with dogs or cats feces. Accordingly, the assessment of potential contamination of these areas with these roundworms eggs is paramount. Methods A duplex quantitative real-time PCR (2qPCR targeting the ribosomal RNA gene internal transcribed spacer (ITS2 has been developed and used for rapid and specific identification of T. canis and T. cati eggs in fecal and soil samples. The assay was set up on DNA samples extracted from 53 adult worms including T. canis, T. cati, T. leonina, Ascaris suum (A. suum and Parascaris equorum (P. equorum. The assay was used to assess the presence of T. cati eggs in several samples, including 12 clean soil samples spiked with eggs of either T. cati or A. suum, 10 actual soil samples randomly collected from playgrounds in Brussels, and fecal samples from cats, dogs, and other animals. 2qPCR results on dogs and cats fecal samples were compared with results from microscopic examination. Results 2qPCR assay allowed specific detection of T. canis and T. cati, whether adult worms, eggs spiked in soil or fecal samples. The 2qPCR limit of detection (LOD in spiked soil samples was 2 eggs per g of soil for a turnaround time of 3 hours. A perfect concordance was observed between 2qPCR assay and microscopic examination on dogs and cats feces. Conclusion The newly developed 2qPCR assay can be useful for high throughput prospective or retrospective detection of T.canis and/or T. cati eggs in fecal samples as well as in soil samples from playgrounds, parks and sandpits.

  1. Duplex real-time qRT-PCR for the detection of hepatitis A virus in water and raspberries using the MS2 bacteriophage as a process control.

    Science.gov (United States)

    Blaise-Boisseau, Sandra; Hennechart-Collette, Catherine; Guillier, Laurent; Perelle, Sylvie

    2010-06-01

    Hepatitis A virus (HAV) infection is the leading worldwide cause of acute viral hepatitis. An important aspect of viral control is rapid diagnosis. Epidemiological studies have linked hepatitis A outbreaks to the consumption of drinking water or soft fruits exposed to faecal contamination. Real-time reverse transcriptase PCR (qRT-PCR) is now widely used for detecting RNA viruses in food samples. Efficiency of viral concentration, nucleic acid extraction and the presence of potential inhibitors of the RT-PCR reaction must be monitored to prevent false negative results. In this study, the MS2 bacteriophage used as a process control was detected simultaneously with HAV in a one-step duplex real-time qRT-PCR. The assay was developed for testing water and raspberries. Adding MS2 showed no loss of sensitivity for HAV detection in water and raspberry samples. The limit of detection of HAV with this new approach was 10PFU for 1.5L of bottled water, 100PFU for 1.5L of tap water, 50PFU for 25g of fresh raspberries and 100PFU for 25g of frozen raspberries. The data show that the MS2 offers a very reliable and simple way to monitor false-negative results, making it a valuable tool in the routine diagnostics laboratory.

  2. Detection, differentiation, and VP1 sequencing of duck hepatitis A virus type 1 and type 3 by a 1-step duplex reverse-transcription PCR assay.

    Science.gov (United States)

    Wen, X J; Cheng, A C; Wang, M S; Jia, R Y; Zhu, D K; Chen, S; Liu, M F; Liu, F; Chen, X Y

    2014-09-01

    Duck hepatitis A virus (DHAV) is an infectious pathogen causing fatal duck viral hepatitis in ducklings. Although both the inactivated vaccines and live attenuated vaccines have been used to protect ducklings, DHAV-1 and DHAV-3 still cause significant serious damage to the duck industry in China and South Korea. For rapid detection, differentiation, and epidemic investigation of DHAV in China, a genotype-specific 1-step duplex reverse-transcription (RT) PCR assay was established in this study. The sensitivity and specificity of the developed RT-PCR assay was evaluated with nucleic acids extracted from 2 DHAV reference strains, and 9 other infectious viruses and bacteria. The genotype-specific primers amplified different size DNA fragments encompassing the complete VP1 gene of the DHAV-1 or DHAV-3. The assay detected the liver samples collected from experimentally infected ducklings and dead ducklings collected from different regions of China. Sequence analysis of these DNA fragments indicated that VP1 sequences of DHAV-1 can be used to distinguish wild type and vaccine strains. The phylogenetic analysis of VP1 sequences indicated that the developed RT-PCR assay can be used for epidemic investigation of DHAV-1 and DHAV-3. The developed RT-PCR assay can be used as a specific molecular tool for simultaneous detection, differentiation, and sequencing the VP1 gene of DHAV-1 and DHAV-3, which can be used for understanding the epidemiology and evolution of DHAV. © 2014 Poultry Science Association Inc.

  3. 双重PCR检测板栗疫病菌的研究%A duplex PCR method for detection of Cryphonectria parasitica

    Institute of Scientific and Technical Information of China (English)

    麻文建; 张静; 郑磊; 朱天辉

    2015-01-01

    Chestnut blight caused by Cyphonectria parasitica is the main disease in Castanea mollissima cultivation area. According to the nucleotide sequence of internal transcribed spacer regions ( ITS) of the ribosomal gene of Cryphonectria in GenBank, a couple of primers CP1/CP2 for C. parasitica were developed. By random amplification polymorphism technology, we identified a specific RAPD fragment of C. parasitica from all the strains tested. After recycling and purifi?cation, the specific fragment was cloned and sequenced. The sequence was used to design two specific primers SC1/SC2 and converted RAPD marker to SCAR marker successfully. Combining the primers CP1/CP2 and SC1/SC2, a duplex PCR method for detecting C. parasitica had been established. The result indicated that the duplex PCR could amplify two unique fragments of 285 bp and 389 bp in size from C. parasitica,but other strains tested didn?t present any fragments. Sensitivity testing showed that the detection limit was 300 fg/μL for genomic DNA. The duplex PCR method also suc?cessfully detected C. parasitica from infected chestnut tissues. Our results suggested that the duplex PCR method would have great significance for accurate identification and detection of C. parasitica.%由板栗疫病菌( Cyphonectria parasitica)引起的板栗疫病是板栗栽培区的主要病害,也是全国林业危险性有害生物. 为建立C. parasitica的快速分子检测技术,根据C. parasitica与GenBank中同属其他物种的ITS序列差异设计了引物 CP1/CP2,扩增了大小为 285 bp 的目的片段. 进一步用 RAPD 技术从供试菌株中标记出C. parasitica的特异性片段,通过对RAPD特异片段克隆、测序后设计引物SC1/SC2,扩增的目的片段大小为389 bp,实现了RAPD标记向SCAR标记的成功转化. 采用引物对组合方式将引物CP1/CP2和SC1/SC2组成双重PCR,优化PCR反应条件并检测引物的特异性和灵敏度. 双重PCR能从C. parasitica扩增出285 bp和389 bp的两

  4. Duplex Quantitative PCR Assay for Detection of Haemophilus influenzae That Distinguishes Fucose- and Protein D-Negative Strains.

    Science.gov (United States)

    de Gier, Camilla; Pickering, Janessa L; Richmond, Peter C; Thornton, Ruth B; Kirkham, Lea-Ann S

    2016-09-01

    We have developed a specific Haemophilus influenzae quantitative PCR (qPCR) that also identifies fucose-negative and protein D-negative strains. Analysis of 100 H. influenzae isolates, 28 Haemophilus haemolyticus isolates, and 14 other bacterial species revealed 100% sensitivity (95% confidence interval [CI], 96% to 100%) and 100% specificity (95% CI, 92% to 100%) for this assay. The evaluation of 80 clinical specimens demonstrated a strong correlation between semiquantitative culture and the qPCR (P < 0.001).

  5. Development of a duplex real-time TaqMan PCR assay with an internal control for the detection of Mycoplasma gallisepticum and Mycoplasma synoviae in clinical samples from commercial and backyard poultry.

    Science.gov (United States)

    Sprygin, A V; Andreychuk, D B; Kolotilov, A N; Volkov, M S; Runina, I A; Mudrak, N S; Borisov, A V; Irza, V N; Drygin, V V; Perevozchikova, N A

    2010-04-01

    In this study, we report the development and validation of a duplex real-time polymerase chain reaction (PCR) assay with an internal control using TaqMan-labelled probes for the detection of Mycoplasma gallisepticum and Mycoplasma synoviae (duplex MGMS PCR). The MGMS PCR was highly specific with a sensitivity of 7 and 1 colony-forming units/ml for M. gallisepticum and M. synoviae, respectively, using dilution of pure culture that corresponds to 34 and 29 DNA copies per reaction. Validation of the assay was completed with 260 and 27 pooled samples (tracheal swabs) from commercial chickens and turkeys, respectively, with potential M. gallisepticum and M. synoviae involvement and 42 samples (palatine cleft swabs) from backyard geese and ducks. Using isolation as the gold standard, the MGMS PCR was more sensitive than isolation and the analytical sensitivity was 0.944 and 0.958 for M. gallisepticum and M. synoviae, respectively. In comparison with a gapA-based assay (gapA PCR) and a 16S rRNA-based assay (16S PCR) for M. gallisepticum and M. synoviae, respectively, the results agreed for 94.5% and 96.6%, respectively. The use of the internal control allowed monitoring of proper extraction and inhibition of amplification that was detected in 12 samples. The duplex MGMS PCR was shown to be superior to the presently reported real-time PCR assays in terms of combination of sensitivity, specificity and capacity of detection of more than one target in a single tube. In conclusion, the duplex MGMS PCR was highly specific, sensitive, and reproducible and could be used on clinical samples from commercial chickens, turkeys and backyard poultry including ducks and geese.

  6. Development, Optimization, and Evaluation of a Duplex Droplet Digital PCR Assay To Quantify the T-nos/hmg Copy Number Ratio in Genetically Modified Maize.

    Science.gov (United States)

    Félix-Urquídez, Dalmira; Pérez-Urquiza, Melina; Valdez Torres, José-Benigno; León-Félix, Josefina; García-Estrada, Raymundo; Acatzi-Silva, Abraham

    2016-01-05

    Certified reference materials (CRMs) are required to guarantee the reliability of analytical measurements. The CRMs available in the field of genetically modified organisms (GMOs) are characterized using real-time polymerase chain reaction (qPCR). This technology has limited application, because of its dependence on a calibrant. The objective of this study was to obtain a method with higher metrological quality, to characterize the CRMs for their contents of T-nos/hmg copy number ratio in maize. A duplex droplet digital PCR (ddPCR) assay was developed and optimized by a central composite design. The developed method achieved an absolute limit of detection (LOD) of 11 cP T-nos, a relative LOD of 0.034%, a limit of quantification (LOQ) of 23 cP (relative LOQ of 0.08%), and a dynamic range of 0.08%-100% T-nos/hmg ratio. The specificity and applicability of the assay were established for the analysis of low T-nos concentrations (0.9%) in several corn varieties. The convenience of DNA digestion to reduce measurement bias in the case of multiple-copy binding was confirmed through an enzymatic restriction assay. Given its overall performance, this method can be used to characterize CRM candidates for their contents of T-nos/hmg ratio.

  7. Development and validation of duplex, triplex, and pentaplex real-time PCR screening assays for the detection of genetically modified organisms in food and feed.

    Science.gov (United States)

    Huber, Ingrid; Block, Annette; Sebah, Daniela; Debode, Frédéric; Morisset, Dany; Grohmann, Lutz; Berben, Gilbert; Stebih, Dejan; Milavec, Mojca; Zel, Jana; Busch, Ulrich

    2013-10-30

    Worldwide, qualitative methods based on PCR are most commonly used as screening tools for genetically modified material in food and feed. However, the increasing number and diversity of genetically modified organisms (GMO) require effective methods for simultaneously detecting several genetic elements marking the presence of transgenic events. Herein we describe the development and validation of a pentaplex, as well as complementary triplex and duplex real-time PCR assays, for the detection of the most common screening elements found in commercialized GMOs: P-35S, T-nos, ctp2-cp4-epsps, bar, and pat. The use of these screening assays allows the coverage of many GMO events globally approved for commercialization. Each multiplex real-time PCR assay shows high specificity and sensitivity with an absolute limit of detection below 20 copies for the targeted sequences. We demonstrate by intra- and interlaboratory tests that the assays are robust as well as cost- and time-effective for GMO screening if applied in routine GMO analysis.

  8. Molecular Prevalence of Babesia bigemina and Trypanosoma evansi in Dairy Animals from Punjab, India, by Duplex PCR: A Step Forward to the Detection and Management of Concurrent Latent Infections

    Directory of Open Access Journals (Sweden)

    Amrita Sharma

    2013-01-01

    Full Text Available Specific duplex polymerase chain reaction (PCR was employed on 411 (386 cattle and 25 buffaloes blood samples of dairy animals from 9 districts of Punjab, India, for simultaneous detection of Babesia bigemina and Trypanosoma evansi. The results were compared and correlated with conventional Giemsa stained thin blood smear (GSTBS examination and haematological alterations to know the clinical status and pathogenicity of infections. The Bg3/Bg4 and TR3/TR4 primers were used in duplex PCR for B. bigemina and T. evansi amplified products of 689 bp and 257 bp, respectively. The overall prevalence by duplex PCR was found to be 36.49, 2.43, and 3.41% for T. evansi, B. bigemina, and dual infection, respectively. A more significant difference was observed for dual infection status (P≤0.005 as compared to T. evansi (P≤0.05 and B. bigemina (P≤0.01 among various districts under study. A very low prevalence of T. evansi (0.73% and B. bigemina (0.48% was seen by GSTBS. The highly sensitive, specific, and cost-effective duplex PCR was able to detect latent T. evansi and B. bigemina infection in cattle and buffaloes. Haematological evaluation revealed marked pathology in B. bigemina infected group and in dual infected group in contrast to that infected with T. evansi alone.

  9. Allele specific expression in worker reproduction genes in the bumblebee Bombus terrestris

    Directory of Open Access Journals (Sweden)

    Harindra E. Amarasinghe

    2015-07-01

    Full Text Available Methylation has previously been associated with allele specific expression in ants. Recently, we found methylation is important in worker reproduction in the bumblebee Bombus terrestris. Here we searched for allele specific expression in twelve genes associated with worker reproduction in bees. We found allele specific expression in Ecdysone 20 monooxygenase and IMP-L2-like. Although we were unable to confirm a genetic or epigenetic cause for this allele specific expression, the expression patterns of the two genes match those predicted for imprinted genes.

  10. Establishment and validation of two duplex one-step real-time RT-PCR assays for diagnosis of foot-and-mouth disease.

    Science.gov (United States)

    Gorna, K; Relmy, A; Romey, A; Zientara, S; Blaise-Boisseau, S; Bakkali-Kassimi, L

    2016-09-01

    Two duplex one-step TaqMan-based RT-PCR protocols for detection of foot-and-mouth disease virus (FMDV) were established and validated. Each RT-PCR test consists of a ready-to-use master mix for simultaneous detection of the well established 3D or IRES FMDV targets and incorporates the host β-actin mRNA as an internal control target, in a single-tube assay. The two real-time RT-PCR 3D/β-actin and IRES/β-actin tests are highly sensitive and able to detect up to 7TCID50/ml of FMDV and 10 copies/1μl of viral RNA. In field epithelium samples, the diagnostic sensitivity was 100% (95% CI; 91-100%) for the 3D/β-actin test and 97% (95% CI; 87-100%) for the IRES/β-actin test. The diagnostic specificity was 100% (95% CI; 95-100%) for both RT-PCRs. In addition, the two protocols proved to be robust, showing inter-assay coefficients of variation ranging from 1.94% to 6.73% for the IRES target and from 2.33% to 5.42% for the 3D target for different RNA extractions and different RT-PCR conditions. The internally controlled one-step real-time RT-PCR protocols described in this study provide a rapid, effective and reliable method for the detection of FMDV and thus may improve the routine diagnosis for foot-and-mouth disease.

  11. Duplex ultrasound

    Science.gov (United States)

    Vascular ultrasound; Peripheral vascular ultrasound ... A duplex ultrasound combines: Traditional ultrasound: This uses sound waves that bounce off blood vessels to create pictures. Doppler ultrasound: This ...

  12. Rapid genetic analysis in the diagnosis of primary Leber's hereditary optic neuropathy by using multiplex allele-specific polymerase chain reaction (MAS-PCR) with whole blood%MAS-PCR法快速诊断分析Leber's遗传性视神经病变原发性致病突变位点

    Institute of Scientific and Technical Information of China (English)

    阳菊华; 童绎; 朱益华; 林宇岚; 陈贻锴; 林建银

    2006-01-01

    目的 探索一种简易、快速、高效的临床基因诊断分析原发性Leber's遗传性视神经病变(Leber's hereditary optic neuropathy,LHON)位点的新方法.方法 根据已知的LHON患者线粒体DNA上的3个原发性LHON致病突变位点(G11778A、T14484C和G3460A),设计3条突变位点特异性聚合酶链反应(polymerase chain reaction,PCR)引物,直接以全血样为模板的等位基因特异性多重PCR(multiplex allelespecific polymerase chain reaction,MAS-PCR)分析3个原发性LHON致病突变位点.以此方法检测24例确诊的LHON患者,其中18例为线粒体DNA G11778A突变,4例为T14484C突变,2例为G3460A突变;同时,以20份正常血样标本作阴性对照.结果 优化的MAS-PCR条件为94℃/3 min,94℃/30 s、59.8℃/30 s、72℃/30 s,30个循环,72℃/3 min;以此方法检测了44份血样标本,其结果与预期结果一致,表明利用该方法筛查3个原发性LHON致病突变位点是可行的.同时,混和血液样本模拟实验结果显示,该方法可检测含多个原发性LHON致病突变位点的血液样本,且整个分析过程只需约2 h.结论 该方法直接以全血样作为PCR的模板,不需从血样中纯化DNA;单管一次性行PCR,可同时筛查3个原发性LHON致病突变位点.因此,该方法具有快速、高效、费用低、特异性好以及只需微量血液样本等优点.

  13. A duplex PCR-based assay for measuring the amount of bacterial contamination in a nucleic acid extract from a culture of free-living protists.

    Directory of Open Access Journals (Sweden)

    Alan O Marron

    Full Text Available BACKGROUND: Cultures of heterotrophic protists often require co-culturing with bacteria to act as a source of nutrition. Such cultures will contain varying levels of intrinsic bacterial contamination that can interfere with molecular research and cause problems with the collection of sufficient material for sequencing. Measuring the levels of bacterial contamination for the purposes of molecular biology research is non-trivial, and can be complicated by the presence of a diverse bacterial flora, or by differences in the relative nucleic acid yield per bacterial or eukaryotic cell. PRINCIPAL FINDINGS: Here we describe a duplex PCR-based assay that can be used to measure the levels of contamination from marine bacteria in a culture of loricate choanoflagellates. By comparison to a standard culture of known target sequence content, the assay can be used to quantify the relative proportions of bacterial and choanoflagellate material in DNA or RNA samples extracted from a culture. We apply the assay to compare methods of purifying choanoflagellate cultures prior to DNA extraction, to determine their effectiveness in reducing bacterial contamination. Together with measurements of the total nucleic acid concentration, the assay can then be used as the basis for determining the absolute amounts of choanoflagellate DNA or RNA present in a sample. CONCLUSIONS: The assay protocol we describe here is a simple and relatively inexpensive method of measuring contamination levels in nucleic acid samples. This provides a new way to establish quantification and purification protocols for molecular biology and genomics in novel heterotrophic protist species. Guidelines are provided to develop a similar protocol for use with any protistan culture. This assay method is recommended where qPCR equipment is unavailable, where qPCR is not viable because of the nature of the bacterial contamination or starting material, or where prior sequence information is insufficient

  14. Simultaneous detection and quantification of Phytophthora nicotianae and P. cactorum, and distribution analyses in strawberry greenhouses by duplex real-time PCR.

    Science.gov (United States)

    Li, Mingzhu; Inada, Minoru; Watanabe, Hideki; Suga, Haruhisa; Kageyama, Koji

    2013-01-01

    Phytophthora nicotianae and P. cactorum cause Phytophthora rot of strawberry. A duplex real-time PCR technique for simultaneous detection and quantification of the two pathogens was developed. Species-specific primers for P. nicotianae and P. cactorum were designed based on the internal transcribed spacer regions (ITS) of rDNA and the ras-related protein gene Ypt1, respectively. TaqMan probes were labeled with FAM for P. nicotianae and HEX for P. cactorum. Specificities were demonstrated using 52 isolates, including various soil-borne pathogens. Sensitivities for P. nicotianae and P. cactorum DNAs were 10 fg and 1 pg, respectively. The technique was applied to naturally infested soil and root samples; the two pathogens were detected and the target DNA concentrations were quantified. Significant correlations of DNA quantities in roots and the surrounding soils were found. The minimum soil DNA concentration predicting the development of disease symptoms was estimated as 20 pg (g soil)(-1). In three strawberry greenhouses examined, the target DNA concentrations ranged from 1 to 1,655 pg (g soil)(-1) for P. nicotianae and from 13 to 233 pg (g soil)(-1) for P. cactorum. The method proved fast and reliable, and provides a useful tool to monitor P. nicotianae and P. cactorum in plants or soils.

  15. Polymorphism analysis of Chinese Theileria sergenti using allele-specific polymerase chain reaction of the major piroplasm surface protein gene.

    Science.gov (United States)

    Liu, Ai Hong; Guan, Gui Quan; Liu, Jun Long; Liu, Zhi Jie; Leblanc, Neil; Li, You Quan; Gao, Jin Liang; Ma, Mi Ling; Niu, Qing Li; Ren, Qiao Yun; Bai, Qi; Yin, Hong; Luo, Jian Xun

    2011-02-01

    Theileria sergenti is a tick-borne parasite found in many parts of the world. The major piroplasm surface protein (MPSP), a conserved protein in all Theileria species, has been used as a marker for epidemiological and phylogenetic studies of benign Theileria species. In this study, Chinese species of T. sergenti were characterized by allele-specific polymerase chain reaction (PCR) and DNA sequence analysis of the MPSP gene. Using universal or allele-specific primer sets for PCR amplification of the MPSP gene, 98 of 288 cattle blood samples, collected from 6 provinces in China, were found to be positive. Among the positive samples, only 3 allelic MPSP gene types (Chitose [C]-, Ikeda [I]-, and buffeli [B]-type) were successfully amplified. Moreover, the results revealed that the majority of the parasites sampled in this study were C- and I-type (prevalence of 84 and 69%, respectively), whereas the B-type was less common (prevalence of 36%). Co-infections with C-, I-, and B-type T. sergenti also were found. An additional known allele, Thai-type, was not detected. Phylogenetic analysis based on the MPSP gene sequences, including 3 standard stocks generated in the laboratory ( T. sergenti Wenchuan, T. sergenti Ningxian, and T. sergenti Liaoyang), revealed that the isolates of Chinese sergenti were comprised of at least 4 allelic MPSP gene types, i.e., C-, I-, B1-, and B2-type, and these parasites with 6 MPSP types 1-5 and 7 were present in China.

  16. Short description of an alternative simplified method for screening recombinant clones within the "AdEasy-System" by Duplex-PCR

    Directory of Open Access Journals (Sweden)

    Kienle Peter

    2005-01-01

    Full Text Available Abstract Background Recombinant adenoviral vectors are highly efficient for in vitro and in vivo gene delivery. They can easily be produced in large numbers, transduce a wide variety of cell types and generate high levels of transgene expression. The AdEasy system is a widely used system for generating recombinant adenoviral vectors, which are created with a minimum of enzymatic manipulations and by employing homologous recombination in E. coli. In this paper we describe an alternative simplified method for screening recombinant DNA within the AdEasy system. This Duplex-PCR-method is independent of the transgene or insert and can be used for the complete AdEasy system. It is characterized by a simple standard protocol and the results can be obtained within a few hours. The PCR is run with two different primer sets. The primers KanaFor and KanaRev hybridizise with the Kanamycin resistence gene and AdFor and AdRev detect the adenoviral backbone. In case of recombinant clones, two diagnostic fragments with a size of 384 bp and 768 bp are generated. Results The practicability of this method was verified with three different transgenes: Cytosin Deaminase (AdCD, p53 (Adp53 and Granulocyte Macrophage Colony Stimulating Factor (AdGM-CSF. Recombinant clones are indicated by two diagnostic fragments and are then suitable for further processing. Conclusion In summary, the presented protocol allows fast detection of recombinants with an easy technique by minimizing the amount of necessary steps for generating a recombinant adenovirus. This method is time sparing and cost-effective.

  17. Self-(in)compatibility inheritance and allele-specific marker development in yellow mustard (Sinapis alba).

    Science.gov (United States)

    Zeng, Fangqin; Cheng, Bifang

    2014-01-01

    Yellow mustard (Sinapis alba) has a sporophytic self-incompatibility reproduction system. Genetically stable self-incompatible (SI) and self-compatible (SC) inbred lines have recently been developed in this crop. Understanding the S haplotype of different inbred lines and the inheritance of the self-(in)compatibility (SI/SC) trait is very important for breeding purposes. In this study, we used the S-locus gene-specific primers in Brassica rapa and Brassica oleracea to clone yellow mustard S-locus genes of SI lines Y514 and Y1130 and SC lines Y1499 and Y1501. The PCR amplification results and DNA sequences of the S-locus genes revealed that Y514 carried the class I S haplotype, while Y1130, Y1499, and Y1501 had the class II S haplotype. The results of our genetic studies indicated that self-incompatibility was dominant over self-compatibility and controlled by a one-gene locus in the two crosses of Y514 × Y1499 and Y1130 × Y1501. Of the five S-locus gene polymorphic primer pairs, Sal-SLGI and Sal-SRKI each generated one dominant marker for the SI phenotype of Y514; Sal-SLGII and Sal-SRKII produced dominant marker(s) for the SC phenotype of Y1501 and Y1499; Sal-SP11II generated one dominant marker for Y1130. These markers co-segregated with the SI/SC phenotype in the F2 populations of the two crosses. In addition, co-dominant markers were developed by mixing the two polymorphic primer pairs specific for each parent in the multiplex PCR, which allowed zygosity to be determined in the F2 populations. The SI/SC allele-specific markers have proven to be very useful for the selection of the desirable SC genotypes in our yellow mustard breeding program.

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

    Science.gov (United States)

    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.

  19. Using Allele-Specific PCR with Molecular Beams as a Means for Genotyping the Diallelic Indels

    Energy Technology Data Exchange (ETDEWEB)

    Doktycz, M.J.; Weber, J.L. (Marshfield Medical Research Foundation)

    2000-06-01

    The first Specific Aim for this grant was to identify and characterize an average of 500 human insertion/deletion polymorphisms per grant year (1500 total). This task was carried out entirely at MMRF. They substantially exceeded this goal by confirming about 2,300 diallelic indels. Complete characterization information for these polymorphisms is available from the Marshfield web site. A manuscript describing results for the first 2,000 diallelic indels was published earlier this year in the American Journal of Human Genetics. The Second Specific Aim of the grant was to investigate and develop improved methods for analysis of diallelic polymorphisms using miniaturized DNA arrays. The initial genotyping technology efforts focused on various hybridization and extension protocols with oligo arrays on flow-through channel glass. Channel glass is a porous material that permits reagents to be passed through the arrays. They devoted roughly 19 months at the beginning of the grant in pursuit of this methodology, but for various technological reasons, progress was limited.

  20. Rapid identification of capybara (Hydrochaeris hydrochaeris) through allele-specific PCR

    OpenAIRE

    2005-01-01

    The capybara is the largest rodent in the world and is widely distributed throughout Central and South America.  It is an animal of economic interest due to the pleasant flavor of its meat and higher protein content in comparison  to beef and pork meat.  The hide, hair and fat also have economic advantages. Thus,  as an animal with such high economic potential, it is the target of hunters, even though  hunting capybara is prohibited by law in Brazil.   Due to their  similarities,  capybara me...

  1. Duplex PCR assay for detecting specific gene sequence from pertussis bacillus%双重PCR快速检测百日咳杆菌

    Institute of Scientific and Technical Information of China (English)

    马卓娅; 王和平; 陈小文; 邓继岿; 张民; 郑跃杰

    2011-01-01

    Objective To establish a rapid assay with high sensitivity and specificity based on the sequence for pertussis toxin SI promoter ptxA-Pr and insertion element sequence IS481. Methods Oligonucleotide primers targeting the ptxA-Pr and IS481 were used to amplify in the same reaction.The amplicons were inserted into the plasmid, sequenced and compared in the GenBank. The sensitivity of pertussis bacillus assay was determined by amplifying pertussis bacillus standard bacterial strain DNA at 10 dilutions. The five other bacterium included Klebsiella pneumoniae,Streptococcus pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and one positive sample were detected in the same time for determining the specificity of this duplex PCR assay. Results The bands of expected size were obtained in standard bacterial strain and positive sample PCR assay(191 bp amplicon in ptxA-Pr and 145 bp amplicon in IS481). The sequence analysis revealed that the amplicons were the same as the sequence in the GenBank. The detection level was approximately 1.65×10-2 ng per reaction. There was no cross reaction with other bacterium.Conclusion The duplex PCR assay can be used to detect pertussis bacillus rapidly with high sensitivity and specificity.%目的:以百日咳毒素S1亚基启动子ptxA-Pr基因和插入序列IS481为目的基因,建立敏感、特异的双重PCR快速检测百日咳杆菌方法.方法:运用百日咳杆菌ptxA-Pr和IS481基因序列特异性引物,采用双重PCR技术同时扩增百日咳杆茵的特异性基因ptxA-Pr和IS481.通过构建目的质粒获得阳性对照,测序并与GenBank比对序列验证扩增产物.百日咳标准茵株DNA 10倍系列稀释为模板.采用此方法扩增双基因,检测此方法敏感性.扩增肺炎克雷伯茵、肺炎链球茵、铜绿假单胞茵、金黄色葡萄球菌、大肠杆菌及阳性样本DNA,检测此方法特异性.结果:百日咳杆菌标准株和阳性样本均能同时扩增ptxA-Pr和IS481

  2. 气单胞菌和嗜水气单胞菌双重PCR检测方法的建立%Development of a duplex PCR for detection of Aeromonas spp.and Aeromonas hydrophila

    Institute of Scientific and Technical Information of China (English)

    潘晓艺; 沈锦玉; 郝贵杰; 姚嘉贇; 徐洋; 尹文林; 孙逢明; 吴颖蕾

    2011-01-01

    Aeromonas is one of the major pathogens which frequently cause the bacterial diseases in freshwater fish terrapin and crustaceans. Aeromonas hydrophila caused economic losses widely. In order to detect Aeromonas spp. And Aeromonas hydrophila, two pairs of specific primers were designed according to the glycerophospholipid-cholesterol acyltransferase (GCAT) gene of Aeromonas spp. And the 16S rRNA gene of Aeromonas hydrophila. So a duplex PCR assay was developed to amplify the 16S rRNA gene and the GCAT gene. The reaction conditions of the duplex PCR were optimized and PCR products were sequenced. Meanwhile, the specificity of duplex PCR was studied. Five Aeromonas hydrophila strains,seven Aeromonas strains and the other five strains of non Aeromonas were tested by duplex PCR. The results showed that the GCAT gene and the 16S rRNA gene could be amplified from Aeromonas hydrophila strains,but only the GCAT gene from the other Aeromonas species and neither from the other five strains. It could be concluded that the duplex PCR was specific and could be used in quick diagnose Aeromonas hydrophila and Aeromonas spp.%针对GenBank中登录的气单胞菌属(Aeromonas)的毒力基因甘油磷脂胆固醇酰基转移酶基因(GCA T)和嗜水气单胞菌(Aeromonash ydrophila)的16S rRNA基因的保守区设计2对特异性引物.通过进行双重PCR反应体系优化,PCR产物的测序鉴定和特异性试验,建立了一种能同时检测气单胞菌(Aeromonas spp.)和嗜水气单胞菌的双重PCR检测方法.用此方法对5株嗜水气单胞菌、7株其他不同种的气单胞菌和5株非气单胞菌属菌株进行双重PCR检测.结果显示,气单胞菌和嗜水气单胞菌在GCAT基因的扩增区都能得到有效扩增,其中嗜水气单胞菌能同时在16S rRNA基因扩增区得到有效扩增,而非气单胞菌属的菌株,在此两扩增区都为阴性,表明此检测方法可靠且可行.

  3. Identification of Atlantic cod (Gadus morhua), ling (Molva molva), and Alaska pollock (Gadus chalcogrammus) by PCR-ELISA using duplex PCR.

    Science.gov (United States)

    Taboada, Ledicia; Sánchez, Ana; Velasco, Amaya; Santaclara, Francisco J; Pérez-Martín, Ricardo I; Sotelo, Carmen G

    2014-06-18

    Species-specific PCR-ELISA assays for the identification of Atlantic cod (Gadus morhua), Alaska pollock (Gadus chalcogrammus), and ling (Molva molva) in food products have been developed. The method, comprising a set of primers common to the first two species, a set of primers for M. molva, and a probe for each species, was designed using ND4 and cytochrome b genes as molecular markers. The sensitivity and selectivity were then determined for each assay. These assays were afterward used to analyze DNA extracted from commercial fish products. The presence of the target species was successfully detected in all analyzed samples, demonstrating the applicability of this method to the analysis of food products.

  4. 贝类包拉米虫和派琴虫二重荧光定量PCR方法的建立%Development of Duplex Real-time PCR Assay for Detection of Bonamia and Perkinsus in Shellfsh

    Institute of Scientific and Technical Information of China (English)

    2013-01-01

    Two pairs of specific oligonucleotide primers for Bonamia and Perkinsus,and two TaqMan probes specific for each protozoan parasite were designed with Primer Express 3.0 software. The reaction parameters such as the concentration of two pairs of primers,two TaqMan probes and the reaction buffer were optimized to develop a duplex real-time PCR assay for the rapid detection of Bonamia and Perkinsus. The sensitivity of the duplex real-time PCR assay was 200 template copies for Bonamia and Perkinsus .The duplex real-time PCR assay was found to be specific and to be able to detect and differentiate Bonamia and Perkinsus,and no positive results were observed when nucleic acids from Haplosporidium,Marteilia refringens,Vibrio parahaemolyticu,V. Alginolyticu,and V.Fluvialisand were used as duplex real-time PCR templates. This duplex real-time PCR assay was a rapid,sensitive,and specific test fo detection of Bonamia and Perkinsus and could be useful for the control of these protozoan parasites in shellfish.%  根据GenBank中包拉米虫和派琴虫保守基因序列,用Primer Express 3.0软件设计合成了两对引物和两条TaqMan探针。对反应条件和试剂浓度进行优化,建立了能够同时检测包拉米虫和派琴虫的二重荧光定量PCR方法。该方法对包拉米虫和派琴虫的检测敏感性达到200个模板拷贝数;此外抗干扰能力强,对包拉米虫和派琴虫不同模板浓度进行组合,仍可有效同时检测到这两个原虫。该方法对单孢子虫、马尔太虫、副溶血弧菌、溶藻弧菌和河弧菌等病原体的检测,结果全为阴性。研究建立的包拉米虫和派琴虫荧光定量PCR具有特异、敏感、快速、定量、重复性好等优点,可用于贝类包拉米虫和派琴虫感染的检测。

  5. A majority of Huntington's disease patients may be treatable by individualized allele-specific RNA interference.

    Science.gov (United States)

    Lombardi, Maria Stella; Jaspers, Leonie; Spronkmans, Christine; Gellera, Cinzia; Taroni, Franco; Di Maria, Emilio; Donato, Stefano Di; Kaemmerer, William F

    2009-06-01

    Use of RNA interference to reduce huntingtin protein (htt) expression in affected brain regions may provide an effective treatment for Huntington disease (HD), but it remains uncertain whether suppression of both wild-type and mutant alleles in a heterozygous patient will provide more benefit than harm. Previous research has shown suppression of just the mutant allele is achievable using siRNA targeted to regions of HD mRNA containing single nucleotide polymorphisms (SNPs). To determine whether more than a minority of patients may be eligible for an allele-specific therapy, we genotyped DNA from 327 unrelated European Caucasian HD patients at 26 SNP sites in the HD gene. Over 86% of the patients were found to be heterozygous for at least one SNP among those tested. Because the sites are genetically linked, one cannot use the heterozygosity rates of the individual SNPs to predict how many sites (and corresponding allele-specific siRNA) would be needed to provide at least one treatment possibility for this percentage of patients. By computing all combinations, we found that a repertoire of allele-specific siRNA corresponding to seven sites can provide at least one allele-specific siRNA treatment option for 85.6% of our sample. Moreover, we provide evidence that allele-specific siRNA targeting these sites are readily identifiable using a high throughput screening method, and that allele-specific siRNA identified using this method indeed show selective suppression of endogenous mutant htt protein in fibroblast cells from HD patients. Therefore, allele-specific siRNA are not so rare as to be impractical to find and use therapeutically.

  6. 羊源肠球菌属与屎肠球菌双重PCR检测方法的建立%Establishment of a Duplex PCR Assay for Simultaneous Detection of Sheep-originated Enterococcus and Enterococcus faecium

    Institute of Scientific and Technical Information of China (English)

    刘昆; 凤英; 高娃; 杨斌; 张月梅; 宋越; 陈伟; 吴树清; 赵世华

    2015-01-01

    Objective]To establish a duplex PCR assay for simultaneous and quick detection of sheep-originated clinical isolates of Enterococcus and Enterococcus faecium. [Methods]The genus specific and species specific primers for Enterococcus and Enterococcus faecium targeting at 16S rDNA and ddl gene were designed respectively, and the reaction system of duplex PCR assay was determined. The specificity and sensitivity of the established duplex PCR assay were evaluated. The simulated bacterial infection blood samples were prepared by using healthy sheep blood and mixed bacterial solution and were used to verify the specificity and sensitivity of the established duplex PCR assay in detection of clinical samples. [Results]The results showed that the duplex PCR assay was able to specifically amplify the DNA fragments of 294 bp from Enterococcus and 557 bp from Enterococcus faecium,but was negative for other common animal-originated pathogenic bacteria. The detection limits of the assay for Enterococcus and Enterococcus faecium were 5.71×10-5 ng/μL and 5.71×10-4 ng/μL, respectively. The duplex PCR assay was able to specifically detect the Enterococcus and Enterococcus faecium in simulated bacterial infection blood samples, and the detection limit for Enterococcus faecium was 1×101 CFU/mL. [Conclusion]It was indicated that the established duplex PCR assay is specific and sensitive for detection of clinical Enterococcus and Enterococcus faecium isolates.%[目的]建立羊源肠球菌属与屎肠球菌的双重PCR检测方法,对临床分离的菌株进行快速检测。[方法]设计肠球菌属特异性和屎肠球菌种特异性引物,建立双重PCR反应体系;评价双重PCR反应的特异性和敏感性;利用混合菌液制备模拟感染样品,确定双重PCR反应对模拟感染样品检测的特异性和敏感性。[结果]建立的双重PCR反应能够特异性的扩增肠球菌属(294 bp)和屎肠球菌(557 bp) DNA片段;该方法对常见的

  7. Allele-specific copy-number discovery from whole-genome and whole-exome sequencing

    Science.gov (United States)

    Wang, WeiBo; Wang, Wei; Sun, Wei; Crowley, James J.; Szatkiewicz, Jin P.

    2015-01-01

    Copy-number variants (CNVs) are a major form of genetic variation and a risk factor for various human diseases, so it is crucial to accurately detect and characterize them. It is conceivable that allele-specific reads from high-throughput sequencing data could be leveraged to both enhance CNV detection and produce allele-specific copy number (ASCN) calls. Although statistical methods have been developed to detect CNVs using whole-genome sequence (WGS) and/or whole-exome sequence (WES) data, information from allele-specific read counts has not yet been adequately exploited. In this paper, we develop an integrated method, called AS-GENSENG, which incorporates allele-specific read counts in CNV detection and estimates ASCN using either WGS or WES data. To evaluate the performance of AS-GENSENG, we conducted extensive simulations, generated empirical data using existing WGS and WES data sets and validated predicted CNVs using an independent methodology. We conclude that AS-GENSENG not only predicts accurate ASCN calls but also improves the accuracy of total copy number calls, owing to its unique ability to exploit information from both total and allele-specific read counts while accounting for various experimental biases in sequence data. Our novel, user-friendly and computationally efficient method and a complete analytic protocol is freely available at https://sourceforge.net/projects/asgenseng/. PMID:25883151

  8. Combined rpoB duplex PCR and hsp65 PCR restriction fragment length polymorphism with capillary electrophoresis as an effective algorithm for identification of Mycobacterial species from clinical isolates

    Directory of Open Access Journals (Sweden)

    Huang Chen-Cheng

    2012-07-01

    Full Text Available Abstract Background Mycobacteria can be quickly and simply identified by PCR restriction-enzyme analysis (PRA, but misidentification can occur because of similarities in band sizes that are critical for discriminating among species. Capillary electrophoresis can provide computer-aided band discrimination. The aim of this research was to develop an algorithm for identifying mycobacteria by combined rpoB duplex PRA (DPRA and hsp65 PRA with capillary electrophoresis. Results Three hundred and seventy-six acid-fast bacillus smear-positive BACTEC cultures, including 200 Mycobacterium tuberculosis complexes (MTC and 176 non-tuberculous mycobacteria (NTM were analyzed. With combined hsp65 and rpoB DPRA, the accuracy rate was 100% (200 isolates for the MTC and 91.4% (161 isolates for the NTM. Among the discordant results (8.6% for the NTM, one isolate of Mycobacterial species and an isolate of M. flavescens were found as new sub-types in hsp65 PRA. Conclusions This effective and novel identification algorithm using combined rpoB DPRA and hsp65 PRA with capillary electrophoresis can rapidly identify mycobacteria and find new sub-types in hsp65 PRA. In addition, it is complementary to 16 S rDNA sequencing.

  9. Using Duplex PCR-RFLP Technology to Detect Mutton and Other Meat Adulteration%利用多重PCR-RFLP同时检测羊肉及掺杂其中的肉类

    Institute of Scientific and Technical Information of China (English)

    韩镌竹; 李欣南; 苏崴艺; 于雷

    2014-01-01

    Pure mutton or the mixture with 2%, 5%and 10%chicken, pork and mouse meat were studied. Genomic DNA was extracted with t DNA extraction kit. Pure mutton and the mixtures were detected simultaneously by duplex polymerase chain reaction(PCR) and restriction fragment length polymorphism(RFLP) techniques. The results showed that method introduced in this study could detect pure mutton , chicken, pork or mouse meat adulterated in it effectively. The detection limitation was 2%.%以纯羊肉为原料,按2%、5%和10%的重量比在羊肉中掺杂鸡肉、猪肉和鼠肉,通过基因组DNA提取试剂盒法提取DNA,利用多重多聚酶链反应(Polymerase Chain Reaction ,PCR)及限制性片段长度多态性(Restriction Fragment Length Polymorphism, RFLP)分析同时检测羊肉及掺杂其中的其他肉类。结果表明,通过多重PCR扩增,可以有效的检测出纯羊肉及其掺杂的鸡肉、猪肉和鼠肉,检出限为2%。

  10. Allele Workbench: transcriptome pipeline and interactive graphics for allele-specific expression.

    Directory of Open Access Journals (Sweden)

    Carol A Soderlund

    Full Text Available Sequencing the transcriptome can answer various questions such as determining the transcripts expressed in a given species for a specific tissue or condition, evaluating differential expression, discovering variants, and evaluating allele-specific expression. Differential expression evaluates the expression differences between different strains, tissues, and conditions. Allele-specific expression evaluates expression differences between parental alleles. Both differential expression and allele-specific expression have been studied for heterosis (hybrid vigor, where the hybrid has improved performance over the parents for one or more traits. The Allele Workbench software was developed for a heterosis study that evaluated allele-specific expression for a mouse F1 hybrid using libraries from multiple tissues with biological replicates. This software has been made into a distributable package, which includes a pipeline, a Java interface to build the database, and a Java interface for query and display of the results. The required input is a reference genome, annotation file, and one or more RNA-Seq libraries with optional replicates. It evaluates allelic imbalance at the SNP and transcript level and flags transcripts with significant opposite directional allele-specific expression. The Java interface allows the user to view data from libraries, replicates, genes, transcripts, exons, and variants, including queries on allele imbalance for selected libraries. To determine the impact of allele-specific SNPs on protein folding, variants are annotated with their effect (e.g., missense, and the parental protein sequences may be exported for protein folding analysis. The Allele Workbench processing results in transcript files and read counts that can be used as input to the previously published Transcriptome Computational Workbench, which has a new algorithm for determining a trimmed set of gene ontology terms. The software with demo files is available

  11. New prediction model for probe specificity in an allele-specific extension reaction for haplotype-specific extraction (HSE) of Y chromosome mixtures.

    Science.gov (United States)

    Rothe, Jessica; Watkins, Norman E; Nagy, Marion

    2012-01-01

    Allele-specific extension reactions (ASERs) use 3' terminus-specific primers for the selective extension of completely annealed matches by polymerase. The ability of the polymerase to extend non-specific 3' terminal mismatches leads to a failure of the reaction, a process that is only partly understood and predictable, and often requires time-consuming assay design. In our studies we investigated haplotype-specific extraction (HSE) for the separation of male DNA mixtures. HSE is an ASER and provides the ability to distinguish between diploid chromosomes from one or more individuals. Here, we show that the success of HSE and allele-specific extension depend strongly on the concentration difference between complete match and 3' terminal mismatch. Using the oligonucleotide-modeling platform Visual Omp, we demonstrated the dependency of the discrimination power of the polymerase on match- and mismatch-target hybridization between different probe lengths. Therefore, the probe specificity in HSE could be predicted by performing a relative comparison of different probe designs with their simulated differences between the duplex concentration of target-probe match and mismatches. We tested this new model for probe design in more than 300 HSE reactions with 137 different probes and obtained an accordance of 88%.

  12. Simultaneous detection of West Nile virus and Chikungunya virus by duplex real-time quantitative PCR%双重荧光定量PCR检测西尼罗病毒和基孔肯雅病毒

    Institute of Scientific and Technical Information of China (English)

    余蓓蓓; 卢亦愚; 谢鑫友; 徐昌平; 张钧

    2013-01-01

    目的 建立同时检测西尼罗病毒(WNV)、基孔肯雅病毒(CHIKV)的双重荧光定量PCR法,为临床疑似病例的诊断提供依据.方法 分别针对WNV CAP基因、CHIKV E1基因保守区设计特异性引物和TaqMan探针,建立并优化双重荧光定量PCR反应体系,评价方法的特异性和灵敏度.结果 建立的双重荧光定量PCR可同时检测WNV、CHIKV核酸,标准曲线相关系数(r)分别达0.999、0.998,灵敏度达10 copies/μL,具有良好的特异性.结论 建立了同时检测WNV、CHIKV的双重荧光定量PCR法,但尚需临床进一步验证.%Objective To develop a duplex real-time quantitative PCR assay for simultaneous detection of West Nile virus and Chikungunya virus.Methods Two sets of primers and TaqMan probes were designed based on highly conserved CAP gene region of West Nile virus and E1 gene region of Chikungunya virus,and the reactive condition of duplex real-time PCR was optimized.The sensitivity and specificity of the assay were evaluated.Results The duplex real-time quantitative PCR assay showed excellent specificity in simultaneous detection of West Nile virus and Chikungunya virus.The sensitivities of the assay were 10 copies/μL for both the virus,and the correlation coefficient of the quantitative curve were 0.999 and 0.998 respectively.Conclusion The duplex fluorescent quantitative PCR assay developed in this study is sensitive and specific for simultaneous detection of West Nile virus and Chikungunya virus.The efficiency of the assay for detecting of clinical samples should be further evaluated.

  13. Simultaneous SNP identification and assessment of allele-specific bias from ChIP-seq data

    Directory of Open Access Journals (Sweden)

    Ni Yunyun

    2012-09-01

    Full Text Available Abstract Background Single nucleotide polymorphisms (SNPs have been associated with many aspects of human development and disease, and many non-coding SNPs associated with disease risk are presumed to affect gene regulation. We have previously shown that SNPs within transcription factor binding sites can affect transcription factor binding in an allele-specific and heritable manner. However, such analysis has relied on prior whole-genome genotypes provided by large external projects such as HapMap and the 1000 Genomes Project. This requirement limits the study of allele-specific effects of SNPs in primary patient samples from diseases of interest, where complete genotypes are not readily available. Results In this study, we show that we are able to identify SNPs de novo and accurately from ChIP-seq data generated in the ENCODE Project. Our de novo identified SNPs from ChIP-seq data are highly concordant with published genotypes. Independent experimental verification of more than 100 sites estimates our false discovery rate at less than 5%. Analysis of transcription factor binding at de novo identified SNPs revealed widespread heritable allele-specific binding, confirming previous observations. SNPs identified from ChIP-seq datasets were significantly enriched for disease-associated variants, and we identified dozens of allele-specific binding events in non-coding regions that could distinguish between disease and normal haplotypes. Conclusions Our approach combines SNP discovery, genotyping and allele-specific analysis, but is selectively focused on functional regulatory elements occupied by transcription factors or epigenetic marks, and will therefore be valuable for identifying the functional regulatory consequences of non-coding SNPs in primary disease samples.

  14. Duplex PCR assay against Cronobacter spp.(Enterobacteria sakazakii)in food samples%食品中克罗诺杆菌(原阪崎肠杆菌)双重PCR检测方法研究

    Institute of Scientific and Technical Information of China (English)

    王翔; 徐幸莲; 祝长青; 周光宏

    2012-01-01

    Duplex PCR assays were developed for the detection of Cronobacter spp. Based on its 16S rRNA gene and partial macromo-lecular synthesis operon. Among all the bacterial strains used in this study, duplex PCR amplified two specific products from 4 strains of Cronobacter spp. But not from others. In pure culture,the sensitivity of single PCR for former gene was 6. 3×101 CFU·mL-1 ,while for the latter was 6. 3 × 103 CFU ·mL-1; whereas for duplex PCR, it was 6. 3 × 103 CFU ? mL-1. The detection limit for duplex PCR assays was 10° CFU·mL-1 or 10° CFU-g-1 for different levels of Cronobacter spp. Inoculated into food samples (milk powder, milk and chicken meat) after 24 h enrichment. The detection limit of the duplex PCR, however, remained unaffected in the presence of Salmonella typhimurium in milk powder. So, the methods described in our study can be adopted to detect Cronobacter spp. In food samples with higher sensitivity and specificity.%根据克罗诺杆菌(Cronobacter spp.)16S rRNA基因以及局部大分子合成(MMS)操纵子特异序列设计2对引物,经反应体系和条件优化,建立了双重PCR检测方法.特异性检测结果显示,Cronobacter spp.菌株PCR扩增均可见2条特异性条带,而其他菌株PCR扩增均为阴性.纯菌检测双重PCR的灵敏度为6.3×103 CFU·mL-1,而相对应单重PCR的灵敏度分别为6.3×101CFU· mL-1和6.3×103 CFU·mL-1;人工污染的食品样品(奶粉、牛奶、鸡肉)在经过24 h增菌后,检测限均可达100 CFU· mL-1或100 CFU·g-1.在鼠伤寒沙门氏菌(Salmonella typhimurium)存在的条件下,双重PCR的检测限没有受到影响.表明本试验建立的双重PCR检测方法具有很好的特异性和灵敏度,能克服食品样品基质及杂菌的干扰,可应用于食品中Cronobacter spp.的检测.

  15. Allele-specific amplification and electrochemiluminescence method for single nucleotide polymorphism analysis

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    A new approach combined the specificity of allele-specific amplification (ASA) with the sensitivity of electrochemiluminescence (ECL) assay for single nucleotide polymorphism (SNP) analysis was proposed. Briefly, target gene was amplified by a biotin-labeled allele-specific forward primer and a Ru(bpy)32+ (TBR)-labeled universal reverse primer. Then, the amplicon was captured onto streptavidin-coated paramagnetic beads through biotin label, and detected by measuring the ECL signal of TBR label. Different genotypes were distinguished according to the ECL values of the amplicons by different genotypic primers. K-ras oncogene was used as a target to validate the feasibility of the method. The experiment results show that the different genotypes can be clearly distinguished by ASA-ECL assay. The method is useful in SNP analysis due to its sensitivity,safety, and simplicity.(C) 2007 Da Xing. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

  16. BaalChIP: Bayesian analysis of allele-specific transcription factor binding in cancer genomes.

    Science.gov (United States)

    de Santiago, Ines; Liu, Wei; Yuan, Ke; O'Reilly, Martin; Chilamakuri, Chandra Sekhar Reddy; Ponder, Bruce A J; Meyer, Kerstin B; Markowetz, Florian

    2017-02-24

    Allele-specific measurements of transcription factor binding from ChIP-seq data are key to dissecting the allelic effects of non-coding variants and their contribution to phenotypic diversity. However, most methods of detecting an allelic imbalance assume diploid genomes. This assumption severely limits their applicability to cancer samples with frequent DNA copy-number changes. Here we present a Bayesian statistical approach called BaalChIP to correct for the effect of background allele frequency on the observed ChIP-seq read counts. BaalChIP allows the joint analysis of multiple ChIP-seq samples across a single variant and outperforms competing approaches in simulations. Using 548 ENCODE ChIP-seq and six targeted FAIRE-seq samples, we show that BaalChIP effectively corrects allele-specific analysis for copy-number variation and increases the power to detect putative cis-acting regulatory variants in cancer genomes.

  17. Allele-specific enzymatic amplification of. beta. -globin genomic DNA for diagnosis of sickle cell anemia

    Energy Technology Data Exchange (ETDEWEB)

    Wu, D.Y.; Ugozzoli, L.; Pal, B.K.; Wallace, B. (Beckman Research Institute of the City of Hope, Duarte, CA (USA))

    1989-04-01

    A rapid nonradioactive approach to the diagnosis of sickle cell anemia is described based on an allele-specific polymerase chain reaction (ASPCR). This method allows direct detection of the normal or the sickle cell {beta}-globin allele in genomic DNA without additional steps of probe hybridization, ligation, or restriction enzyme cleavage. Two allele-specific oligonucleotide primers, one specific for the sickle cell allele and one specific for the normal allele, together with another primer complementary to both alleles were used in the polymerase chain reaction with genomic DNA templates. The allele-specific primers differed from each other in their terminal 3{prime} nucleotide. Under the proper annealing temperature and polymerase chain reaction conditions, these primers only directed amplification on their complementary allele. In a single blind study of DNA samples from 12 individuals, this method correctly and unambiguously allowed for the determination of the genotypes with no false negatives or positives. If ASPCR is able to discriminate all allelic variation (both transition and transversion mutations), this method has the potential to be a powerful approach for genetic disease diagnosis, carrier screening, HLA typing, human gene mapping, forensics, and paternity testing.

  18. 应用单一和双重荧光定量PCR法快速检测军团菌%Single and duplex fluorescence quantitative PCR for rapid detection of Legionella

    Institute of Scientific and Technical Information of China (English)

    莫自耀; 秦建强; 赵红波; 关文达; 秦笙; 王玉涛; 杨子峰

    2011-01-01

    Objective To develop the single and duplex fluorescence quantitative PCR for detection of Legionella and Legionella pneumophila respectively.Methods Specific primers and probes were designed according to the 16 S rRNA gene of Legionella and mip gene of Legionella pneumophila.The probes were labeled with reporter genes, FAM (for 16 S rRNA) and HEX (for mip gene) respectively.Moreover, the reaction systems and conditions for PCR were optimized.Then the single gene probe (single fluorescence quantitative PCR) and duplex gene probe (duplex fluorescence quantitative PCR) were used to detect the pneumophila and non-Legionella pneumophila strains, as well as other non-Legionella, in order to validate the specificity and sensitivity of these two methods.In addition, duplex fluorescence quantitative PCR was employed to detect Legionella on filtering membrane and extracted Legionella DNA in water samples from air - conditioners, to compare the consistency between the both.Results The 16 S rRNA genes of Legionella and mip genes of Legionella pneumophila were well detected with fluorescence quantitative PCR,with the lowest detectable templates being 8 and 10 copies respectively.An ideal reaction system after optimization of conditions was established successfully.Single fluorescence quantitative PCR demonstrated positive expression of 16 S rRNA gene in all of 8 Legionella pneumophila and 4 nonLegionella pneumophila strains, and positive expression of mip gene in Legionella pneumophila but not in non- Legionella pneumophila strains.Duplex fluorescence quantitative PCR demonstrated 2 false negative strains among 23 Legionella pneumophila strains and 1 false positive strain among 9 nonLegionella pneumophila and non - Legionella strains.The results of DNA detection were consistent between 49 filtering membranes and extracted DNA in water samples from air-conditioners.There were 26 water samples tested positive for Legionella, including 20 for Legionella pneumophila and 6 for non

  19. Enhancement of allele discrimination by introduction of nucleotide mismatches into siRNA in allele-specific gene silencing by RNAi.

    Directory of Open Access Journals (Sweden)

    Yusuke Ohnishi

    Full Text Available Allele-specific gene silencing by RNA interference (RNAi is therapeutically useful for specifically inhibiting the expression of disease-associated alleles without suppressing the expression of corresponding wild-type alleles. To realize such allele-specific RNAi (ASP-RNAi, the design and assessment of small interfering RNA (siRNA duplexes conferring ASP-RNAi is vital; however, it is also difficult. In a previous study, we developed an assay system to assess ASP-RNAi with mutant and wild-type reporter alleles encoding the Photinus and Renilla luciferase genes. In line with experiments using the system, we realized that it is necessary and important to enhance allele discrimination between mutant and corresponding wild-type alleles. Here, we describe the improvement of ASP-RNAi against mutant alleles carrying single nucleotide variations by introducing base substitutions into siRNA sequences, where original variations are present in the central position. Artificially mismatched siRNAs or short-hairpin RNAs (shRNAs against mutant alleles of the human Prion Protein (PRNP gene, which appear to be associated with susceptibility to prion diseases, were examined using this assessment system. The data indicates that introduction of a one-base mismatch into the siRNAs and shRNAs was able to enhance discrimination between the mutant and wild-type alleles. Interestingly, the introduced mismatches that conferred marked improvement in ASP-RNAi, appeared to be largely present in the guide siRNA elements, corresponding to the 'seed region' of microRNAs. Due to the essential role of the 'seed region' of microRNAs in their association with target RNAs, it is conceivable that disruption of the base-pairing interactions in the corresponding seed region, as well as the central position (involved in cleavage of target RNAs, of guide siRNA elements could influence allele discrimination. In addition, we also suggest that nucleotide mismatches at the 3'-ends of sense

  20. Quantification of allele-specific expression of a gene encoding strawberry polygalacturonase-inhibiting protein (PGIP) using Pyrosequencing((TM))

    NARCIS (Netherlands)

    Schaart, J.G.; Mehli, L.; Schouten, H.J.

    2005-01-01

    Recent studies indicate that allele-specific differences in gene expression are a common phenomenon. The extent to which differential allelic expression exists might be underestimated, due to the limited accuracy of the methods used so far. To demonstrate allele-specific expression, we investigated

  1. Allele-specific polymerase chain reaction for detection of a mutation in the relax circular DNA and the covalently closed circular DNA of hepatitis B virus.

    Science.gov (United States)

    Pan, Wan-Long; Hu, Jie-Li; Fang, Yan; Luo, Qiang; Xu, Ge; Xu, Lei; Jing, Zhou-Hong; Shan, Xue-Feng; Zhu, Yan-Ling; Huang, Ai-Long

    2013-12-01

    The relax circle DNA (rcDNA) sequence and the covalently closed circle DNA (cccDNA) sequence in hepatitis B virus (HBV) are crucial regions for HBV infections. To analyze mutations in rcDNA and cccDNA, DNA sequencing is often used, although it is time-consuming and expensive. Herein, we report a simple, economic, albeit accurate allele-specific polymerase chain reaction (AS-PCR) to detect mutations in these regions of HBV. This method can be extensively used to screen for mutations at specific positions of HBV genome.

  2. Sequenza: allele-specific copy number and mutation profiles from tumor sequencing data.

    Science.gov (United States)

    Favero, F; Joshi, T; Marquard, A M; Birkbak, N J; Krzystanek, M; Li, Q; Szallasi, Z; Eklund, A C

    2015-01-01

    Exome or whole-genome deep sequencing of tumor DNA along with paired normal DNA can potentially provide a detailed picture of the somatic mutations that characterize the tumor. However, analysis of such sequence data can be complicated by the presence of normal cells in the tumor specimen, by intratumor heterogeneity, and by the sheer size of the raw data. In particular, determination of copy number variations from exome sequencing data alone has proven difficult; thus, single nucleotide polymorphism (SNP) arrays have often been used for this task. Recently, algorithms to estimate absolute, but not allele-specific, copy number profiles from tumor sequencing data have been described. We developed Sequenza, a software package that uses paired tumor-normal DNA sequencing data to estimate tumor cellularity and ploidy, and to calculate allele-specific copy number profiles and mutation profiles. We applied Sequenza, as well as two previously published algorithms, to exome sequence data from 30 tumors from The Cancer Genome Atlas. We assessed the performance of these algorithms by comparing their results with those generated using matched SNP arrays and processed by the allele-specific copy number analysis of tumors (ASCAT) algorithm. Comparison between Sequenza/exome and SNP/ASCAT revealed strong correlation in cellularity (Pearson's r = 0.90) and ploidy estimates (r = 0.42, or r = 0.94 after manual inspecting alternative solutions). This performance was noticeably superior to previously published algorithms. In addition, in artificial data simulating normal-tumor admixtures, Sequenza detected the correct ploidy in samples with tumor content as low as 30%. The agreement between Sequenza and SNP array-based copy number profiles suggests that exome sequencing alone is sufficient not only for identifying small scale mutations but also for estimating cellularity and inferring DNA copy number aberrations. © The Author 2014. Published by Oxford University Press on behalf of

  3. Allele-specific silencing of mutant Ataxin-7 in SCA7 patient-derived fibroblasts.

    Science.gov (United States)

    Scholefield, Janine; Watson, Lauren; Smith, Danielle; Greenberg, Jacquie; Wood, Matthew J A

    2014-12-01

    Polyglutamine (polyQ) disorders are inherited neurodegenerative conditions defined by a common pathogenic CAG repeat expansion leading to a toxic gain-of-function of the mutant protein. Consequences of this toxicity include activation of heat-shock proteins (HSPs), impairment of the ubiquitin-proteasome pathway and transcriptional dysregulation. Several studies in animal models have shown that reducing levels of toxic protein using small RNAs would be an ideal therapeutic approach for such disorders, including spinocerebellar ataxia-7 (SCA7). However, testing such RNA interference (RNAi) effectors in genetically appropriate patient cell lines with a disease-relevant phenotype has yet to be explored. Here, we have used primary adult dermal fibroblasts from SCA7 patients and controls to assess the endogenous allele-specific silencing of ataxin-7 by two distinct siRNAs. We further identified altered expression of two disease-relevant transcripts in SCA7 patient cells: a twofold increase in levels of the HSP DNAJA1 and a twofold decrease in levels of the de-ubiquitinating enzyme, UCHL1. After siRNA treatment, the expression of both genes was restored towards normal levels. To our knowledge, this is the first time that allele-specific silencing of mutant ataxin-7, targeting a common SNP, has been demonstrated in patient cells. These findings highlight the advantage of an allele-specific RNAi-based therapeutic approach, and indicate the value of primary patient-derived cells as useful models for mechanistic studies and for measuring efficacy of RNAi effectors on a patient-to-patient basis in the polyQ diseases.

  4. Genome-wide survey of allele-specific splicing in humans

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    Scheffler Konrad

    2008-06-01

    Full Text Available Abstract Background Accurate mRNA splicing depends on multiple regulatory signals encoded in the transcribed RNA sequence. Many examples of mutations within human splice regulatory regions that alter splicing qualitatively or quantitatively have been reported and allelic differences in mRNA splicing are likely to be a common and important source of phenotypic diversity at the molecular level, in addition to their contribution to genetic disease susceptibility. However, because the effect of a mutation on the efficiency of mRNA splicing is often difficult to predict, many mutations that cause disease through an effect on splicing are likely to remain undiscovered. Results We have combined a genome-wide scan for sequence polymorphisms likely to affect mRNA splicing with analysis of publicly available Expressed Sequence Tag (EST and exon array data. The genome-wide scan uses published tools and identified 30,977 SNPs located within donor and acceptor splice sites, branch points and exonic splicing enhancer elements. For 1,185 candidate splicing polymorphisms the difference in splicing between alternative alleles was corroborated by publicly available exon array data from 166 lymphoblastoid cell lines. We developed a novel probabilistic method to infer allele-specific splicing from EST data. The method uses SNPs and alternative mRNA isoforms mapped to EST sequences and models both regulated alternative splicing as well as allele-specific splicing. We have also estimated heritability of splicing and report that a greater proportion of genes show evidence of splicing heritability than show heritability of overall gene expression level. Our results provide an extensive resource that can be used to assess the possible effect on splicing of human polymorphisms in putative splice-regulatory sites. Conclusion We report a set of genes showing evidence of allele-specific splicing from an integrated analysis of genomic polymorphisms, EST data and exon array

  5. Allele-specific locus binding and genome editing by CRISPR at the p16INK4a locus.

    Science.gov (United States)

    Fujita, Toshitsugu; Yuno, Miyuki; Fujii, Hodaka

    2016-07-28

    The clustered regularly interspaced short palindromic repeats (CRISPR) system has been adopted for a wide range of biological applications including genome editing. In some cases, dissection of genome functions requires allele-specific genome editing, but the use of CRISPR for this purpose has not been studied in detail. In this study, using the p16INK4a gene in HCT116 as a model locus, we investigated whether chromatin states, such as CpG methylation, or a single-nucleotide gap form in a target site can be exploited for allele-specific locus binding and genome editing by CRISPR in vivo. First, we showed that allele-specific locus binding and genome editing could be achieved by targeting allele-specific CpG-methylated regions, which was successful for one, but not all guide RNAs. In this regard, molecular basis underlying the success remains elusive at this stage. Next, we demonstrated that an allele-specific single-nucleotide gap form could be employed for allele-specific locus binding and genome editing by CRISPR, although it was important to avoid CRISPR tolerance of a single nucleotide mismatch brought about by mismatched base skipping. Our results provide information that might be useful for applications of CRISPR in studies of allele-specific functions in the genomes.

  6. A novel SYBR-based duplex qPCR for the detection of gene dosage: detection of an APC large deletion in a familial adenomatous polyposis patient with an unusual phenotype

    Directory of Open Access Journals (Sweden)

    Torrezan Giovana

    2012-07-01

    Full Text Available Abstract Background Familial adenomatous polyposis (FAP is a hereditary colorectal cancer syndrome caused by a loss of function of the APC gene. Large deletions in APC are a common cause of FAP; despite the existence of a variety of gene dosage detection methodologies, most are labor intensive and time and resource consuming. Methods We describe a new duplex qPCR method for gene dosage analysis based on the coamplification of a target and a reference gene in a SYBR Green reaction, followed by a comparison of the ratio between the target and the reference peaks of the melting curve for the test (patient and control samples. The reliability of the described duplex qPCR was validated for several genes (APC, HPRT1, ATM, PTEN and BRCA1. Results Using this novel gene dosage method, we have identified an APC gene deletion in a FAP patient undergoing genetic testing. Comparative genomic hybridization based on microarrays (aCGH was used to confirm and map the extent of the deletion, revealing a 5.2 MB rearrangement (5q21.3-q22.3 encompassing the entire APC and 19 additional genes. Conclusion The novel assay accurately detected losses and gains of one copy of the target sequences, representing a reliable and flexible alternative to other gene dosage techniques. In addition, we described a FAP patient harboring a gross deletion at 5q21.3-q22.3 with an unusual phenotype of the absence of mental impairment and dysmorphic features.

  7. Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples using a duplex real-time fluorescence resonance energy transfer PCR and melting curve analysis.

    Science.gov (United States)

    Sanpool, Oranuch; Intapan, Pewpan M; Thanchomnang, Tongjit; Janwan, Penchom; Lulitanond, Viraphong; Doanh, Pham Ngoc; Van Hien, Hoang; Dung, Do Trung; Maleewong, Wanchai; Nawa, Yukifumi

    2012-07-01

    We developed a single step duplex real-time fluorescence resonance energy transfer (FRET) PCR merged with melting curve analysis for the fast detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples. Two species of mitochondrial NADH dehydrogenase subunit 2 (nad2) DNA elements, the 165-bp nad2 product of C. sinensis and the 209-bp nad2 product of O. viverrini, were amplified by species-specific primers, and the fluorescence melting curve analyses were generated from hybrid of amplicons and two pairs of species-specific fluorophore-labeled probes. By their different fluorescence channels and melting temperatures, both C. sinensis and O. viverrini eggs in infected human fecal samples were detected and differentiated with high (100%) sensitivity and specificity. Detection limit was as little as a single C. sinensis egg and two O. viverrini eggs in 100 mg of fecal sample. The assay could distinguish the DNA of both parasites from the DNA of negative fecal samples and fecal samples with other parasitosis, as well as from the well-defined genomic DNA of human leukocytes and other parasites. It can reduce labor time of microscopic examination and is not prone to carry over contamination of agarose electrophoresis. Our duplex real-time FRET PCR method would be useful to determine the accurate range of endemic areas and/or to discover the co-endemic areas of two liver flukes, C. sinensis and O. viverrini, in Asia. This method also would be helpful for the differential diagnosis of the suspected cases of liver fluke infections among travelers who had visited the endemic countries of those parasites.

  8. Germline allele-specific expression of DAPK1 in chronic lymphocytic leukemia.

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    Quan-Xiang Wei

    Full Text Available We previously reported a rare germline variant (c.1-6531 that resulted in allele-specific expression (ASE of death-associated protein kinase 1 (DAPK1 and predisposition to chronic lymphocytic leukemia (CLL. We investigated a cohort of CLL patients lacking this mutation for the presence of ASE of DAPK1. We developed a novel strategy that combines single-nucleotide primer extension (SNuPE with MALDI-TOF mass spectrometry, and detected germline DAPK1 ASE in 17 out of 120 (14.2% CLL patients associated with a trend towards younger age at diagnosis. ASE was absent in 63 healthy controls. Germline cells of CLL patients with ASE showed increased levels of DNA methylation in the promoter region, however, neither genetic nor further epigenetic aberrations could be identified in the DAPK1 5' upstream regulatory region, within distinct exons or in the 3'-UTR. We identified B-lymphoid malignancy related cell line models harboring allelic imbalance and found that allele-specific methylation in DAPK1 is associated with ASE. Our data indicate that ASE at the DAPK1 gene locus is a recurrent event, mediated by epigenetic mechanisms and potentially predisposing to CLL.

  9. Therapy for dominant inherited diseases by allele-specific RNA interference: successes and pitfalls.

    Science.gov (United States)

    Trochet, Delphine; Prudhon, Bernard; Vassilopoulos, Stéphane; Bitoun, Marc

    2015-01-01

    RNA interference (RNAi) is a conserved mechanism for post-transcriptional gene silencing mediated by messenger RNA (mRNA) degradation. RNAi is commonly induced by synthetic siRNA or shRNA which recognizes the targeted mRNA by base pairing and leads to target-mRNA degradation. RNAi may discriminate between two sequences only differing by one nucleotide conferring a high specificity of RNAi for its target mRNA. This property was used to develop a particular therapeutic strategy called "allele-specific-RNA interference" devoted to silence the mutated allele of genes causing dominant inherited diseases without affecting the normal allele. Therapeutic benefit was now demonstrated in cells from patients and animal models, and promising results of the first phase Ib clinical trial using siRNA-based allele-specific therapy were reported in Pachyonychia Congenita, an inherited skin disorder due to dominant mutations in the Keratin 6 gene. Our purpose is to review the successes of this strategy aiming to treat dominant inherited diseases and to highlight the pitfalls to avoid.

  10. Comparison of two extraction methods for the detection of hepatitis A virus in semi-dried tomatoes and murine norovirus as a process control by duplex RT-qPCR.

    Science.gov (United States)

    Martin-Latil, Sandra; Hennechart-Collette, Catherine; Guillier, Laurent; Perelle, Sylvie

    2012-09-01

    Enteric viruses are important agents of foodborne diseases. Due to their low infectious doses and low concentrations in food samples, an efficient and rapid virus concentration method is required for routine control. Because of the absence of a reliable cell culture method for most of the enteric viruses involved in outbreaks, reverse transcription quantitative real-time PCR (RT-qPCR) is now widely used for the detection of RNA viruses in food samples. One of the general requirements for viral diagnosis concerns the use of a process control to monitor the efficiency of viral particle concentration, nucleic acid extraction and the presence of potential inhibitors of the RT-PCR reaction. Recent epidemiological studies have linked hepatitis A outbreaks to the consumption of semi-dried tomatoes (SDT) in Australia, the Netherlands and France. In this study, the virus concentration reference method proposed by the CEN/TC275/WG6/TAG4 working group for samples of soft fruit and salad vegetables was compared to a method including an ultracentrifugation step to recover hepatitis A virus (HAV) in SDT. Murine norovirus (MNV-1) was used as a process control and detected simultaneously with HAV in a one-step duplex RT-qPCR in both procedures. The LOD of HAV was 10 PFU and 1 PFU of HAV/25 g of SDT in the presence or absence of MNV-1 respectively, whatever the method used. We conclude that both methods achieved an identical limit of detection and that the MNV-1 offers a very reliable and simple way to monitor the quality of the extraction procedures and the presence of RT-qPCR inhibitors.

  11. Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia.

    OpenAIRE

    1989-01-01

    A rapid nonradioactive approach to the diagnosis of sickle cell anemia is described based on an allele-specific polymerase chain reaction (ASPCR). This method allows direct detection of the normal or the sickle cell beta-globin allele in genomic DNA without additional steps of probe hybridization, ligation, or restriction enzyme cleavage. Two allele-specific oligonucleotide primers, one specific for the sickle cell allele and one specific for the normal allele, together with another primer co...

  12. DEMETER DNA glycosylase establishes MEDEA polycomb gene self-imprinting by allele-specific demethylation.

    Science.gov (United States)

    Gehring, Mary; Huh, Jin Hoe; Hsieh, Tzung-Fu; Penterman, Jon; Choi, Yeonhee; Harada, John J; Goldberg, Robert B; Fischer, Robert L

    2006-02-10

    MEDEA (MEA) is an Arabidopsis Polycomb group gene that is imprinted in the endosperm. The maternal allele is expressed and the paternal allele is silent. MEA is controlled by DEMETER (DME), a DNA glycosylase required to activate MEA expression, and METHYLTRANSFERASE I (MET1), which maintains CG methylation at the MEA locus. Here we show that DME is responsible for endosperm maternal-allele-specific hypomethylation at the MEA gene. DME can excise 5-methylcytosine in vitro and when expressed in E. coli. Abasic sites opposite 5-methylcytosine inhibit DME activity and might prevent DME from generating double-stranded DNA breaks. Unexpectedly, paternal-allele silencing is not controlled by DNA methylation. Rather, Polycomb group proteins that are expressed from the maternal genome, including MEA, control paternal MEA silencing. Thus, DME establishes MEA imprinting by removing 5-methylcytosine to activate the maternal allele. MEA imprinting is subsequently maintained in the endosperm by maternal MEA silencing the paternal allele.

  13. Allele-specific interactions between CAST AWAY and NEVERSHED control abscission in Arabidopsis flowers

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    William D. Groner

    2016-10-01

    Full Text Available An advantage of analyzing abscission in genetically tractable model plants is the ability to make use of classic genetic tools such as suppression analysis. We have investigated the regulation of organ abscission by carrying out suppression analysis in Arabidopsis flowers. Plants carrying mutations in the NEVERSHED (NEV gene, which encodes an ADP-ribosylation factor GTPase-activating protein, retain their outer floral organs after fertilization. Mutant alleles of CAST AWAY (CST, which encodes a receptor-like cytoplasmic kinase, were found to restore organ abscission in nev flowers in an allele-specific manner. To further explore the basis of the interactions between CST and NEV, we tested whether the site of a nev mutation is predictive of its ability to be suppressed. Our results suggest instead that the strength of a nev allele influences whether organ abscission can be rescued by a specific allele of CST.

  14. Establishment and application of duplex PCR method for pathogenic cocci detection in raw pork%生鲜猪肉中致病性球菌的双重PCR检测方法的建立与应用

    Institute of Scientific and Technical Information of China (English)

    李英英; 邵刘云; 刘肖; 张秀方; 胡慧; 王亚宾; 陈丽颖

    2014-01-01

    以金黄色葡萄球菌clfa基因和溶血性链球菌sdy基因为靶基因设计引物.通过对单个基因PCR和多重基因PCR扩增进行特异性、敏感性试验以及优化反应体系,建立了快速检测溶血性链球菌和金黄色葡萄球菌的双重PCR方法.在郑州市不同地区随机抽检126份生鲜猪肉样品,分别进行了双重PCR检测和常规微生物学检验.结果表明,建立的双重PCR方法特异性好,抗干扰能力强,灵敏度可达到102 ng·L-1.在126个样品中检测出溶血性链球菌的样品数为7份,检出率为5.55%;检出金黄色葡萄球菌阳性的样品数为8份,检出率为6.35%.%Primersfor clfa gene of Staphylococcus aureus and sdy gene of Streptococcus hemolyticus were specifically designed.Through reaction optimization and specificity and sensitivity tests,singular and duplex PCR methods were set up,and were then applied to detection of the target bacteria.126 raw pork samples collected from different places of Zhengzhou city,were analyzed by the PCR method,and conventional microbiological examination was also taken as a control.The results show that the duplex PCR protocol turned out to be specific,effective with a sensitivity of 102 ng· L-1.The examination of the raw pork samples showed that 7 out of 126 were detected as Streptococcus hemolyticus positive (5.55%),whilst Staphylococcus aureus was detected among 8 samples,showing a detection rate of 6.35%.

  15. A duplex nested PCR assay detecting of Vibrio cholerae and its application on environmental spechnens%双重套式PCR方法在霍乱弧菌外环境监测中的应用

    Institute of Scientific and Technical Information of China (English)

    陈爱平; 董新平; 徐海滨; 杨劲松; 严延生; 郭维植; 王多春; 阚飙

    2009-01-01

    Objective To establish a duplex nested PCR assay system which is capable for detecting O1 and O139 groups of Vibrio cholerae simultaneously, and is applicable to environmental specimens from routine cholera surveillance. Methods Based on nucleic acid sequences available in GenBank, six sets of primers were designed by PrimerSelect program of DNAStar, targeting the rib gene that encodes the O antigens of O1 and O139 V. cholerae, respectively. The specificity of several primer combinations was tested. A duplex nested PCR assay system for simultaneously detecting O1 and O139 V. cholerae was established, subsequently,its sensitivity, specificity, reproducibility and field evaluation were tested. The sensitivity of this assay was evaluated by comparing detection limits of nested PCR and conventional PCR Its reproducibility was tested by 32 positive samples ( 11 samples positive for 01,21 samples positive for 0139 ) from environmental surveillance. In addition ,the selected amplicons from positive samples were sequenced and analyzed with relevant sequences. Results This newly-established duplex nested PCR assay might distinguish O1 V. cholerae from O139 V. cholerae, based on fragment lengths of amplieons,with reliable reproducibility, and no specific amplification was observed as compared with other vibrio species. The sensitivity of this nested PCR was ( 15 000) higher than conventional PCR,and there was no interference observed with multiple primers and complicated templates in the same vial In its field evaluation,32 positive DNA samples were detected and be further confirmed with double or triple tests,implying reliable reproducibility and consistency of this system. These results indicated that this assay had reliable reproducibility. No amplification was observed in all negative specimens and also suggested the acceptable specificity of this assay. Sequence analysis of the selected amplification products revealed 100% homogeneous with relevant genes from

  16. Oncogene mutations, copy number gains and mutant allele specific imbalance (MASI frequently occur together in tumor cells.

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    Junichi Soh

    Full Text Available BACKGROUND: Activating mutations in one allele of an oncogene (heterozygous mutations are widely believed to be sufficient for tumorigenesis. However, mutant allele specific imbalance (MASI has been observed in tumors and cell lines harboring mutations of oncogenes. METHODOLOGY/PRINCIPAL FINDINGS: We determined 1 mutational status, 2 copy number gains (CNGs and 3 relative ratio between mutant and wild type alleles of KRAS, BRAF, PIK3CA and EGFR genes by direct sequencing and quantitative PCR assay in over 400 human tumors, cell lines, and xenografts of lung, colorectal, and pancreatic cancers. Examination of a public database indicated that homozygous mutations of five oncogenes were frequent (20% in 833 cell lines of 12 tumor types. Our data indicated two major forms of MASI: 1 MASI with CNG, either complete or partial; and 2 MASI without CNG (uniparental disomy; UPD, due to complete loss of wild type allele. MASI was a frequent event in mutant EGFR (75% and was due mainly to CNGs, while MASI, also frequent in mutant KRAS (58%, was mainly due to UPD. Mutant: wild type allelic ratios at the genomic level were precisely maintained after transcription. KRAS mutations or CNGs were significantly associated with increased ras GTPase activity, as measured by ELISA, and the two molecular changes were synergistic. Of 237 lung adenocarcinoma tumors, the small number with both KRAS mutation and CNG were associated with shortened survival. CONCLUSIONS: MASI is frequently present in mutant EGFR and KRAS tumor cells, and is associated with increased mutant allele transcription and gene activity. The frequent finding of mutations, CNGs and MASI occurring together in tumor cells indicates that these three genetic alterations, acting together, may have a greater role in the development or maintenance of the malignant phenotype than any individual alteration.

  17. Detection of pathogenic Listonella anguillarum isolated from Cynoglossus semilaevis by duplex PCR%半滑舌鳎病原鳗利斯顿氏菌双重PCR检测方法的研究

    Institute of Scientific and Technical Information of China (English)

    张晓君; 阎斌伦; 梁利国; 秦国民; 毕可然

    2011-01-01

    L anguillarum is the cause of the high mortalities of cultured half-smooth tongue sole (Cynoglossus semilaevis) during cultivation in Lian Yungang, Jiangsu province. In this study, the detection of the pathogenic L anguillarum by duplex PCR based on hemolysin and metalloprotease genes were conducted. Two pairs of specific primers were designed based on hemolysin and metalloprotease gene sequence, and these two primers could simultaneously amplify 493 bp and 248 bp gene fragments from chromosomal DNA of L. Anguillarum in one PCR reaction, and no cross reaction was detected in 5 other pathogenic bacteria tested. The results of sensitivity of dulplex PCR showed that the two primers could detect L anguillarum as low as 8 × 102CFU/mL, and detect purified chromosomal DNA as low as 0.171 875 ng/μL. Dominant L. Anguillarum could be isolated from the positive sample of duplex PCR through detecting ulcer tissues and liver of diseased Cynoglossus semilaevis. The specific PCR method for detection of L. Anguillarum was established, and the PCR protocol simultaneously amplifying different gene fragments of the L. Anguillarum could be useful in the specific, accurate and rapid detection of pathogenic L anguillarum.%对引起江苏连云港工厂化养殖半滑舌鳎大量死亡的病原鳗利斯顿氏菌,进行了基于溶血素和金属蛋白酶两种基因的双重PCR检测.根据鳗利斯顿氏菌溶血素基因和金属蛋白酶基因序列设计2对特异性引物,在同一PCR反应体系中,鳗利斯顿氏菌可同时扩增出上述2种基因片段,扩增片段大小分别为493 bp和248bp,两对引物对5种其他水产动物病原菌无交叉反应,敏感性检测结果为该双重PCR最低能检测8× 102 CFU/mL的鳗利斯顿氏菌,最低能检测0.171 875 ng/μL的鳗利斯顿氏菌基因组DNA.对发病半滑舌鳎溃疡组织、肝脏及养殖用水进行双重PCR检测,呈阳性反应的样品可分离出优势生长的鳗利斯顿氏菌.该实验表明两种不

  18. 鸭新城疫病毒和鸭圆环病毒二重PCR检测方法的建立%Development of a Duplex PCR Assay for Detection of Duck Newcastle Disease Virus and Duck Circovirus in Duck

    Institute of Scientific and Technical Information of China (English)

    许宗丽; 谢芝勋; 谢丽基; 刘加波; 谢志勤; 邓显文; 范晴

    2012-01-01

    According to the sequences of duck NDV F gene and DuCV V1/rep gene in GenBank, two pairs of specific primers were designed, and the reaction conditions were optimized, and then a duplex PCR assay was developed for detection of Newcastle disease virus and circovirus in ducks. All samples containing Newcastle disease virus and circovirus could be amplified into two specific bands, 493 bp for duck Newcastle disease virus and 218 bp for duck circovirus by this duplex PCR, but no specific bands of the same sizes were amplified from other duck pathogens, such as Muscovy duck parvovirus, duck plague virus, duck hepatitis virus, gosling plague virus, duck H9 subtype avian influenza virus, Riemerella anatipestifer, E.coli, avian Pasteurella multocida. As little as 40 fg of duck NDV and 20 fg ofDuCV DNA could be detected.%本研究根据GenBank中鸭新城疫病毒(NDV)的F基因和鸭圆环病毒(DuCV)的V1/rep基因的保守序列,各设计一对特异性引物,并对二重PCR的扩增条件进行优化,建立了鸭NDV和DuCV的二重PCR检测方法。对混合样品进行扩增,得到2条大小为493bp(鸭NDV)和218bp(DuCV)的特异性条带,与预扩增片段相符。而对番鸭细小病毒、鸭瘟病毒、鸭肝炎病毒、鸭源小鹅瘟病毒、鸭H9亚型流感病毒、鸭疫里氏杆菌、大肠杆菌、禽多杀性巴氏杆菌等病原检测,结果为阴性。该方法的敏感性试验表明,鸭NDV的核酸最小量为40fg,DuCV为20龟。

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

    Science.gov (United States)

    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. Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  20. Molecular characterization and a multiplex allele-specific PCR method for detection of thiabendazole resistance in Penicillium expansum from apple

    Science.gov (United States)

    Thiabendazole (TBZ) is commonly used as a postharvest treatment for control of blue mold in apples caused by Penicillium expansum. Different point mutations in the ß-tubulin gene conferring benzimidazole resistance have been reported in plant pathogens, but molecular mechanisms of TBZ resistance in ...

  1. Allele-specific deposition of macroH2A1 in Imprinting Control Regions

    Energy Technology Data Exchange (ETDEWEB)

    Choo, J H; Kim, J D; Chung, J H; Stubbs, L; Kim, J

    2006-01-13

    In the current study, we analyzed the deposition patterns of macroH2A1 at a number of different genomic loci located in X chromosome and autosomes. MacroH2A1 is preferentially deposited at methylated CpG CpG-rich regions located close to promoters. The macroH2A1 deposition patterns at the methylated CpG islands of several imprinted domains, including the Imprinting Control Regions (ICRs) of Xist, Peg3, H19/Igf2 Igf2, Gtl2/Dlk1, and Gnas domains, show consistent allele-specificity towards inactive, methylated alleles. The macroH2A1 deposition levels at the ICRs and other Differentially Methylated Regions (DMRs) of these domains are also either higher or comparable to those observed at the inactive X chromosome of female mammals. Overall, our results indicate that besides DNA methylation macroH2A1 is another epigenetic component in the chromatin of ICRs displaying differential association with two parental alleles.

  2. Utilising polymorphisms to achieve allele-specific genome editing in zebrafish

    Directory of Open Access Journals (Sweden)

    Samuel J. Capon

    2017-01-01

    Full Text Available The advent of genome editing has significantly altered genetic research, including research using the zebrafish model. To better understand the selectivity of the commonly used CRISPR/Cas9 system, we investigated single base pair mismatches in target sites and examined how they affect genome editing in the zebrafish model. Using two different zebrafish strains that have been deep sequenced, CRISPR/Cas9 target sites containing polymorphisms between the two strains were identified. These strains were crossed (creating heterozygotes at polymorphic sites and CRISPR/Cas9 complexes that perfectly complement one strain injected. Sequencing of targeted sites showed biased, allele-specific editing for the perfectly complementary sequence in the majority of cases (14/19. To test utility, we examined whether phenotypes generated by F0 injection could be internally controlled with such polymorphisms. Targeting of genes bmp7a and chordin showed reduction in the frequency of phenotypes in injected ‘heterozygotes’ compared with injecting the strain with perfect complementarity. Next, injecting CRISPR/Cas9 complexes targeting two separate sites created deletions, but deletions were biased to selected chromosomes when one CRISPR/Cas9 target contained a polymorphism. Finally, integration of loxP sequences occurred preferentially in alleles with perfect complementarity. These experiments demonstrate that single nucleotide polymorphisms (SNPs present throughout the genome can be utilised to increase the efficiency of in cis genome editing using CRISPR/Cas9 in the zebrafish model.

  3. Allele-specific methylation occurs at genetic variants associated with complex disease.

    Directory of Open Access Journals (Sweden)

    John N Hutchinson

    Full Text Available We hypothesize that the phenomenon of allele-specific methylation (ASM may underlie the phenotypic effects of multiple variants identified by Genome-Wide Association studies (GWAS. We evaluate ASM in a human population and document its genome-wide patterns in an initial screen at up to 380,678 sites within the genome, or up to 5% of the total genomic CpGs. We show that while substantial inter-individual variation exists, 5% of assessed sites show evidence of ASM in at least six samples; the majority of these events (81% are under genetic influence. Many of these cis-regulated ASM variants are also eQTLs in peripheral blood mononuclear cells and monocytes and/or in high linkage-disequilibrium with variants linked to complex disease. Finally, focusing on autoimmune phenotypes, we extend this initial screen to confirm the association of cis-regulated ASM with multiple complex disease-associated variants in an independent population using next-generation bisulfite sequencing. These four variants are implicated in complex phenotypes such as ulcerative colitis and AIDS progression disease (rs10491434, Celiac disease (rs2762051, Crohn's disease, IgA nephropathy and early-onset inflammatory bowel disease (rs713875 and height (rs6569648. Our results suggest cis-regulated ASM may provide a mechanistic link between the non-coding genetic changes and phenotypic variation observed in these diseases and further suggests a route to integrating DNA methylation status with GWAS results.

  4. Assessing allele-specific expression across multiple tissues from RNA-seq read data

    Science.gov (United States)

    Pirinen, Matti; Lappalainen, Tuuli; Zaitlen, Noah A.; Dermitzakis, Emmanouil T.; Donnelly, Peter; McCarthy, Mark I.; Rivas, Manuel A.

    2015-01-01

    Motivation: RNA sequencing enables allele-specific expression (ASE) studies that complement standard genotype expression studies for common variants and, importantly, also allow measuring the regulatory impact of rare variants. The Genotype-Tissue Expression (GTEx) project is collecting RNA-seq data on multiple tissues of a same set of individuals and novel methods are required for the analysis of these data. Results: We present a statistical method to compare different patterns of ASE across tissues and to classify genetic variants according to their impact on the tissue-wide expression profile. We focus on strong ASE effects that we are expecting to see for protein-truncating variants, but our method can also be adjusted for other types of ASE effects. We illustrate the method with a real data example on a tissue-wide expression profile of a variant causal for lipoid proteinosis, and with a simulation study to assess our method more generally. Availability and implementation: http://www.well.ox.ac.uk/~rivas/mamba/. R-sources and data examples http://www.iki.fi/mpirinen/ Contact: matti.pirinen@helsinki.fi or rivas@well.ox.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25819081

  5. H19-DMR allele-specific methylation analysis reveals epigenetic heterogeneity of CTCF binding site 6 but not of site 5 in head-and-neck carcinomas

    DEFF Research Database (Denmark)

    De Castro Valente Esteves, Leda Isabel; De Karla Cervigne, Nilva; Do Carmo Javaroni, Afonso

    2006-01-01

    Aberrant methylation of seven potential binding sites of the CTCF factor in the differentially methylated region upstream of the H19 gene (H19-DMR) has been suggested as critical for the regulation of IGF2 and H19 imprinted genes. In this study, we analyzed the allele-specific methylation pattern...... of CTCF binding sites 5 and 6 using methylation-sensitive restriction enzyme PCR followed by RFLP analysis in matched tumoral and lymphocyte DNA from head-and-neck squamous cell carcinoma (HNSCC) patients, as well as in lymphocyte DNA from control individuals who were cancer-free. The monoallelic...... methylation pattern was maintained in CTCF binding site 5 in 22 heterozygous out of 91 samples analyzed. Nevertheless, a biallelic methylation pattern was detected in CTCF binding site 6 in a subgroup of HNSCC patients as a somatic acquired feature of tumor cells. An atypical biallelic methylation was also...

  6. A single-tube allele specific-polymerase chain reaction to detect T315I resistant mutation in chronic myeloid leukemia patients

    Directory of Open Access Journals (Sweden)

    Auewarakul Chirayu U

    2011-02-01

    Full Text Available Abstract Background BCR-ABL kinase domain (KD mutation is the major mechanism contributing to suboptimal response to tyrosine kinase inhibitors (TKI in BCR-ABL-positive chronic myeloid leukemia (CML patients. T315I mutation, as one of the most frequent KD mutations, has been shown to be strongly associated with TKI resistance and subsequent therapeutic failure. A simple and sensitive method is thus required to detect T315I mutation at the earliest stage. Methods A single-tube allele specific-polymerase chain reaction (AS-PCR method was developed to detect T315I mutation in a mixture of normal and mutant alleles of varying dilutions. Denaturing high performance liquid chromatography (DHPLC and direct sequencing were performed as a comparison to AS-PCR. Results T315I mutant bands were observed in the mixtures containing as low as 0.5-1% of mutant alleles by AS-PCR. The detection sensitivity of DHPLC was around 1.5-3% dilution whereas sequencing analysis was unable to detect below 6.25% dilution. Conclusion A single-tube AS-PCR is a rapid and sensitive screening method for T315I mutation. Detection of the most resistant leukemic clone in CML patients undergoing TKI therapy should be feasible with this simple and inexpensive method.

  7. Measurement of Mycobacterium tuberculosis mRNA with duplex real-time PCR%结核分枝杆菌双重实时RT-PCR检测方法的研究

    Institute of Scientific and Technical Information of China (English)

    古莉冰; 朱玉兰; 王佃鹏; 董瑞玲; 李微; 孙杰; 刘胜牙

    2012-01-01

    目的 建立检测结核分枝杆菌mRNA表达水平的双重实时RT-PCR反应体系,准确快速鉴定结核分枝杆菌.方法 根据GenBank上已发表的结核分枝杆菌表达85B抗原的fbpB mRNA基因序列,进行对比分析,设计合成fbpB引物和探针,其中探针以CY5为报告基团,BHQ3为淬灭基团.根据WHO文献报道合成人类RNase P基因引物和探针,其中探针以FAM为报告基团,BHQ1为淬灭基团.经条件优化后,建立检测结核分枝杆菌mRNA双重实时RT-PCR方法,在同一体系内同时扩增结核分枝杆菌fbpB基因和人类RNase P基因,通过检测10份肺结核患者痰液和10份健康无症状人群痰液,检验方法的特异性、重复性和检测限性.结果 肺结核患者痰液fbpB和RNase P基因均扩增阳性,健康无症状人群痰液fbpB均无扩展曲线,RNase P均扩增阳性.重复性检测显示fbpB基因重复检测的变异系数在1%以下,fbpB检测CT值与痰涂片抗酸染色分枝杆菌的数量呈良好的相关性.结论 建立了一种快速双重实时RT-PCR方法能同时对结核分枝杆菌fbpB mRNA基因和人类RNase P基因进行检测,在检测fbpBmRNA基因的同时,通过检测RNase P基因监测RNA提取效果和PCR反应扩增效果.%Objective To establish a real-time RT-PCR method to identify Mycobacterium tuberculosis mRNA quickly and correctly. Methods According to the gene sequences of the Mycobacterium tuberculosis fbpB mRNA from the GenBank, primers and probe were designed. RNase P primers and probe were cited from the WHO recommendation. FbpB probe was labelled by CY5-BHQ3 while RNase P probe was labelled by FAM- BHQ1. The reaction condition was optimized. A duplex real-time PCR method was established to detect fbpB and RNase P genes accordingly. The sputum of 10 TB patients and 10 healthy controls were tested to identify the specificity, reproducibility and sensitivity of the method. Results 10 sputum from the subjects was tested positive for both fbpB and

  8. Efficient and allele-specific genome editing of disease loci in human iPSCs.

    Science.gov (United States)

    Smith, Cory; Abalde-Atristain, Leire; He, Chaoxia; Brodsky, Brett R; Braunstein, Evan M; Chaudhari, Pooja; Jang, Yoon-Young; Cheng, Linzhao; Ye, Zhaohui

    2015-03-01

    Efficient and precise genome editing is crucial for realizing the full research and therapeutic potential of human induced pluripotent stem cells (iPSCs). Engineered nucleases including CRISPR/Cas9 and transcription activator like effector nucleases (TALENs) provide powerful tools for enhancing gene-targeting efficiency. In this study, we investigated the relative efficiencies of CRISPR/Cas9 and TALENs in human iPSC lines for inducing both homologous donor-based precise genome editing and nonhomologous end joining (NHEJ)-mediated gene disruption. Significantly higher frequencies of NHEJ-mediated insertions/deletions were detected at several endogenous loci using CRISPR/Cas9 than using TALENs, especially at nonexpressed targets in iPSCs. In contrast, comparable efficiencies of inducing homologous donor-based genome editing were observed at disease-associated loci in iPSCs. In addition, we investigated the specificity of guide RNAs used in the CRISPR/Cas9 system in targeting disease-associated point mutations in patient-specific iPSCs. Using myeloproliferative neoplasm patient-derived iPSCs that carry an acquired JAK2-V617F point mutation and α1-antitrypsin (AAT) deficiency patient-derived iPSCs that carry an inherited Z-AAT point mutation, we demonstrate that Cas9 can specifically target either the mutant or the wild-type allele with little disruption at the other allele differing by a single nucleotide. Overall, our results demonstrate the advantages of the CRISPR/Cas9 system in allele-specific genome targeting and in NHEJ-mediated gene disruption.

  9. Establishment and primary application of duplex PCR for detection of Vibrio parahaemolytious and Vibrio alginolyticus%副溶血弧菌和溶藻弧菌双重PCR检测方法的建立与初步应用

    Institute of Scientific and Technical Information of China (English)

    刘阳; 孔繁德; 徐淑菲; 吴德峰; 林立

    2012-01-01

    In this study, a duplex PCR (dPCR) was established and optimized to detect Vibrio parahaemolytious (VP) and Vibrio alginolyticus (VA) simultaneously, two sets of specific primers were designed according to the toxR sequence of VP and VA available in GenBank. The results showed that the detection limits of the dPCR assay were 2.32 × 103 cfu/mL and 2.56 × 103 cfu/mL for VP and VA with pure bacteria culture, and 2 cfu and 3 cfu for VP and VA with clinic samples, respectively. The method possessed a better specificity and no cross-reaction with other enterobacteria such as Citro Bacter, Salmonella, V. damsela, V. cholera, V. mets-chnikovii. The method could provide a useful assay for identification and inspection of VP or VA from fishery product in import and export.%为建立同时快速检测海产品中的副溶血弧菌(VP)和溶藻孤菌(VA)的双重PCR方法,本研究根据VP和VA的toxR基因序列设计针对这两种细菌的两对特异性引物,建立能够快速同时检测这两种细菌的双重PCR方法,并对该反应体系的特异性和灵敏度进行检测.结果显示纯培养细菌VP和VA的检测灵敏度分别为2.32×103cfu/mL和2.56×103cfu/mL,临床病料检测灵敏度分别为2cfu和3cfu;与枸橼酸杆菌、沙门氏菌、美人鱼弧菌、霍乱孤菌、麦氏孤菌无交叉反应.研究表明本实验方法操作简便快速、特异性强、灵敏度高、稳定性好,并且经济实惠,值得推广应用.

  10. Duplex real-time PCR assay using SYBR Green to detect and quantify Malayan box turtle (Cuora amboinensis) materials in meatballs, burgers, frankfurters and traditional Chinese herbal jelly powder.

    Science.gov (United States)

    Asing; Ali, Eaqub; Hamid, Sharifah Bee Abd; Hossain, Motalib; Ahamad, Mohammad Nasir Uddin; Hossain, S M Azad; Naquiah, Nina; Zaidul, I S M

    2016-11-01

    The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected species widely used in exotic foods and traditional medicines. Currently available polymerase chain reaction (PCR) assays to identify MBT lack automation and involve long targets which break down in processed or denatured tissue. This SYBR Green duplex real-time PCR assay has addressed this research gap for the first time through the combination of 120- and 141-bp targets from MBT and eukaryotes for the quantitative detection of MBT DNA in food chain and herbal medicinal preparations. This authentication ensures better security through automation, internal control and short targets that were stable under the processing treatments of foods and medicines. A melting curve clearly demonstrated two peaks at 74.63 ± 0.22 and 78.40 ± 0.31°C for the MBT and eukaryotic products, respectively, under pure, admixed and commercial food matrices. Analysis of 125 reference samples reflected a target recovery of 93.25-153.00%, PCR efficiency of 99-100% and limit of detection of 0.001% under various matrices. The quantification limits were 0.00001, 0.00170 ± 0.00012, 0.00228 ± 0.00029, 0.00198 ± 0.00036 and 0.00191 ± 0.00043 ng DNA for the pure meat, binary mixtures, meatball, burger and frankfurter products, respectively. The assay was used to screen 100 commercial samples of traditional Chinese herbal jelly powder from eight different brands; 22% of them were found to be MBT-positive (5.37 ± 0.50-7.00 ± 0.34% w/w), which was reflected through the Ct values (26.37 ± 0.32-28.90 ± 0.42) and melting curves (74.63-78.65 ± 0.22°C) of the amplified MBT target (120 bp), confirming the speculation that MBT materials are widely used in Chinese herbal desserts, exotic dishes consumed with the hope of prolonging life and youth.

  11. Development of allele-specific therapeutic siRNA in Meesmann epithelial corneal dystrophy.

    Directory of Open Access Journals (Sweden)

    Haihui Liao

    Full Text Available BACKGROUND: Meesmann epithelial corneal dystrophy (MECD is an inherited eye disorder caused by dominant-negative mutations in either keratins K3 or K12, leading to mechanical fragility of the anterior corneal epithelium, the outermost covering of the eye. Typically, patients suffer from lifelong irritation of the eye and/or photophobia but rarely lose visual acuity; however, some individuals are severely affected, with corneal scarring requiring transplant surgery. At present no treatment exists which addresses the underlying pathology of corneal dystrophy. The aim of this study was to design and assess the efficacy and potency of an allele-specific siRNA approach as a future treatment for MECD. METHODS AND FINDINGS: We studied a family with a consistently severe phenotype where all affected persons were shown to carry heterozygous missense mutation Leu132Pro in the KRT12 gene. Using a cell-culture assay of keratin filament formation, mutation Leu132Pro was shown to be significantly more disruptive than the most common mutation, Arg135Thr, which is associated with typical, mild MECD. A siRNA sequence walk identified a number of potent inhibitors for the mutant allele, which had no appreciable effect on wild-type K12. The most specific and potent inhibitors were shown to completely block mutant K12 protein expression with negligible effect on wild-type K12 or other closely related keratins. Cells transfected with wild-type K12-EGFP construct show a predominantly normal keratin filament formation with only 5% aggregate formation, while transfection with mutant K12-EGFP construct resulted in a significantly higher percentage of keratin aggregates (41.75%; p<0.001 with 95% confidence limits. The lead siRNA inhibitor significantly rescued the ability to form keratin filaments (74.75% of the cells contained normal keratin filaments; p<0.001 with 95% confidence limits. CONCLUSIONS: This study demonstrates that it is feasible to design highly potent si

  12. 二重实时PCR快速检测耐甲氧西林金黄色葡萄球菌%Rapid detection of methicillin-resistant Staphylococcus aureus with duplex real-time PCR assay

    Institute of Scientific and Technical Information of China (English)

    王艳; 叶冬青; 李家斌; 张胜权

    2007-01-01

    目的 建立二重实时聚合酶链反应(duplex real-time PCR) 快速检测耐甲氧西林金黄色葡萄球菌(MRSA).方法 采用二重SYBR Green实时PCR快速检测MRSA的决定基因mecA和金葡菌的种特异性基因nuc,经熔解曲线分析鉴定产物.结果 所有MRSA菌株的熔解曲线均呈现mecA、nuc基因特异性的峰,甲氧西林敏感的金葡菌仅有nuc峰,耐甲氧西林的表葡菌仅有mecA峰,甲氧西林敏感的表葡菌与其他菌种的菌株无特异峰出现;当MRSA菌浓度达102cfu/ml时就可检出.在131株金葡菌中,30.53%(40/131)耐药;二重实时PCR扩增mecA基因29.01%(38/131)阳性,nuc基因100%阳性.单引物与二重实时PCR两者阳性扩增符合率为100%.结论 对mecA、nuc基因进行二重实时PCR能准确、快速地鉴定耐甲氧西林的金葡菌和凝固酶阴性的葡萄球菌.

  13. Allele-specific oligonucleotide polymerase chain reaction for the determination of Rh C/c and Rh E/e antigens in thalassaemic patients

    Science.gov (United States)

    Hojjati, Mohammad Taher; Einollahi, Nahid; Nabatchian, Fariba; Pourfathollah, Ali Akbar; Mahdavi, Mohammad Reza

    2011-01-01

    Background Thalassaemia is a genetic disease in which there is a relative or complete lack of alpha or beta globin chains. Patients with moderate to severe forms of thalassaemia need transfusions from the early years of life. Antibody production against blood group antigens may cause many problems in preparing compatible blood units for transfusion. The identification of definite blood group phenotypes by the haemagglutination method can be difficult because of the mixed population of red blood cells from the donor and recipient. Materials and methods Forty multiply transfused thalassaemic patients and ten healthy controls with no history of blood transfusion were enrolled in this study. Allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) and haemagglutination methods were used to determine the presence of Rhesus (Rh) C, c, E and e antigens. Results In this study four primer sets were used for ASO-PCR amplification of RhC/c and RhE/e. Although PCR assays for RhC/c and RHE/e genotyping have been described previously, in this study we used a new condition for PCR by decreasing the annealing temperature from 63 °C to 58 °C in order to amplify all four genes in the same condition. In order to evaluate this single run molecular method, we used the haemagglutination test as the standard method and compared the results from the two methods. We found discrepancies between phenotype and genotype results among patients with beta thalassaemia, but complete agreement between phenotype and genotype in the control group. Conclusions The advantage of this new ASO-PCR method compared to a restriction fragment length polymorphism (RFLP) PCR method is that with the former all four genes can be amplified at the same time by PCR, and electrophoresis can be performed immediately to determine individual antigen profiles. The simplicity of the ASO-PCR method makes it suitable for routine use in medical centres and it is also cheaper than RFLP-PCR. Furthermore, as shown

  14. Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia.

    Science.gov (United States)

    Wu, D Y; Ugozzoli, L; Pal, B K; Wallace, R B

    1989-04-01

    A rapid nonradioactive approach to the diagnosis of sickle cell anemia is described based on an allele-specific polymerase chain reaction (ASPCR). This method allows direct detection of the normal or the sickle cell beta-globin allele in genomic DNA without additional steps of probe hybridization, ligation, or restriction enzyme cleavage. Two allele-specific oligonucleotide primers, one specific for the sickle cell allele and one specific for the normal allele, together with another primer complementary to both alleles were used in the polymerase chain reaction with genomic DNA templates. The allele-specific primers differed from each other in their terminal 3' nucleotide. Under the proper annealing temperature and polymerase chain reaction conditions, these primers only directed amplification on their complementary allele. In a single blind study of DNA samples from 12 individuals, this method correctly and unambiguously allowed for the determination of the genotypes with no false negatives or positives. If ASPCR is able to discriminate all allelic variation (both transition and transversion mutations), this method has the potential to be a powerful approach for genetic disease diagnosis, carrier screening, HLA typing, human gene mapping, forensics, and paternity testing.

  15. Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi.

    Science.gov (United States)

    Kaulich, Manuel; Lee, Yeon J; Lönn, Peter; Springer, Aaron D; Meade, Bryan R; Dowdy, Steven F

    2015-04-20

    Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms.

  16. Detection and Differentiation of Vaccine and Wild-type Strains of PPRV by Duplex Real-time RT-PCR%实时荧光RT-PCR鉴别小反刍兽疫病毒疫苗株和野毒株

    Institute of Scientific and Technical Information of China (English)

    李林; 吴晓东; 刘春菊; 王清华; 包静月; 王志亮

    2016-01-01

    To differentiate vaccine virus and wild-type peste des petits ruminants virus(PPRV)by duplex real-time RT-PCR,one pair of primers and two TaqMan probes were designed based on the comparison of PPRV genome sequences available in GenBank. The duplex real-time RT-PCR results showed that the detection limits for vaccine strain and wild-type strains were both 10 copies per reaction. The developed assay also showed high ampliifcation efifciency,linearity and speciifcity in discriminating the two PPRV types. A total of 136 clinical samples tested by the conventional RT-PCR were run again by the duplex real-time RT-PCR. The results were conifrmed subsequently by sequencing the positive conventional RT-PCR products. This reliable and rapid duplex real-time RT-PCR assay would be feasible and attractive for diagnostic laboratories,both for clinical purpose in active surveillance and for epidemiological investigations.%为快速鉴别小反刍兽疫病毒的疫苗株和野毒株,本研究通过分析GenBank中已发布的小反刍兽疫病毒全基因组序列,设计了1对通用引物以及疫苗株和野毒株特异性探针各1条,建立了小反刍兽疫病毒鉴别实时荧光RT-PCR方法。结果显示,该方法对疫苗株和野毒株的检测灵敏度均可以检测至10拷贝/反应,在108至101拷贝/反应之间具有良好的线性关系,扩增效率均接近1。该方法具有良好的特异性,能够区分不同谱系的野毒株株和疫苗株。使用该方法对136份临床疑似样品检测,检出的阳性数量与普通RT-PCR相同,并且测序后的区分结果也相同。本方法的建立为该病的实验室鉴别检测和流行病学调查提供了技术支持。

  17. PrimerSNP: a web tool for whole-genome selection of allele-specific and common primers of phylogenetically-related bacterial genomic sequences

    Directory of Open Access Journals (Sweden)

    Lemos Eliana

    2008-10-01

    Full Text Available Abstract Background The increasing number of genomic sequences of bacteria makes it possible to select unique SNPs of a particular strain/species at the whole genome level and thus design specific primers based on the SNPs. The high similarity of genomic sequences among phylogenetically-related bacteria requires the identification of the few loci in the genome that can serve as unique markers for strain differentiation. PrimerSNP attempts to identify reliable strain-specific markers, on which specific primers are designed for pathogen detection purpose. Results PrimerSNP is an online tool to design primers based on strain specific SNPs for multiple strains/species of microorganisms at the whole genome level. The allele-specific primers could distinguish query sequences of one strain from other homologous sequences by standard PCR reaction. Additionally, PrimerSNP provides a feature for designing common primers that can amplify all the homologous sequences of multiple strains/species of microorganisms. PrimerSNP is freely available at http://cropdisease.ars.usda.gov/~primer. Conclusion PrimerSNP is a high-throughput specific primer generation tool for the differentiation of phylogenetically-related strains/species. Experimental validation showed that this software had a successful prediction rate of 80.4 – 100% for strain specific primer design.

  18. Detection of Salmonella spp. and Shigella spp. in fresh pork using duplex real-time PCR based on immunemagnetic separation%基于免疫磁分离-双重荧光定量PCR的鲜猪肉中沙门氏菌和志贺氏菌的检测

    Institute of Scientific and Technical Information of China (English)

    马凯; 高丽娟; 武会娟; 杜美红; 白羽; 陈尔凝; 刘杰; 刘悦

    2015-01-01

    ABSTRACT:Objective To develop a rapid and simultaneous detection for Salmonella spp. and Shigella spp. from fresh pork samples by combining immunomagnetic separation (IMS) with duplex real-time PCR (RT-PCR). Methods Magnetic beads coated with specific antibodies were used to capture the pathogens from a 250 mL cycling system at 37℃condition. Then, duplex RT-PCR was applied to detect Salmonella spp. and Shigella spp. with 2 sets of specific primers and probes. Results The limit of detections were 3.4 cfu/g for Salmonella spp., and 9.4 cfu/g for Shigella spp.. The sensitivity, specificity, and accuracy of IMS-multiplex RT-PCR method were all 100%. Moreover, the results of new method were consistent to those of traditional method by detecting 30 samples. Conclusion IMS-duplex RT-PCR method is more rapid and effective than GB method, and which is suitable for the rapid detection of Salmonella spp. and Shigella spp. in fresh pork.%目的:使用免疫磁分离-双重荧光定量PCR方法实现对鲜猪肉中金黄色葡萄球菌和志贺氏菌的同时、快速检测。方法在37℃的条件下,利用特异性免疫磁球从250 mL循环体系中快速、有效地捕获目标菌。再通过特异性的引物与探针,对沙门氏菌和志贺氏菌进行双重荧光定量 PCR 检测。结果本研究方法针对鲜猪肉中沙门氏菌和志贺氏菌的检测限分别达到3.4 cfu/g和9.4 cfu/g。方法总体灵敏度、特异性和准确度均达到100%。此外,通过对30组实际样品的检测,该方法与传统标准方法的结果保持一致。结论本研究建立的免疫磁分离-双重荧光定量PCR方法比国标方法更快速和高效,适用于鲜猪肉中沙门氏菌和志贺氏菌的快速检测。

  19. Allele-specific up-regulation of FGFR2 increases susceptibility to breast cancer.

    Directory of Open Access Journals (Sweden)

    Kerstin B Meyer

    2008-05-01

    Full Text Available The recent whole-genome scan for breast cancer has revealed the FGFR2 (fibroblast growth factor receptor 2 gene as a locus associated with a small, but highly significant, increase in the risk of developing breast cancer. Using fine-scale genetic mapping of the region, it has been possible to narrow the causative locus to a haplotype of eight strongly linked single nucleotide polymorphisms (SNPs spanning a region of 7.5 kilobases (kb in the second intron of the FGFR2 gene. Here we describe a functional analysis to define the causative SNP, and we propose a model for a disease mechanism. Using gene expression microarray data, we observed a trend of increased FGFR2 expression in the rare homozygotes. This trend was confirmed using real-time (RT PCR, with the difference between the rare and the common homozygotes yielding a Wilcox p-value of 0.028. To elucidate which SNPs might be responsible for this difference, we examined protein-DNA interactions for the eight most strongly disease-associated SNPs in different breast cell lines. We identify two cis-regulatory SNPs that alter binding affinity for transcription factors Oct-1/Runx2 and C/EBPbeta, and we demonstrate that both sites are occupied in vivo. In transient transfection experiments, the two SNPs can synergize giving rise to increased FGFR2 expression. We propose a model in which the Oct-1/Runx2 and C/EBPbeta binding sites in the disease-associated allele are able to lead to an increase in FGFR2 gene expression, thereby increasing the propensity for tumour formation.

  20. Use of allele-specific FAIRE to determine functional regulatory polymorphism using large-scale genotyping arrays

    DEFF Research Database (Denmark)

    Smith, Frank Andrew; Howard, Philip; Shah, Sonia;

    2012-01-01

    identified an allele-specific regulatory polymorphism within NR1H3 (coding for LXR-α), rs7120118, coinciding with a previously GWAS-identified SNP for HDL-C levels. This finding was confirmed using FAIRE-gen with the 200,000 SNP Illumina Metabochip and verified with the established method of TaqMan allelic...... variants, we describe the technique of allele-specific FAIRE, utilising large-scale genotyping technology (FAIRE-gen) to determine allelic effects on chromatin accessibility and regulatory potential. FAIRE-gen was explored using lymphoblastoid cells and the 50,000 SNP Illumina CVD BeadChip. The technique...... discrimination. Examination of this SNP in two prospective Caucasian cohorts comprising 15,000 individuals confirmed the association with HDL-C levels (combined beta = 0.016; p = 0.0006), and analysis of gene expression identified an allelic association with LXR-α expression in heart tissue. Using increasingly...

  1. Detection of HIV proviral DNA by a duplex fluorescence PCR for early diagnosis of HIV infection in infants%双重荧光PCR检测HIV前病毒DNA及其在婴幼儿HIV感染早期诊断中的应用

    Institute of Scientific and Technical Information of China (English)

    张佳峰; 郭志宏; 黄晶晶; 丁晓贝; 黄蓓

    2013-01-01

    Objective To establish a duplex fluorescence PCR for detection of HIV proviral DNA and to evaluate its application for early diagnosis of HIV infection in infants .Methods A duplex fluores-cence PCR system was set up based on TaqMan technology for detection of human ribonuclease P ( RNase P) gene and long terminal repeat ( LTR) region of HIV.A recombinant plasmid containing the targeted gene fragment , pTG19-T, was constructed by TA cloning technique and used as the template for evaluation of sen -sitivity of the assay .Blood samples from 11 healthy individuals and 98 HIV-infected patients were collected and detected to validate the assay specificity .The assay of duplex fluorescence PCR was then carried out to detect 96 infant blood samples collected from several maternal and child health hospitals in Zhejiang province from January 2011 to September 2012 for early diagnosis of HIV infection .The results were compared with those by using the Roche HIV DNA qualitative detection kit .Results The established duplex fluorescence PCR could specifically detect HIV proviral DNA with a specificity of 100%and a detection sensitivity of 100 cps per reaction .The coincidence rate between the established assay and the Roche HIV DNA qualitative de -tection kit was 100%in the detection of 96 blood samples .Conclusion The duplex fluorescence PCR as-say showed advantages of cost-effectiveness , convenience , good specificity and accuracy with high sensitivi-ty.It could be used for early diagnosis of HIV infection in infants and also as a general technical platform for the detection of HIV proviral DNA .%  目的建立双重荧光PCR检测HIV前病毒DNA的方法,并应用于婴幼儿HIV感染的早期诊断。方法采用TaqMan技术,组建针对人类核糖核酸酶P( RNase P)和HIV的长末端重复序列( LTR)基因的双重荧光PCR体系;采用TA克隆技术构建pTG19-T重组质粒作为模板进行该方法灵敏度的评价;采用11

  2. DEVELOPMENT OF DUPLEX TAQMAN REAL-TIME PCR TO DETECT CTX AND TDH%霍乱毒素基因(ctx)和耐热直接溶血素基因(tdh)双重TaqMan实时PCR检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    韩辉; 李海山; 胡群; 姚李四; 谭绪良; 贾琳

    2011-01-01

    [目的]建立霍乱毒素和耐热直接溶血素基因双重TaqMan实时-PCR实验室检测方法.[方法]根据霍乱毒素基因(Cholera toxin gene,ctx)和耐热直接溶血素基因(thermostable direct hemolysin,tdh)的保守序列设计引物和TaqMan探针,建立检测霍乱毒素和耐热直接溶血素两种毒力基因的双重TaqMan实时PCR方法.对所建立的霍乱毒素基因和耐热直接溶血素基因双重TaqMan实时PCR检测方法进行灵敏度和特异度评价.[结果]建立了霍乱毒素基因和酎热直接溶血素基因双重TaqMan实时PCR的实验室检测方法.优化的tdh和ct双重TaqMan实时PCR反应体系中,ct引物和探针的浓度分别为200 nmol/L和100 nmol/L;tdh引物和探针的浓度分别为200 nmoL/L和100 nmol/L.反应体系的灵敏度和特异度均为100%.优化的反应体系对两种质粒模板的检测下限均为1.0x10(2)拷贝/μl,扩增效率分别为94%和97.7%.[结论]本研究建立了基于TaqMan探针的tdh和ct双重实时PCR检测方法,具有令人满意的灵敏度和特异度.检测下限能达到1.0x10(2)拷贝/μl,高于普通PCR 100倍.并且双重实时PCR能够在一个反应体系中同时检测两种毒力基因,这为费时又繁琐的传统检测方法提供了一种可靠又快速的替代选择.%[Objective] To develop a duplex TaqMan real-time PCR for the detection of ctx and tdh. [Methods] The conserved region of TDH and CTX gene were used to design primers and probes, and the duplex TaqMan real-time PCR system of detecting ctx and tdh was established. The sensitivity and specificity of duplex TaqMan real-time PCR system was evaluated. [Results] The duplex TaqMan real-time PCR system detecting ctx and tdh-was established. In the optimized reaction system of ctx and tdh duplex TaqMan real-time PCR, the concentrations of ctx primers and probe were 200 nmol/L and 100 nmol/L, respectively; the concentrations of tdh primers and probe were 200 nmol/L and 100 nmol/L, respectively. The

  3. An allele-specific polymerase chain reaction assay for the differentiation of members of the Anopheles culicifacies complex

    Indian Academy of Sciences (India)

    O P Singh; Geeta Goswami; N Nanda; K Raghavendra; D Chandra; S K Subbarao

    2004-09-01

    Anopheles culicifacies, the principal vector of malaria in India, is a complex of five cryptic species which are morphologically indistinguishable at any stage of life. In view of the practical difficulties associated with classical cytotaxonomic method for the identification of members of the complex, an allele-specific polymerase chain reaction (ASPCR) assay targeted to the D3 domain of 28S ribosomal DNA was developed. The assay discriminates An. culicifacies species A and D from species B, C and E. The assay was validated using chromosomally-identified specimens of An. culicifacies from different geographical regions of India representing different sympatric associations. The assay correctly differentiates species A and D from species B, C and E. The possible use of this diagnostic assay in disease vector control programmes is discussed.

  4. A Novel Real-time Fluorescence Mutant-allele-specific Amplification Method for Rapid Single Nucleotide Polymorphism Analysis

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Current methods for single nucleotide polymorphism (SNP) analysis are timeconsuming and complicated. We aimed at development of one-step real-time fluorescence mutant-allele-specific amplification (MASA) method for rapid SNP analysis. The method is a marriage of two technologies: MASA primers for target DNA and a double-stranded DNA-selective fluorescent dye, SYBR Green I. Genotypes are separated according to the different threshold cycles of the wild-type and mutant primers. K-ras oncogene was used as a target to validate the feasibility of the method. The experimental results showed that the different genotypes can be clearly discriminated by the assay. The real-time fluorescence MASA method will have an enormous potential for fast and reliable SNP analysis due to its simplicity and low cost.

  5. Powerful identification of cis-regulatory SNPs in human primary monocytes using allele-specific gene expression.

    Directory of Open Access Journals (Sweden)

    Jonas Carlsson Almlöf

    Full Text Available A large number of genome-wide association studies have been performed during the past five years to identify associations between SNPs and human complex diseases and traits. The assignment of a functional role for the identified disease-associated SNP is not straight-forward. Genome-wide expression quantitative trait locus (eQTL analysis is frequently used as the initial step to define a function while allele-specific gene expression (ASE analysis has not yet gained a wide-spread use in disease mapping studies. We compared the power to identify cis-acting regulatory SNPs (cis-rSNPs by genome-wide allele-specific gene expression (ASE analysis with that of traditional expression quantitative trait locus (eQTL mapping. Our study included 395 healthy blood donors for whom global gene expression profiles in circulating monocytes were determined by Illumina BeadArrays. ASE was assessed in a subset of these monocytes from 188 donors by quantitative genotyping of mRNA using a genome-wide panel of SNP markers. The performance of the two methods for detecting cis-rSNPs was evaluated by comparing associations between SNP genotypes and gene expression levels in sample sets of varying size. We found that up to 8-fold more samples are required for eQTL mapping to reach the same statistical power as that obtained by ASE analysis for the same rSNPs. The performance of ASE is insensitive to SNPs with low minor allele frequencies and detects a larger number of significantly associated rSNPs using the same sample size as eQTL mapping. An unequivocal conclusion from our comparison is that ASE analysis is more sensitive for detecting cis-rSNPs than standard eQTL mapping. Our study shows the potential of ASE mapping in tissue samples and primary cells which are difficult to obtain in large numbers.

  6. The genetic association of RUNX3 with ankylosing spondylitis can be explained by allele-specific effects on IRF4 recruitment that alter gene expression.

    Science.gov (United States)

    Vecellio, Matteo; Roberts, Amity R; Cohen, Carla J; Cortes, Adrian; Knight, Julian C; Bowness, Paul; Wordsworth, B Paul

    2016-08-01

    To identify the functional basis for the genetic association of single nucleotide polymorphisms (SNP), upstream of the RUNX3 promoter, with ankylosing spondylitis (AS). We performed conditional analysis of genetic association data and used ENCODE data on chromatin remodelling and transcription factor (TF) binding sites to identify the primary AS-associated regulatory SNP in the RUNX3 region. The functional effects of this SNP were tested in luciferase reporter assays. Its effects on TF binding were investigated by electrophoretic mobility gel shift assays and chromatin immunoprecipitation. RUNX3 mRNA levels were compared in primary CD8+ T cells of AS risk and protective genotypes by real-time PCR. The association of the RUNX3 SNP rs4648889 with AS (p<7.6×10(-14)) was robust to conditioning on all other SNPs in this region. We identified a 2 kb putative regulatory element, upstream of RUNX3, containing rs4648889. In reporter gene constructs, the protective rs4648889 'G' allele increased luciferase activity ninefold but significantly less activity (4.3-fold) was seen with the AS risk 'A' allele (p≤0.01). The binding of Jurkat or CD8+ T-cell nuclear extracts to the risk allele was decreased and IRF4 recruitment was reduced. The AS-risk allele also affected H3K4Me1 histone methylation and associated with an allele-specific reduction in RUNX3 mRNA (p<0.05). We identified a regulatory region upstream of RUNX3 that is modulated by rs4648889. The risk allele decreases TF binding (including IRF4) and reduces reporter activity and RUNX3 expression. These findings may have important implications for understanding the role of T cells and other immune cells in AS. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  7. Overall and allele-specific expression of the SMC1A gene in female Cornelia de Lange syndrome patients and healthy controls.

    Science.gov (United States)

    Parenti, Ilaria; Rovina, Davide; Masciadri, Maura; Cereda, Anna; Azzollini, Jacopo; Picinelli, Chiara; Limongelli, Giuseppe; Finelli, Palma; Selicorni, Angelo; Russo, Silvia; Gervasini, Cristina; Larizza, Lidia

    2014-07-01

    Cornelia de Lange syndrome (CdLS) is a rare multisystem disorder characterized by facial dysmorphisms, limb anomalies, and growth and cognitive deficits. Mutations in genes encoding subunits (SMC1A, SMC3, RAD21) or regulators (NIPBL, HDAC8) of the cohesin complex account for approximately 65% of clinically diagnosed CdLS cases. The SMC1A gene (Xp11.22), responsible for 5% of CdLS cases, partially escapes X chromosome inactivation in humans and the allele on the inactive X chromosome is variably expressed. In this study, we evaluated overall and allele-specific SMC1A expression. Real-time PCR analysis conducted on 17 controls showed that SMC1A expression in females is 50% higher than in males. Immunoblotting experiments confirmed a 44% higher protein level in healthy females than in males, and showed no significant differences in SMC1A protein levels between controls and patients. Pyrosequencing was used to assess the reciprocal level of allelic expression in six female carriers of different SMC1A mutations and 15 controls who were heterozygous at a polymorphic transcribed SMC1A locus. The two alleles were expressed at a 1:1 ratio in the control group and at a 2:1 ratio in favor of the wild type allele in the test group. Since a dominant negative effect is considered the pathogenic mechanism in SMC1A-defective female patients, the level of allelic preferential expression might be one of the factors contributing to the wide phenotypic variability observed in these patients. An extension of this study to a larger cohort containing mild to borderline cases could enhance our understanding of the clinical spectrum of SMC1A-linked CdLS.

  8. Allelic diversity of a beer haze active protein gene in cultivated and Tibetan wild barley and development of allelic specific markers.

    Science.gov (United States)

    Ye, Lingzhen; Dai, Fei; Qiu, Long; Sun, Dongfa; Zhang, Guoping

    2011-07-13

    The formation of haze is a serious quality problem in beer production. It has been shown that the use of silica elute (SE)-ve malt (absence of molecular weight (MW) ∼14000 Da) for brewing can improve haze stability in the resultant beer, and the protein was identified as a barley trypsin inhibitor of the chloroform/methanol type (BTI-CMe). The objectives of this study were to determine (1) the allelic diversity of the gene controlling BTI-CMe in cultivated and Tibetan wild barley and (2) allele-specific (AS) markers for screening SE protein type. A survey of 172 Tibetan annual wild barley accessions and 71 cultivated barley genotypes was conducted, and 104 wild accessions and 35 cultivated genotypes were identified as SE+ve and 68 wild accessions and 36 cultivated genotypes as SE-ve. The allelic diversity of the gene controlling BTI-CMe was investigated by cloning, alignment, and association analysis. It was found that there were significant differences between the SE+ve and SE-ve types in single-nucleotide polymorphisms at 234 (SNP(234)), SNP(313), and SNP(385.) Furthermore, two sets of AS markers were developed to screen SE protein type based on SNP(313). AS-PCR had results very similar to those obtained by immunoblot method. Mapping analysis showed that the gene controlling the MW∼14 kDa band was located on the short arm of chromosome 3H, at the position of marker BPB-0527 (33.302 cM) in the Franklin/Yerong DH population.

  9. Establishment and application of duplex real-time PCR quantitative determination method on latent infection of wheat stripe rust%双重real-time PCR定量测定小麦条锈菌潜伏侵染方法的建立与应用

    Institute of Scientific and Technical Information of China (English)

    潘阳; 谷医林; 骆勇; 马占鸿

    2016-01-01

    小麦条锈病是我国小麦生产上的重要病害之一.为高效准确地定量检测处于潜伏侵染阶段的小麦条锈菌,本研究根据已发表的小麦条锈菌和寄主小麦的引物,设计了各自的探针,建立了双重real-time PCR检测方法.为排除多个引物互作造成的干扰,对小麦条锈菌引物探针体系、小麦体系以及二者的双重real-time PCR体系进行了比较.CT值相关线性回归分析证明,引物之间互作很小,对定量检测无影响.已知浓度样品经梯度稀释,进行灵敏度检测,确定了双重real-time PCR对小麦条锈菌DNA和小麦DNA准确定量测定的最小检测限为0.4 pg和0.5 ng.同时建立了小麦条锈菌和小麦各自的标准曲线.用此方法检测来自两个不同地区的田间样本,得到的分子病情指数(MDI)与随后的发病趋势一致.本研究建立的双重real-time PCR分析方法可靠、高效、低误差,是对本实验室已有小麦条锈菌潜伏期分子检测方法的进一步优化.%Wheat stripe rust,caused by Puccinia striiformis f.sp.tritici (PST),is one of the most important wheat diseases in China.Based on two pairs of published primers (from PST and wheat respectively),we designed specific probes respectively and developed a duplex real-time PCR approach for the quantification of PST in latent period.In order to determine if the primers would interact in the duplex PCR system,and affect the system subsequently,we designed two real-time PCR reaction systems with a single pair of the primers and one probe from PST and wheat respectively for comparison.The linear regression analysis of CT values showed that duplex real-time PCR was hardly affected by the interaction of the primers.Serial ten-fold dilutions of samples to the determined concentration were performed for sensitivity test;the minimum genomic DNA concentrations of PST and wheat that could be accurately quantified were 0.4 pg and 0.5 ng respectively.In the mean time,two standard

  10. 单核细胞增生李斯特菌与志贺菌双重实时PCR检测技术的建立%The development of duplex real-time PCR for detection of Listeria monocytogenes and Shigella

    Institute of Scientific and Technical Information of China (English)

    徐伟; 李素芳; 刘军; 胡巅

    2008-01-01

    Objective To develop a rapid,sensitive,specific and accurate quantitative duplex real-time PCR assay for detection of Listeria monocytogenes and Shigella.Methods Two sets of specific primers and probes were selected according to Listeria monocytogencs hly gene and Shigella ipaH gene.The target hly and iPaH fragments were amplified by PCR,and used to construct recombinant pGEM-T-hly and pGEM-T-ipaH respectively.The two recombinant circular plasmid DNAs were linearized with EcoR I that did not cut within the target DNA fragment.The ten-fold dilutions of plasmid were subjected to the standard quantitation curve in duplex real-time PCR assay.Various genomic DNAs of Listeria innocua,Listeria weshimeri,Salmonella,Staphylococcus aureus,Bacillus subtilis,Escherichia coli and Proteus were used as negative controls to confirm the specificity of duplex real-time PCR assay.The assay was also used to detect Listeria monocytogenes and Shigella in artificially contaminated sterilized skim milk.Results The recombinant plasmids were constructed successfully,hly probe(rAM and TAMRA double labelled)and ipaH probe (HEX and TAMRA double labelled)were used to develop an optimized PCR successfuliv.Conclusion The selected primers and probes showed high specificity for these two target bacteria,the linear range of the assay was good(105-101 copies/μl,R2≥0.998)and sensitivity Was 10 copies/PCR.Following a DNA extraction method which combined EZ Spin Colum Genomic DNA Isolation Kit(BBI)/Phenol-chloroform,the sensitivity of assay Was 102CFU/ml for both Listeria monocytogenes and Shigella in artificially contaminated sterilized skim milk,which equivalents to 10 CFU/PCR.%目的 建立一种快速、特异、灵敏、准确定量的单核细胞增生(单增)李斯特菌(Listeriamonocytogenes)与志贺菌(Shigella)同步检测方法.方法 分别根据单增李斯特菌溶血素O基因hly与志贺菌侵袭性质粒抗原H基因ipaH设计合成引物和探针.构建重组质粒pGEM-T-hly

  11. High-throughput analysis of candidate imprinted genes and allele-specific gene expression in the human term placenta

    Directory of Open Access Journals (Sweden)

    Clark Taane G

    2010-04-01

    Full Text Available Abstract Background Imprinted genes show expression from one parental allele only and are important for development and behaviour. This extreme mode of allelic imbalance has been described for approximately 56 human genes. Imprinting status is often disrupted in cancer and dysmorphic syndromes. More subtle variation of gene expression, that is not parent-of-origin specific, termed 'allele-specific gene expression' (ASE is more common and may give rise to milder phenotypic differences. Using two allele-specific high-throughput technologies alongside bioinformatics predictions, normal term human placenta was screened to find new imprinted genes and to ascertain the extent of ASE in this tissue. Results Twenty-three family trios of placental cDNA, placental genomic DNA (gDNA and gDNA from both parents were tested for 130 candidate genes with the Sequenom MassArray system. Six genes were found differentially expressed but none imprinted. The Illumina ASE BeadArray platform was then used to test 1536 SNPs in 932 genes. The array was enriched for the human orthologues of 124 mouse candidate genes from bioinformatics predictions and 10 human candidate imprinted genes from EST database mining. After quality control pruning, a total of 261 informative SNPs (214 genes remained for analysis. Imprinting with maternal expression was demonstrated for the lymphocyte imprinted gene ZNF331 in human placenta. Two potential differentially methylated regions (DMRs were found in the vicinity of ZNF331. None of the bioinformatically predicted candidates tested showed imprinting except for a skewed allelic expression in a parent-specific manner observed for PHACTR2, a neighbour of the imprinted PLAGL1 gene. ASE was detected for two or more individuals in 39 candidate genes (18%. Conclusions Both Sequenom and Illumina assays were sensitive enough to study imprinting and strong allelic bias. Previous bioinformatics approaches were not predictive of new imprinted genes

  12. Allele-specific Gene Silencing of Mutant mRNA Restores Cellular Function in Ullrich Congenital Muscular Dystrophy Fibroblasts

    Directory of Open Access Journals (Sweden)

    Satoru Noguchi

    2014-01-01

    Full Text Available Ullrich congenital muscular dystrophy (UCMD is an inherited muscle disorder characterized clinically by muscle weakness, distal joint hyperlaxity, and proximal joint contractures. Sporadic and recessive mutations in the three collagen VI genes, COL6A1, COL6A2, and COL6A3, are reported to be causative. In the sporadic forms, a heterozygous point mutation causing glycine substitution in the triple helical domain has been identified in higher rate. In this study, we examined the efficacy of siRNAs, which target point mutation site, on specific knockdown toward transcripts from mutant allele and evaluated consequent cellular phenotype of UCMD fibroblasts. We evaluated the effect of siRNAs targeted to silence-specific COL6A1 alleles in UCMD fibroblasts, where simultaneous expression of both wild-type and mutant collagen VI resulted in defective collagen localization. Addition of mutant-specific siRNAs allowed normal extracellular localization of collagen VI surrounding fibroblasts, suggesting selective inhibition of mutant collagen VI. Targeting the single-nucleotide COL6A1 c.850G>A (p.G284R mutation responsible a sporadic autosomal dominant form of UCMD can potently and selectively block expression of mutant collagen VI. These results suggest that allele-specific knockdown of the mutant mRNA can potentially be considered as a therapeutic procedure in UCMD due to COL6A1 point mutations.

  13. Ribosomal protein genes are highly enriched among genes with allele-specific expression in the interspecific F1 hybrid catfish.

    Science.gov (United States)

    Chen, Ailu; Wang, Ruijia; Liu, Shikai; Peatman, Eric; Sun, Luyang; Bao, Lisui; Jiang, Chen; Li, Chao; Li, Yun; Zeng, Qifan; Liu, Zhanjiang

    2016-06-01

    Interspecific hybrids provide a rich source for the analysis of allele-specific expression (ASE). In this work, we analyzed ASE in F1 hybrid catfish using RNA-Seq datasets. While the vast majority of genes were expressed with both alleles, 7-8 % SNPs exhibited significant differences in allele ratios of expression. Of the 66,251 and 177,841 SNPs identified from the datasets of the liver and gill, 5420 (8.2 %) and 13,390 (7.5 %) SNPs were identified as significant ASE-SNPs, respectively. With these SNPs, a total of 1519 and 3075 ASE-genes were identified. Gene Ontology analysis revealed that genes encoding cytoplasmic ribosomal proteins (RP) were highly enriched among ASE genes. Parent-of-origin was determined for 27 and 30 ASE RP genes in the liver and gill, respectively. The results indicated that genes from both channel catfish and blue catfish were involved in ASE. However, each RP gene appeared to be almost exclusively expressed from only one parent, indicating that ribosomes in the hybrid catfish were in the "hybrid" form. Overall representation of RP transcripts among the transcriptome appeared lower in the F1 hybrid catfish than in channel catfish or blue catfish, suggesting that the "hybrid" ribosomes may work more efficiently for translation in the F1 hybrid catfish.

  14. Visualizing allele-specific expression in single cells reveals epigenetic mosaicism in an H19 loss-of-imprinting mutant.

    Science.gov (United States)

    Ginart, Paul; Kalish, Jennifer M; Jiang, Connie L; Yu, Alice C; Bartolomei, Marisa S; Raj, Arjun

    2016-03-01

    Imprinting is a classic mammalian epigenetic phenomenon that results in expression from a single parental allele. Imprinting defects can lead to inappropriate expression from the normally silenced allele, but it remains unclear whether every cell in a mutant organism follows the population average, which would have profound implications for human imprinting disorders. Here, we apply a new fluorescence in situ hybridization method that measures allele-specific expression in single cells to address this question in mutants exhibiting aberrant H19/Igf2 (insulin-like growth factor 2) imprinting. We show that mutant primary embryonic mouse fibroblasts are comprised of two subpopulations: one expressing both H19 alleles and another expressing only the maternal copy. Only in the latter cell population is Igf2 expression detected. Furthermore, the two subpopulations are stable in that cells do not interconvert between the two expression patterns. Combined small input methylation analysis and transcriptional imaging revealed that these two mutant subpopulations exhibit distinct methylation patterns at their imprinting control regions. Consistently, pharmacological inhibition of DNA methylation reduced the proportion of monoallelic cells. Importantly, we observed that the same two subpopulations are also present in vivo within murine cardiac tissue. Our results establish that imprinting disorders can display striking single-cell heterogeneity in their molecular phenotypes and suggest that such heterogeneity may underlie epigenetic mosaicism in human imprinting disorders.

  15. Allele-specific suppression of mutant huntingtin using antisense oligonucleotides: providing a therapeutic option for all Huntington disease patients.

    Directory of Open Access Journals (Sweden)

    Niels H Skotte

    Full Text Available Huntington disease (HD is an inherited, fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The mutant protein causes neuronal dysfunction and degeneration resulting in motor dysfunction, cognitive decline, and psychiatric disturbances. Currently, there is no disease altering treatment, and symptomatic therapy has limited benefit. The pathogenesis of HD is complicated and multiple pathways are compromised. Addressing the problem at its genetic root by suppressing mutant huntingtin expression is a promising therapeutic strategy for HD. We have developed and evaluated antisense oligonucleotides (ASOs targeting single nucleotide polymorphisms that are significantly enriched on HD alleles (HD-SNPs. We describe our structure-activity relationship studies for ASO design and find that adjusting the SNP position within the gap, chemical modifications of the wings, and shortening the unmodified gap are critical for potent, specific, and well tolerated silencing of mutant huntingtin. Finally, we show that using two distinct ASO drugs targeting the two allelic variants of an HD-SNP could provide a therapeutic option for all persons with HD; allele-specifically for roughly half, and non-specifically for the remainder.

  16. Validation of a Multiplex Allele-Specific Polymerase Chain Reaction Assay for Detection of KRAS Gene Mutations in Formalin-Fixed, Paraffin-Embedded Tissues from Colorectal Cancer Patients.

    Directory of Open Access Journals (Sweden)

    Sirirat Seekhuntod

    Full Text Available Patients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC. The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR assay to detect KRAS mutations.We developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D. We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay.Overall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%, G12D (25.83% and G12V (12.50% in codon 12.The MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues.

  17. PCR amplfication on a microarray of gel-immobilized oligonucleotides : detection of bacterial toxin- and drug-resistent genes and their mutations.

    Energy Technology Data Exchange (ETDEWEB)

    Strizhkov, B. N.; Drobyshev, A. L.; Mikhailovich, V. M.; Mirzabekov, A. D.; Biochip Technology Center; Engelhardt Inst. of Molecular Biology

    2000-10-01

    PCR amplification on a microarray of gel-immobilized primers (microchip) has been developed. One of a pair of PCR primers was immobilized inside a separate microchip polyacrylamide porous gel pad of 0.1 x 0.1 x 0.02 (or 0.04) micron in size and 0.2 (or 0.4) nL in volume. The amplification was carried out simultaneously both in solution covering the microchip array and inside gel pads. Each gel pad contained the immobilized forward primers, while the fluorescently labeled reverse primers, as well as all components of the amplification reaction, diffused into the gel pads from the solution. To increase the amplification efficiency, the forward primers were also added into the solution. The kinetics of amplification was measured in real time in parallel for all gel pads with a fluorescent microscope equipped with a charge-coupled device (CCD) camera. The accuracy of the amplification was assessed by using the melting curves obtained for the duplexes formed by the labeled amplification product and the gel-immobilized primers during the amplification process; alternatively, the duplexes were produced by hybridization of the extended immobilized primers with labeled oligonucleotide probes. The on-chip amplification was applied to detect the anthrax toxin genes and the plasmid-borne beta-lactamase gene responsible for bacterial ampicillin resistance. The allele-specific type of PCR amplification was used to identify the Shiga toxin gene and discriminate it from the Shiga-like one. The genomic mutations responsible for rifampicin resistance of the Mycobacterium tuberculosis strains were detected by the same type of PCR amplification of the rpoB gene fragment isolated from sputum of tuberculosis patients. The on-chip PCR amplification has been shown to be a rapid, inexpensive and powerful tool to test genes responsible for bacterial toxin production and drug resistance, as well as to reveal point nucleotide mutations.

  18. Development of a Duplex RT-PCR Assay for Detection of Duck Hepatitis Virus Type I and Muscovy Duck Parvovirus%鸭I型肝炎病毒和番鸭细小病毒二重RT-PCR方法的建立

    Institute of Scientific and Technical Information of China (English)

    谢丽基; 谢芝勋; 刘加波; 庞耀珊; 邓显文; 谢志勤; 范晴

    2012-01-01

      根据基因库中鸭I型肝炎病毒和番鸭细小病毒的基因序列,分别设计了两对特异性引物,通过对二重RT-PCR扩增条件的优化,研究建立了可同时鉴别检测鸭I型肝炎病毒和番鸭细小病毒的二重RT-PCR方法.该方法对同一样品中的鸭I型肝炎病毒和番鸭细小病毒模板进行扩增,结果均同时得到2条大小与实验设计相符的202 bp(鸭I型肝炎病毒)和474 bp(番鸭细小病毒)的特异性扩增条带,对鸭圆环病毒、鹅细小病毒、鸭副黏病毒、鸭瘟病毒和禽流感病毒等病原体的检测全为阴性.敏感性试验结果表明,该技术最低能检测到100 fg的鸭I型肝炎病毒RNA和番鸭细小病毒DNA.研究建立的鸭I型肝炎病毒和番鸭细小病毒的二重RT-PCR方法,具有快速、敏感、特异、定量和重复性好等优点,可用于临床上鸭I型肝炎病毒和番鸭细小病毒感染的检测.%  A duplex reverse polymerase chain reaction assay(duplex RT-PCR)was developed and optimized to simultaneously detect duck hepatitis virus Type I and Muscovy duck parvovirus in one reaction.Two pairs of specific primers were designed according to the conserved regions on the sequences of duck hepatitis virus Type I and Muscovy duck parvovirus in GenBank.It was shown that samples containg duck hepatitis virus Type I and Muscovy duck parvovirus could be amplified into the specific bands,202 bp for duck hepatitis virus Type I and 474 bp for Muscovy duck parvovirus by this duplex PCR,but no specific bands of the same sizes were amplified from duck circovirus, gosling plague virus,duck paramyxovirus,duck plague virus and avian influenza virus.As little as 100 fg of duck hepatitis virus Type I RNA and Muscovy duck parvovirus DNA could be detected.This duplex RT-PCR assay was a quick,sensitive,and specific test for detection of duck hepatitis virus Type I and Muscovy duck parvovirus,and could be useful for the control of these viruses in

  19. Identification of transcriptome SNPs between Xiphophorus lines and species for assessing allele specific gene expression within F1 interspecies hybrids☆

    Science.gov (United States)

    Shen, Yingjia; Catchen, Julian; Garcia, Tzintzuni; Amores, Angel; Beldroth, Ion; Wagner, Jonathon R; Zhang, Ziping; Postlethwait, John; Warren, Wes; Schartl, Manfred; Walter, Ronald B.

    2011-01-01

    Variations in gene expression are essential for the evolution of novel phenotypes and for speciation. Studying allelic specific gene expression (ASGE) within interspecies hybrids provides a unique opportunity to reveal underlying mechanisms of genetic variation. Using Xiphophorus interspecies hybrid fishes and high-throughput next generation sequencing technology, we were able to assess variations between two closely related vertebrate species, X. maculatus and X. couchianus, and their F1 interspecies hybrids. We constructed transcriptome-wide SNP polymorphism sets between two highly inbred X. maculatus lines (JP 163 A and B), and between X. maculatus and a second species, X. couchianus. The X. maculatus JP 163 A and B parental lines have been separated in the laboratory for ≈ 70 years and we were able to identify SNPs at a resolution of 1 SNP per 49 kb of transcriptome. In contrast, SNP polymorphisms between X. couchianus and X. maculatus species, which diverged ≈ 5–10 million years ago, were identified about every 700 bp. Using 6,524 transcripts with identified SNPs between the two parental species (X. maculatus and X. couchianus), we mapped RNA-seq reads to determine ASGE within F1 interspecies hybrids. We developed an in silico X. couchianus transcriptome by replacing 90,788 SNP bases for X. maculatus transcriptome with the consensus X. couchianus SNP bases and provide evidence that this procedure overcomes read mapping biases. Employment of the insilico reference transcriptome and tolerating 5 mismatches during read mapping allow direct assessment of ASGE in the F1 interspecies hybrids. Overall, these results show that Xiphophorus is a tractable vertebrate experimental model to investigate how genetic variations that occur during speciation may affect gene interactions and the regulation of gene expression. PMID:21466860

  20. Identification of transcriptome SNPs between Xiphophorus lines and species for assessing allele specific gene expression within F₁ interspecies hybrids.

    Science.gov (United States)

    Shen, Yingjia; Catchen, Julian; Garcia, Tzintzuni; Amores, Angel; Beldorth, Ion; Wagner, Jonathan; Zhang, Ziping; Postlethwait, John; Warren, Wes; Schartl, Manfred; Walter, Ronald B

    2012-01-01

    Variations in gene expression are essential for the evolution of novel phenotypes and for speciation. Studying allelic specific gene expression (ASGE) within interspecies hybrids provides a unique opportunity to reveal underlying mechanisms of genetic variation. Using Xiphophorus interspecies hybrid fishes and high-throughput next generation sequencing technology, we were able to assess variations between two closely related vertebrate species, Xiphophorus maculatus and Xiphophorus couchianus, and their F(1) interspecies hybrids. We constructed transcriptome-wide SNP polymorphism sets between two highly inbred X. maculatus lines (JP 163 A and B), and between X. maculatus and a second species, X. couchianus. The X. maculatus JP 163 A and B parental lines have been separated in the laboratory for ≈70 years and we were able to identify SNPs at a resolution of 1 SNP per 49 kb of transcriptome. In contrast, SNP polymorphisms between X. couchianus and X. maculatus species, which diverged ≈5-10 million years ago, were identified about every 700 bp. Using 6524 transcripts with identified SNPs between the two parental species (X. maculatus and X. couchianus), we mapped RNA-seq reads to determine ASGE within F(1) interspecies hybrids. We developed an in silico X. couchianus transcriptome by replacing 90,788 SNP bases for X. maculatus transcriptome with the consensus X. couchianus SNP bases and provide evidence that this procedure overcomes read mapping biases. Employment of the in silico reference transcriptome and tolerating 5 mismatches during read mapping allow direct assessment of ASGE in the F(1) interspecies hybrids. Overall, these results show that Xiphophorus is a tractable vertebrate experimental model to investigate how genetic variations that occur during speciation may affect gene interactions and the regulation of gene expression.

  1. Identification of transcriptome SNPs for assessing allele-specific gene expression in a super-hybrid rice Xieyou9308.

    Directory of Open Access Journals (Sweden)

    Rongrong Zhai

    Full Text Available Hybridization, a common process in nature, can give rise to a vast reservoir of allelic variants. Combination of these allelic variants may result in novel patterns of gene action and is thought to contribute to heterosis. In this study, we analyzed genome-wide allele-specific gene expression (ASGE in the super-hybrid rice variety Xieyou9308 using RNA sequencing technology (RNA-Seq. We identified 9325 reliable single nucleotide polymorphisms (SNPs distributed throughout the genome. Nearly 68% of the identified polymorphisms were CT and GA SNPs between R9308 and Xieqingzao B, suggesting the existence of DNA methylation, a heritable epigenetic mark, in the parents and their F1 hybrid. Of 2793 identified transcripts with consistent allelic biases, only 480 (17% showed significant allelic biases during tillering and/or heading stages, implying that trans effects may mediate most transcriptional differences in hybrid offspring. Approximately 67% and 62% of the 480 transcripts showed R9308 allelic expression biases at tillering and heading stages, respectively. Transcripts with higher levels of gene expression in R9308 also exhibited R9308 allelic biases in the hybrid. In addition, 125 transcripts were identified with significant allelic expression biases at both stages, of which 74% showed R9308 allelic expression biases. R9308 alleles may tend to preserve their characteristic states of activity in the hybrid and may play important roles in hybrid vigor at both stages. The allelic expression of 355 transcripts was highly stage-specific, with divergent allelic expression patterns observed at different developmental stages. Many transcripts associated with stress resistance were differently regulated in the F1 hybrid. The results of this study may provide valuable insights into molecular mechanisms of heterosis.

  2. Integrative analysis of hereditary nonpolyposis colorectal cancer: the contribution of allele-specific expression and other assays to diagnostic algorithms.

    Directory of Open Access Journals (Sweden)

    Laura De Lellis

    Full Text Available The identification of germline variants predisposing to hereditary nonpolyposis colorectal cancer (HNPCC is crucial for clinical management of carriers, but several probands remain negative for such variants or bear variants of uncertain significance (VUS. Here we describe the results of integrative molecular analyses in 132 HNPCC patients providing evidences for improved genetic testing of HNPCC with traditional or next generation methods. Patients were screened for: germline allele-specific expression (ASE, nucleotide variants, rearrangements and promoter methylation of mismatch repair (MMR genes; germline EPCAM rearrangements; tumor microsatellite instability (MSI and immunohistochemical (IHC MMR protein expression. Probands negative for pathogenic variants of MMR genes were screened for germline APC and MUTYH sequence variants. Most germline defects identified were sequence variants and rearrangements of MMR genes. Remarkably, altered germline ASE of MMR genes was detected in 8/22 (36.5% probands analyzed, including 3 cases negative at other screenings. Moreover, ASE provided evidence for the pathogenic role and guided the characterization of a VUS shared by 2 additional probands. No germline MMR gene promoter methylation was observed and only one EPCAM rearrangement was detected. In several cases, tumor IHC and MSI diverged from germline screening results. Notably, APC or biallelic MUTYH germline defects were identified in 2/19 probands negative for pathogenic variants of MMR genes. Our results show that ASE complements gDNA-based analyses in the identification of MMR defects and in the characterization of VUS affecting gene expression, increasing the number of germline alterations detected. An appreciable fraction of probands negative for MMR gene variants harbors APC or MUTYH variants. These results indicate that germline ASE analysis and screening for APC and MUTYH defects should be included in HNPCC diagnostic algorithms.

  3. Imprinted chromosomal domains revealed by allele-specific replication timing of the GABRB3 and GABRA5 genes

    Energy Technology Data Exchange (ETDEWEB)

    LaSalle, J.; Flint, A.; Lalande, M. [Harvard Medical School, Boston, MA (United States)] [and others

    1994-09-01

    The GABRB3 and GABRA5 genes are organized as a cluster in chromosome 15q11-q13. The genes are separated by around 100 kb and arranged in opposite transcriptional orientations. The GABA{sub A} receptor cluster lies near the Angelman and Prader-Willi loci and displays asynchronous DNA replication, suggesting that this region is subject to parental imprinting. In order to further study the association between DNA replication and imprinting, allele-specific replication was assayed by fluorescence in situ hybridization with {lambda}-phage probes from the GABRB3/A5 region and a D15Z1 satellite probe to identify the parental origin of each chromosome. The replication kinetics of each allele was determined by using a flow sorter to fractionate mitogen-stimulated lymphocytes on the basis of cell cycle progression prior to FISH analysis. These kinetic studies reveal a 50-150 kb chromosomal domain extending from the middle of the GABRB3/A5 intergenic region into the GABRA5 5{prime}-UTR which displays maternal replication in early S with paternal replication delayed until the end of S. In contrast, genomic regions on either side of this maternal early replication domain exhibit the opposite pattern with paternal before maternal replication and both alleles replicating in the latter half of S. These results indicate that the GABRB3/A5 region is divided into domains in which replication timing is determined by parental origin. In addition to a loss of asynchronous replication, organization into replication timing domains is also lost in lymphocytes from maternal and paternal uniparental disomy 15 patients suggesting that a chromosome contribution from both parents is required for the establishment of the imprinted replication domains.

  4. Infrequent detection of germline allele-specific expression of TGFBR1 in lymphoblasts and tissues of colon cancer patients.

    LENUS (Irish Health Repository)

    Guda, Kishore

    2009-06-15

    Recently, germline allele-specific expression (ASE) of the gene encoding for transforming growth factor-beta type I receptor (TGFBR1) has been proposed to be a major risk factor for cancer predisposition in the colon. Germline ASE results in a lowered expression of one of the TGFBR1 alleles (>1.5-fold), and was shown to occur in approximately 20% of informative familial and sporadic colorectal cancer (CRC) cases. In the present study, using the highly quantitative pyrosequencing technique, we estimated the frequency of ASE in TGFBR1 in a cohort of affected individuals from familial clusters of advanced colon neoplasias (cancers and adenomas with high-grade dysplasia), and also from a cohort of individuals with sporadic CRCs. Cases were considered positive for the presence of ASE if demonstrating an allelic expression ratio <0.67 or >1.5. Using RNA derived from lymphoblastoid cell lines, we find that of 46 informative Caucasian advanced colon neoplasia cases with a family history, only 2 individuals display a modest ASE, with allelic ratios of 1.65 and 1.73, respectively. Given that ASE of TGFBR1, if present, would likely be more pronounced in the colon compared with other tissues, we additionally determined the allele ratios of TGFBR1 in the RNA derived from normal-appearing colonic mucosa of sporadic CRC cases. We, however, found no evidence of ASE in any of 44 informative sporadic cases analyzed. Taken together, we find that germline ASE of TGFBR1, as assayed in lymphoblastoid and colon epithelial cells of colon cancer patients, is a relatively rare event.

  5. Advantage of whole exome sequencing over allele-specific and targeted segment sequencing in detection of novel TULP1 mutation in leber congenital amaurosis

    DEFF Research Database (Denmark)

    Guo, Yiran; Prokudin, Ivan; Yu, Cong

    2015-01-01

    Background: Leber congenital amaurosis (LCA) is a severe form of retinal dystrophy with marked underlying genetic heterogeneity. Until recently, allele-specific assays and Sanger sequencing of targeted segments were the only available approaches for attempted genetic diagnosis in this condition. ...

  6. Detection of Fusarium oxysporum f. sp. vasinfectum race 3 by single-base extension method and allele-specific polymerase chain reaction

    Science.gov (United States)

    We developed allele specific (AS) SNP primers for rapid detection of Fusarium oxysporum f.sp vasinfectum (FOV) race 3. FOV_BT_SNP_R3 and FOV_BT_AS_R3 primers were designed based on single nucleotide polymorphisms of partial sequence alignment of the ß-tubulin (BT) gene from several FOV races. These ...

  7. 转基因大豆和玉米加工产品的双重精确定量PCR检测方法%Improved quantification accuracy for duplex real-time PCR detection of genetically modified soybean and maize in heat processed foods

    Institute of Scientific and Technical Information of China (English)

    程芳; 沈平; 张大兵; 李建粤; 杨立桃

    2013-01-01

    Real-time PCR technique has been widely used in quantitative GMO detection in recent years.The accuracy of GMOs quantification based on the real-time PCR methods is still a difficult problem,especially for the quantification of high processed samples.To develop the suitable and accurate real-time PCR system for high processed GM samples,we made ameliorations to several realtime PCR parameters,including re-designed shorter target DNA fragment,similar lengths of amplified endogenous and exogenous gene targets,similar GC contents and melting temperatures of PCR primers and TaqMan probes.Also,one Heat-Treatment Processing Model (HTPM) was established using soybean flour samples containing GM soybean GTS 40-3-2 to validate the effectiveness of the improved real-time PCR system.Tested results showed that the quantitative bias of GM content in heat processed samples were lowered using the new PCR system.The improved duplex real-time PCR wasfurther validated using processed foods derived from GM soybean,and more accurate GM content values in these foods was also achieved.These results demonstrated that the improved duplex real-time PCR would be quite suitable in quantitative detection of high processed food products.%在很多国家,转基因生物(GMOs)及其衍生产品必须标有精确的转基因含量.最近研究中,实时定量PCR技术广泛应用于转基因成分的检测.然而,转基因生物的实时定量PCR方法的精确度仍然是一个难以解决的问题,尤其是对于高温处理过的样品.为了更好地准确定量高温处理样品中转基因的含量,对普通的实时定量PCR体系做了一些改进,包括重新设计内源基因和外源基因的引物,使得扩增较短并且大小接近的目标DNA片段,同时引物的GC含量和溶解温度也都相近.此外,采用热处理加工模型(HTPM)的方法,制备了含有转基因大豆GTS 40-3-2的样品,并验证了改进后的实时定量PCR系统.实验结果表明:使用改进后的

  8. Duplex RT-PCR detection and sequences comparison of Macrobrachium rosenbergii nodavirus and Macrobrachium rosenbergii dicistrovirus%罗氏沼虾野田村病毒和双顺反子病毒双重RT-PCR检测方法与序列分析

    Institute of Scientific and Technical Information of China (English)

    潘晓艺; 袁雪梅; 刘杜鹃; 沈锦玉; 张宇飞; 蔺凌云; 王军毅; 郝贵杰; 姚嘉赟; 徐洋

    2012-01-01

    罗氏沼虾野田村病毒(Macrobrachium rosenbergii nodavirus,MrNV)和双顺反子病毒(M.rosenbergii dicistrovirus,MrDV)是已报道对罗氏沼虾易感的主要致病性病毒,该研究通过建立双重RT-PCR方法对MrDV和MrNV两种病毒同时进行检测。根据MrDV和MrNV基因组序列的保守区分别设计特异性引物,并对双重PCR的退火温度和引物浓度进行优化,在获得优化反应体系和反应条件后,对罗氏沼虾样品进行检测。结果表明,双重PCR最佳退火温度为60℃,反应体系最佳引物终浓度MrNV384为0.1μmol/L,MrDV472为0.05μmol/L,对病样总RNA的最低检测限为360fg。引物的特异性检测表明,该检测方法对TSV、WSSV、IHHNV和嗜水气单胞菌TPS-30基因组无交叉反应。对阳性样品的病毒扩增序列分析表明,MrDV RNA依赖性RNA聚合酶编码区序列无变异,MrNV-RNA2序列存在较多变异,进化树结果表明2011年长三角的MrNV病毒主要来自于中国基因型和东南亚基因型。该方法的建立为罗氏沼虾病毒性疾病的预防和种苗的繁育奠定了基础。%Macrobrachium rosenbergii nodavirus(MrNV) and Macrobrachium rosenbergii dicistrovirus(MrDV) are the main viral pathogens of Macrobrachium rosenbergii.In order to detect the two viruses,two pairs of primers targeted sequences located within the MrNV capsid protein gene and MrDV RNA-dependent RNA polymerase(RDRP) gene amplified amplicons of 384 bp and 472 bp,respectively.The reaction conditions of the duplex RT-PCR were optimized and PCR products were sequenced.Meanwhile,the specificity and sensitivity of duplex RT-PCR were studied.Results reveal that optimum annealing temperature of duplex RT-PCR is 60 ℃ and the minimum concentrations of primers are 0.1 μmol/L of MrNV384 and 0.05 μmol/L of MrDV472.The detection limit of duplex RT-PCR was determined to be 360 fg of tissue total RNA for both the viruses.The two pairs of primers were found to be specific to MrNV and Mr

  9. [Carotid duplex ultrasonography for neurosurgeons].

    Science.gov (United States)

    Sadahiro, Hirokazu; Ishihara, Hideyuki; Oka, Fumiaki; Suzuki, Michiyasu

    2011-12-01

    Carotid duplex ultrasonography (CDU) is one of the most well-known imaging methods for arteriosclerosis and ischemic stroke. For neurosurgeons, it is very important for the details of carotid plaque to be thoroughly investigated by CDU. Symptomatic carotid plaque is very fragile and easily changes morphologically, and so requires frequent CDU examination. Furthermore, after carotid endarterectomy (CEA) and carotid artery stenting (CAS), restenosis is evaluated with CDU. CDU facilitates not only morphological imaging in the B mode, but also allows a flow study with color Doppler and duplex imaging. So, CDU can help assess the presence of proximal and intracranial artery lesions in spite of only having a cervical view, and the patency of the extracranial artery to intracranial artery bypass is revealed with CDU, which shows a rich velocity and low pulsatility index (PI) in duplex imaging. For the examiner, it is necessary to ponder on what duplex imaging means in examinations, and to summarize all imaging finding.

  10. Impact of pre-existing MSP142-allele specific immunity on potency of an erythrocytic Plasmodium falciparum vaccine

    Directory of Open Access Journals (Sweden)

    Bergmann-Leitner Elke S

    2012-09-01

    Full Text Available Abstract Background MSP1 is the major surface protein on merozoites and a prime candidate for a blood stage malaria vaccine. Preclinical and seroepidemiological studies have implicated antibodies to MSP1 in protection against blood stage parasitaemia and/or reduced parasite densities, respectively. Malaria endemic areas have multiple strains of Plasmodium falciparum circulating at any given time, giving rise to complex immune responses, an issue which is generally not addressed in clinical trials conducted in non-endemic areas. A lack of understanding of the effect of pre-existing immunity to heterologous parasite strains may significantly contribute to vaccine failure in the field. The purpose of this study was to model the effect of pre-existing immunity to MSP142 on the immunogenicity of blood-stage malaria vaccines based on alternative MSP1 alleles. Methods Inbred and outbred mice were immunized with various recombinant P. falciparum MSP142 proteins that represent the two major alleles of MSP142, MAD20 (3D7 and Wellcome (K1, FVO. Humoral immune responses were analysed by ELISA and LuminexTM, and functional activity of induced MSP142-specific antibodies was assessed by growth inhibition assays. T-cell responses were characterized using ex vivo ELISpot assays. Results Analysis of the immune responses induced by various immunization regimens demonstrated a strong allele-specific response at the T cell level in both inbred and outbred mice. The success of heterologous regimens depended on the degree of homology of the N-terminal p33 portion of the MSP142, likely due to the fact that most T cell epitopes reside in this part of the molecule. Analysis of humoral immune responses revealed a marked cross-reactivity between the alleles. Functional analyses showed that some of the heterologous regimens induced antibodies with improved growth inhibitory activities. Conclusion The development of a more broadly efficacious MSP1 based vaccine may be

  11. Identifying breast cancer risk loci by global differential allele-specific expression (DASE analysis in mammary epithelial transcriptome

    Directory of Open Access Journals (Sweden)

    Gao Chuan

    2012-10-01

    Full Text Available Abstract Background The significant mortality associated with breast cancer (BCa suggests a need to improve current research strategies to identify new genes that predispose women to breast cancer. Differential allele-specific expression (DASE has been shown to contribute to phenotypic variables in humans and recently to the pathogenesis of cancer. We previously reported that nonsense-mediated mRNA decay (NMD could lead to DASE of BRCA1/2, which is associated with elevated susceptibility to breast cancer. In addition to truncation mutations, multiple genetic and epigenetic factors can contribute to DASE, and we propose that DASE is a functional index for cis-acting regulatory variants and pathogenic mutations, and that global analysis of DASE in breast cancer precursor tissues can be used to identify novel causative alleles for breast cancer susceptibility. Results To test our hypothesis, we employed the Illumina® Omni1-Quad BeadChip in paired genomic DNA (gDNA and double-stranded cDNA (ds-cDNA samples prepared from eight BCa patient-derived normal mammary epithelial lines (HMEC. We filtered original array data according to heterozygous genotype calls and calculated DASE values using the Log ratio of cDNA allele intensity, which was normalized to the corresponding gDNA. We developed two statistical methods, SNP- and gene-based approaches, which allowed us to identify a list of 60 candidate DASE loci (DASE ≥ 2.00, P ≤ 0.01, FDR ≤ 0.05 by both methods. Ingenuity Pathway Analysis of DASE loci revealed one major breast cancer-relevant interaction network, which includes two known cancer causative genes, ZNF331 (DASE = 2.31, P = 0.0018, FDR = 0.040 and USP6 (DASE = 4.80, P = 0.0013, FDR = 0.013, and a breast cancer causative gene, DMBT1 (DASE=2.03, P = 0.0017, FDR = 0.014. Sequence analysis of a 5′ RACE product of DMBT1 demonstrated that rs2981745, a putative breast cancer risk locus, appears to be one of the causal variants leading to DASE

  12. Genotyping of the 19-bp insertion/deletion polymorphism in the 5' flank of beta-hydroxylase gene by dissociation analysis of allele-specific PCR products

    DEFF Research Database (Denmark)

    Rasmussen, Henrik Berg; Werge, Thomas

    2005-01-01

    DNA fragments. Mistyping of heterozygote samples due to preferential allele amplification was prevented by use of an optimized concentration of Mg(2+), addition of dimethyl sulfoxide and annealing/extension at an appropriate temperature. Comparison of results achieved by the closed-tube assay...

  13. 双重荧光定量PCR检测沙门菌和单核细胞增生李斯特菌方法的建立%Development of a duplex real-time PCR method for the detection of Salmonella and Listeria monocytogenes

    Institute of Scientific and Technical Information of China (English)

    王振东; 吉尚志; 杨宇; 王静

    2011-01-01

    Objective To develop a duplex real-time PCR array for rapid detection of Salmonella and Listeria monocytogenes. Methods The specific primers and probes were designed to amplify the fimY gene of Salmonella and the hly gene of Listeria monocytogenes. The sensitivity of the system was detected by a multiple proportional dilution method. In order to examine the specificity of the system, other seven intestinal bacteria strains were assayed simultaneously. Salmonella and Listeria monocytogenes infected mice model were reproduced for evaluating the applicability of the method. Results A highly sensitive and specific duplex real-time PCR assay for the detection of Salmonella and Listeria monocytogenes was established. The sensitivity was 11 copies/gl for Salmonella and 12.8 copies/gl for Listeria monocytogenes. The specificity was 100% and the accuracy was 93.3%. The whole detection procedure can be finished within 2. 5 h. Conclusion The duplex real-time PCR detection method is efficient in detecting Salmonella and Listeria monocytogenes in foods with good sensitivity and specificity. Due to the simultaneous detection of these two pathogens, the detection time was reduced significantly. There is a good prospect of this method applying in disease prevention and food industry.%目的 建立双重荧光定量PCR方法,快速检测沙门菌和单核细胞增生李斯特菌.方法 通过设计特异性引物和探针,扩增沙门菌的fimY基因和单核细胞增生李斯特菌的hly基因,采用倍比梯度稀释法检测该体系的灵敏度,以另外7株肠道致病菌评价检测体系的特异性;建立了沙门菌和单核细胞增生李斯特菌感染小鼠的检测模型以验证方法的适用性.结果 建立了同时检测沙门菌和单核细胞增生李斯特菌的双重荧光定量PCR方法,从DNA提取到检测完毕仅需2.5 h.检测两种病原菌的灵敏度分别为11和12.8 copies/μl,特异性为100%,符合率为93.3%.结论 该法缩短了检测时

  14. A duplex polymerase chain reaction assay for the identification of goat cashmere and sheep wool.

    Science.gov (United States)

    Geng, Qing-Rong

    2016-05-01

    In this article attempts were made to establish one-step duplex PCR assay for the identification of goat cashmere and sheep wool. Primers were selected from published papers or designed in the well-conserved region of mitochondrial D-loop genes after alignment of the available sequences in the GenBank database. A fragment of 294 bp from cashmere goat was amplified and three PCR fragments including a bright main band of approximately 404 bp in length were obtained from sheep. The duplex PCR was found to be effective in detecting mixed samples precisely when sheep wool was mixed to goat cashmere with the relative proportion of over 9.09%. The duplex PCR could be considered as a simple and promising method in identification of goat cashmere and sheep wool.

  15. A genome-wide screen in human embryonic stem cells reveals novel sites of allele-specific histone modification associated with known disease loci

    LENUS (Irish Health Repository)

    Prendergast, James G D

    2012-05-19

    AbstractBackgroundChromatin structure at a given site can differ between chromosome copies in a cell, and such imbalances in chromatin structure have been shown to be important in understanding the molecular mechanisms controlling several disease loci. Human genetic variation, DNA methylation, and disease have been intensely studied, uncovering many sites of allele-specific DNA methylation (ASM). However, little is known about the genome-wide occurrence of sites of allele-specific histone modification (ASHM) and their relationship to human disease. The aim of this study was to investigate the extent and characteristics of sites of ASHM in human embryonic stem cells (hESCs).ResultsUsing a statistically rigorous protocol, we investigated the genomic distribution of ASHM in hESCs, and their relationship to sites of allele-specific expression (ASE) and DNA methylation. We found that, although they were rare, sites of ASHM were substantially enriched at loci displaying ASE. Many were also found at known imprinted regions, hence sites of ASHM are likely to be better markers of imprinted regions than sites of ASM. We also found that sites of ASHM and ASE in hESCs colocalize at risk loci for developmental syndromes mediated by deletions, providing insights into the etiology of these disorders.ConclusionThese results demonstrate the potential importance of ASHM patterns in the interpretation of disease loci, and the protocol described provides a basis for similar studies of ASHM in other cell types to further our understanding of human disease susceptibility.

  16. Development of an allele-specific, loop-mediated, isothermal amplification method (AS-LAMP to detect the L1014F kdr-w mutation in Anopheles gambiae s. l.

    Directory of Open Access Journals (Sweden)

    Badolo Athanase

    2012-07-01

    Full Text Available Abstract Background Malaria control relies heavily on treated bed nets and indoor residual spraying with pyrethroid insecticides. Unfortunately, the resistance to pyrethroid insecticides, mainly due to the kdr mutation, is spreading in the main malaria vector Anopheles gambiae s.l., decreasing the insecticides’ efficacy. To manage the insecticide resistance rapidly and flexibly, simple and effective tools for the early detection of resistant mosquitoes are needed. This study aimed to develop an allele-specific, loop-mediated, isothermal amplification (AS-LAMP method to detect the West African-type kdr mutation (kdr-w; L1014F in field-collected mosquitoes. Methods DNA fragments of the wild-type and the mutated kdr gene were used to select the primers and develop the method. The primers were designed with the mutation at the 5’ end of the backward inner primer (BIP. The AS-LAMP method was compared to the AS-PCR method using the genomic DNA of 120 field-collected mosquitoes. Results The AS-LAMP method could discriminate between the wild-type homozygote, the heterozygote, and the kdr-w homozygote within 75 min. The AS-LAMP method has the advantage of being faster and at least as sensitive and specific as the AS-PCR method. Conclusions The AS-LAMP method can be used to detect the kdr mutation for quick decision-making, even in less well-equipped laboratories.

  17. HLA-DQA1 typing in Danes by two polymerase chain reaction (PCR) based methods

    DEFF Research Database (Denmark)

    Cowland, J B; Madsen, H O; Morling, N

    1995-01-01

    A total of 280 persons were HLA-DQA1 typed by two different polymerase chain reaction (PCR) based methods; (i) a reverse dot-blot (RDB) method, which can differentiate between six alleles, and (ii) a combined PCR-restriction fragment length polymorphism (PCR-RFLP) and allele-specific amplification...

  18. 猪繁殖与呼吸综合征病毒和牛病毒性腹泻病毒双重RT-PCR检测方法的建立%Establishment of duplex RT-PCR assay for detection of bovine viral diarrhea virus and porcine reproductive and respiratory syndrome virus

    Institute of Scientific and Technical Information of China (English)

    王克伟; 李玉峰; 王先炜; 马涛; 张国龙; 姜平

    2011-01-01

    根据牛病毒性腹泻病毒(BVDV)5'端非编码区基因序列,设计合成了1对特异性引物,参考本实验室针对猪繁殖与呼吸综合征病毒(PRRSV)N蛋白设计的引物,经过PCR反应条件的优化,建立了BVDV和PRRSV双重RT-PCR的检测方法.对于PRRSV和BVDV的cDNA最低检测量分别为3.8×10-4 ng和7×1014 ng,对于猪瘟病毒(CFSV)、脑心肌炎病毒(EMCV)和猪圆环病毒2型(PCV-2)的PCR扩增结果均为阴性;用该方法对江苏省不同地区采集的75份仔猪的肺脏、脾脏和淋巴结等病料进行了检测,结果PRRSV有55份阳性,BVDV有14份阳性,PRRSV和BVDV混合感染的有12份,与PRRSV和BVDV单一RT-PCR的检测结果符合率分别为89.3%和92%.证明建立的双重RT-PCR检测方法可用于临床样品中BVDV和PRRSV的检测.%According to the sequence of 5' untranslated region (5'UTR) of the bovine viral diarrhea virus ( BVDV) genome, a pair of primers was designed and synthesized. Combined with the established primers of the N protein of porcine reproductive and respiratory syndrome vims ( PRRSV) , a duplex RT-PCR was established for detection of the two viruses simultaneously based on the optimization of the reaction condition. The specific PCR products were 290 bp for BVDV and 374 bp for PRRSV in length. The detection limit of cDNA template in the RT-PCR was 3. 8×1O-4 ng for PRRSV and 7×10-4 ng for BVDV. The high specificity of the method was demonstrated by detecting classical swine fever virus ( CSFV) , encephalomyocarditis virus ( FMCV) and porcine circovirus 2 ( PCV-2). Among 75 clinical samples,there were 55 samples with PRRSV positive, 14 samples with BVDV positive, and 12 samples with coinfection of BVDV and PRRSV. The coincidence rates were 89. 3% and 92% with the single RT-PCR for PRRSV and BVDV, respectively. It suggested that the duplex RT-PCR assay in this study could be applied for the detection of BVDV and PRRSV in clinical samples.

  19. 大鼠细小病毒 H-1株和 KRV 株双重 PCR检测方法的建立及应用%Development and application of duplex PCR for detection of H-1 and KRV strains

    Institute of Scientific and Technical Information of China (English)

    李晓波; 付瑞; 王吉; 卫礼; 王淑菁; 岳秉飞; 贺争鸣

    2015-01-01

    Objective To develop a duplex PCR assay for detection of rat parvovirus H-1 and KRV and its application.Methods To design specific primers on the basis of H-1 ( NC_001358 ) and KRV ( U790330 ) genome sequences published in NCBI and establish a duplex PCR assay using H-1 and KRV DNA as templates.To verify the sensitivity and specificity of the method after optimizing PCR.The rats were infected by oral inoculation.The rats were divided into three groups:H-1 infection, KRV infection and mixed infection groups.To collect feces at the 4th, 6th, 8th and 10th days postinfection.Rats were euthanized on the 10th day and samples from heart, liver, spleen, lung, kidney and cecal contents were collected from each rat, then all the samples were screened with the duplex PCR.Results The 183 bp and 302 bp bands were amplified using H-1 and KRV as templates.The sensitivity test showed that the PCR method can detect as low as 3.8 pg/mL H-1 and 0.73 pg/mL KRV.There were no bands amplified when mouse minus virus, canine parvovirus and feline parvovirus were used as templates, showing that the specificity of the duplex PCR assay is very good. The nucleic acids of H-1 or KRV were detected in all rat feces on the 2th day postinfection and there was no obvious clinical symptoms in all the infected rats.The positive rates of H-1 were as follows:50%(4/8) heart tissues, 50%(4/8) liver tissues, 62.5%(5/8) spleen tissues, 50%(4/8) lung tissues, 37.5%(3/8) kidney tissues and 62.5%(5/8) cecum contents, and the positive rate of single infection group was higher than that of mixed infection group.The positive rates of KRV were as follows:0 (0/8) heart tissues, 25% (2/8) liver tissues, 87.5% (7/8) spleen tissues, 12.5% (1/8) lung tissues, 25%(2/8) kidney tissues and 62.5%(5/8) cecum contents, and the positive rate of mixed infection group was higher than that of single infection group.Conclusions The duplex PCR assay for H-1 and KRV established in this study can effectively detect H-1 or KRV

  20. A novel quantitative real-time PCR assay using self-reporting duplex mutation primers for detection of Chlamydia trachomatis%新型二聚体突变荧光引物定量检测沙眼衣原体

    Institute of Scientific and Technical Information of China (English)

    夏乾峰; 覃西; 钱士匀; 涂植光

    2011-01-01

    目的 以二聚体突变荧光引物技术为基础建立对沙眼衣原体进行定量检测的新方法.方法 以沙眼衣原体主要外膜蛋白(MOMP)基因构建重组质粒作为DNA标准品,设计二聚体突变荧光引物,优化定量PCR体系并进行性能评价;同时对148例临床生殖道标本进行检测.结果 建立的二聚体突变荧光引物的定量PCR方法,其线性范围为101~109copies/μL,灵敏度为10 copies/μL;低浓度样品的批内变异系数(CV)为4.71%,批间CV为5.57%;高浓度样品的批内CV为3.20%,批间CV为3.66%;常见生殖道病原菌检测结果均为阴性;对确诊的148例患者标本检测准确率为90.32%.结论 建立的沙眼衣原体定量PCR检测方法具有快速、准确、结果可靠等特点,可为沙眼衣原体感染的诊断、治疗监测和流行病学调查提供较好的技术支持.%Objective To establish a novel real-time fluorescence PCR method to detect Chlamydia trachomatis (CT) using self-reporting duplex mutation primers. Methods The recombinant vector was constructed with major outer membrane protein ( MOMP) gene of CT and was used as the standard template. The self-reporting duplex mutation primers were designed according to the cloned gene sequence. The quantitative PCR reaction system was optimized and the experimental performance of real-time PCR was evaluated. A total of 148 clinical samples from tractus genitalis were detected. Results The developed method showed a wide range of linearity from 101 to 109 copies/μL and high sensitivity (10 copies/μL). The intra-assay and inter-assay coefficient of variation (CV) in the low concentration of samples were 4.71% and 5.57% respectively, while they were 3.20% and 3. 66% in the high concentration of samples. The results of common pathogenic bacteria in tractus genitalis detected by the established method were all negative, while 99.32% were positive in 148 clinical samples which were identified as Chlamydia trachomatis infection

  1. A组轮状病毒G1和G9亚型的二重qPCR检测%The duplex qPCR assay for G1 and G9 subtypes of group A Rotavirius

    Institute of Scientific and Technical Information of China (English)

    徐垚; 曹以诚; 李晖; 方苓; 祖冬梅

    2013-01-01

    目的 建立快速检测A组轮状病毒G1和G9亚型的二重实时荧光定量PCR(qPCR)检测法.方法 针对A组轮状病毒G1和G9亚型VP7基因的保守区域与高变区域,设计特异的引物、探针.构建含A组轮状病毒G1和G9亚型VP7基因的质粒,将其作为该检测系统的阳性标准品,优化A组轮状病毒G1和G9亚型的qPCR检测系统.结果 该方法检测A组轮状病毒G1和G9亚型阳性样本的灵敏度为103 copies/μL,对其他亚型的标准质粒检测呈阴性.结论 本实验建立的A组轮状病毒G1和G9亚型的二重qPCR检测方法具有良好的特异度、敏感度和重复性,该方法的应用有助于A组轮状病毒G1与G9亚型感染的早期快速诊断.

  2. Allele-specific chromatin remodeling in the ZPBP2/GSDMB/ORMDL3 locus associated with the risk of asthma and autoimmune disease.

    Science.gov (United States)

    Verlaan, Dominique J; Berlivet, Soizik; Hunninghake, Gary M; Madore, Anne-Marie; Larivière, Mathieu; Moussette, Sanny; Grundberg, Elin; Kwan, Tony; Ouimet, Manon; Ge, Bing; Hoberman, Rose; Swiatek, Marcin; Dias, Joana; Lam, Kevin C L; Koka, Vonda; Harmsen, Eef; Soto-Quiros, Manuel; Avila, Lydiana; Celedón, Juan C; Weiss, Scott T; Dewar, Ken; Sinnett, Daniel; Laprise, Catherine; Raby, Benjamin A; Pastinen, Tomi; Naumova, Anna K

    2009-09-01

    Common SNPs in the chromosome 17q12-q21 region alter the risk for asthma, type 1 diabetes, primary biliary cirrhosis, and Crohn disease. Previous reports by us and others have linked the disease-associated genetic variants with changes in expression of GSDMB and ORMDL3 transcripts in human lymphoblastoid cell lines (LCLs). The variants also alter regulation of other transcripts, and this domain-wide cis-regulatory effect suggests a mechanism involving long-range chromatin interactions. Here, we further dissect the disease-linked haplotype and identify putative causal DNA variants via a combination of genetic and functional analyses. First, high-throughput resequencing of the region and genotyping of potential candidate variants were performed. Next, additional mapping of allelic expression differences in Yoruba HapMap LCLs allowed us to fine-map the basis of the cis-regulatory differences to a handful of candidate functional variants. Functional assays identified allele-specific differences in nucleosome distribution, an allele-specific association with the insulator protein CTCF, as well as a weak promoter activity for rs12936231. Overall, this study shows a common disease allele linked to changes in CTCF binding and nucleosome occupancy leading to altered domain-wide cis-regulation. Finally, a strong association between asthma and cis-regulatory haplotypes was observed in three independent family-based cohorts (p = 1.78 x 10(-8)). This study demonstrates the requirement of multiple parallel allele-specific tools for the investigation of noncoding disease variants and functional fine-mapping of human disease-associated haplotypes.

  3. Achieving single channel, full duplex wireless communication

    KAUST Repository

    Choi, Jung Il

    2010-01-01

    This paper discusses the design of a single channel full-duplex wireless transceiver. The design uses a combination of RF and baseband techniques to achieve full-duplexing with minimal effect on link reliability. Experiments on real nodes show the full-duplex prototype achieves median performance that is within 8% of an ideal full-duplexing system. This paper presents Antenna Cancellation, a novel technique for self-interference cancellation. In conjunction with existing RF interference cancellation and digital baseband interference cancellation, antenna cancellation achieves the amount of self-interference cancellation required for full-duplex operation. The paper also discusses potential MAC and network gains with full-duplexing. It suggests ways in which a full-duplex system can solve some important problems with existing wireless systems including hidden terminals, loss of throughput due to congestion, and large end-to-end delays. Copyright 2010 ACM.

  4. Investigation of parameters that affect the success rate of microarray-based allele-specific hybridization assays.

    Directory of Open Access Journals (Sweden)

    Lena Poulsen

    Full Text Available BACKGROUND: The development of microarray-based genetic tests for diseases that are caused by known mutations is becoming increasingly important. The key obstacle to developing functional genotyping assays is that such mutations need to be genotyped regardless of their location in genomic regions. These regions include large variations in G+C content, and structural features like hairpins. METHODS/FINDINGS: We describe a rational, stable method for screening and combining assay conditions for the genetic analysis of 42 Phenylketonuria-associated mutations in the phenylalanine hydroxylase gene. The mutations are located in regions with large variations in G+C content (20-75%. Custom-made microarrays with different lengths of complementary probe sequences and spacers were hybridized with pooled PCR products of 12 exons from each of 38 individual patient DNA samples. The arrays were washed with eight buffers with different stringencies in a custom-made microfluidic system. The data were used to assess which parameters play significant roles in assay development. CONCLUSIONS: Several assay development methods found suitable probes and assay conditions for a functional test for all investigated mutation sites. Probe length, probe spacer length, and assay stringency sufficed as variable parameters in the search for a functional multiplex assay. We discuss the optimal assay development methods for several different scenarios.

  5. Development of a duplex real-time PCR assay for detection of bacillus anthracis%炭疽杆菌双重实时荧光PCR检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    谭维国; 陈文琦; 吕恒; 刘玉; 王平; 陈凤娟; 王长军; 张锦海

    2013-01-01

    目的 建立同时检测炭疽杆菌capA基因、PA基因的双重实时定量荧光PCR方法,用于应对突发传染病疫情和流行病学调查、突发公共卫生事件应急处置的早期检测及防范生物恐怖威胁.方法 设计和合成分别针对炭疽杆菌capA基因、PA基因的引物对和荧光双标记探针,构建质粒标准品,通过优化探针、引物浓度、反应体系试剂组分等参数,建立可同时检测capA基因、PA基因的双重实时荧光PCR方法,测试方法的灵敏度和特异性,并在实战检测中应用.结果 双重实时荧光定量PCR的炭疽杆菌capA基因、PA基因检测的灵敏度分别可达到每反应5和50拷贝,并具有良好的特异性,在实战应用中亦经过考验.结论 双重实时荧光定量PCR技术具有可以同时筛查、经济、快速、特异性强等优点,在炭疽杆菌检测方面有良好应用价值.

  6. 双重胶体金层析法快速检测番茄环斑病毒和烟草环斑病毒RT-PCR扩增子%Duplex Detection of Tomato Ringspot Virus and Tobacco Ringspot Virus RT-PCR Amplicons by Dipstick Assay

    Institute of Scientific and Technical Information of China (English)

    曹成; 魏梅生; 张永江; 李桂芬; 吴兴泉

    2011-01-01

    Both tomato ringspot virus(ToRSV)and tobacco ringspot virus(TRSV) are quarantine pests for China. We have developed dipstick assay for detection of RT-PCR amplicons of the two viruses within 15 minutes. The single dipstick assay is performed by labeling primers of the virus with biotin and fluorescein (or digoxigenin). Monoclonal antibody against fluorescein (or digoxigenin) is spotted on nitrocellulose membrane as T dot. The dual-labeled(biotin-fluorescein or biotin-digoxigenin) RT-PCR amplicons can be detected on the test dot(T dot). The duplex dipstick assay is performed by labeling primers of ToRSV with biotin and fluorescein (or digoxigenin) and primers of TRSV with biotin and digoxigenin(or fluorescein). Monoclonal antibody against fluorescein and digoxigenin is spotted on nitrocellulose membrane respectively as T1 and T2 dot . The dual-labeled(biotin-fluorescein and biotin-digoxigenin) RT-PCR amplicons of the two viruses can be detected on the Tl and T2 dot simultaneously.%番茄环斑病毒和烟草环斑病毒是中国进境检疫性有害生物,此研究建立了单个和双重胶体金层析快速检测方法,在15 min即可获得对双标记PCR产物的检测结果.单个胶体金层析是在一张层析膜上点上荧光素(或地高辛)的单克隆抗体作为T检测点,经生物素-荧光素(或生物素-地高辛)双标记的RT-PCR扩增产物可在T点上被检测到.双重胶体金层析是在一张层析膜上分别点上荧光素和地高辛的单克隆抗体作为T1和T2检测点,经生物素-荧光素和生物素-地高辛双标记的两种病毒的RT-PCR扩增产物可分别在T1和T2点上被检测到.

  7. The − 5 A/G single-nucleotide polymorphism in the core promoter region of MT2A and its effect on allele-specific gene expression and Cd, Zn and Cu levels in laryngeal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Starska, Katarzyna, E-mail: katarzyna.starska@umed.lodz.pl [I Department of Otolaryngology and Laryngological Oncology, Medical University of Łódź, Kopcinskiego 22, 90-153 Łódź (Poland); Krześlak, Anna; Forma, Ewa [Department of Cytobiochemistry, University of Łódź, Pomorska 142/143, 90-236 Łódź (Poland); Olszewski, Jurek [II Department of Otolaryngology and Laryngological Oncology, Medical University of Łódź, Żeromskiego 113, 90-549 Łódź (Poland); Morawiec-Sztandera, Alina [Department of Head and Neck Surgery, Medical University of Łódź, Paderewskiego 4, 93-509 Łódź (Poland); Aleksandrowicz, Paweł [Department of Otolaryngology and Laryngological Oncology, Medical University of Lublin, Jaczewskiego 8, 20-954 Lublin (Poland); Lewy-Trenda, Iwona [Department of Pathology, Medical University of Łódź, Pomorska 251, 92-213 Łódź (Poland); and others

    2014-10-15

    Metallothioneins (MTs) are low molecular weight, cysteine-rich heavy metal-binding proteins which participate in the mechanisms of Zn homeostasis, and protect against toxic metals. MTs contain metal-thiolate cluster groups and suppress metal toxicity by binding to them. The aim of this study was to determine the − 5 A/G (rs28366003) single-nucleotide polymorphism (SNP) in the core promoter region of the MT2A gene and to investigate its effect on allele-specific gene expression and Cd, Zn and Cu content in squamous cell laryngeal cancer (SCC) and non-cancerous laryngeal mucosa (NCM) as a control. The MT2A promoter region − 5 A/G SNP was determined by restriction fragment length polymorphism using 323 SCC and 116 NCM. MT2A gene analysis was performed by quantitative real-time PCR. The frequency of A allele carriage was 94.2% and 91.8% in SCC and NCM, respectively, while G allele carriage was detected in 5.8% and 8.2% of SCC and NCM samples, respectively. As a result, a significant association was identified between the − 5 A/G SNP in the MT2A gene with mRNA expression in both groups. Metal levels were analyzed by flame atomic absorption spectrometry. The significant differences were identified between A/A and both the A/G and G/G genotypes, with regard to the concentration of the contaminating metal. The Spearman rank correlation results showed that the MT2A expression and Cd, Zn, Cu levels were negatively correlated. Results obtained in this study suggest that − 5 A/G SNP in MT2A gene may have an effect on allele-specific gene expression and accumulation of metal levels in laryngeal cancer. - Highlights: • MT2A gene expression and metal content in laryngeal cancer tissues • Association between SNP (rs28366003) and expression of MT2A • Significant associations between the SNP and Cd, Zn and Cu levels • Negative correlation between MT2A gene expression and Cd, Zn and Cu levels.

  8. Effect of metallothionein 2A gene polymorphism on allele-specific gene expression and metal content in prostate cancer

    Energy Technology Data Exchange (ETDEWEB)

    Krześlak, Anna; Forma, Ewa [Department of Cytobiochemistry, University of Łódź, Pomorska 141/143, 90-236 Łódź (Poland); Chwatko, Grażyna [Department of Environmental Chemistry, University of Łódź, Pomorska 163, 90-236 Łódź (Poland); Jóźwiak, Paweł; Szymczyk, Agnieszka [Department of Cytobiochemistry, University of Łódź, Pomorska 141/143, 90-236 Łódź (Poland); Wilkosz, Jacek; Różański, Waldemar [2nd Department of Urology, Medical University of Łódź, Pabianicka 62, 93-513 Łódź (Poland); Bryś, Magdalena, E-mail: zreg@biol.uni.lodz.pl [Department of Cytobiochemistry, University of Łódź, Pomorska 141/143, 90-236 Łódź (Poland)

    2013-05-01

    Metallothioneins (MTs) are highly conserved, small molecular weight, cysteine rich proteins. The major physiological functions of metallothioneins include homeostasis of essential metals Zn and Cu and protection against cytotoxicity of heavy metals. The aim of this study was to determine whether there is an association between the − 5 A/G single nucleotide polymorphism (SNP; rs28366003) in core promoter region and expression of metallothionein 2A (MT2A) gene and metal concentration in prostate cancer tissues. MT2A polymorphism was determined by the polymerase chain reaction–restriction fragment length polymorphism technique (PCR–RFLP) using 412 prostate cancer tissue samples. MT2A gene expression analysis was performed by real-time RT-PCR method. A significant association between rs28366003 genotype and MT2A expression level was found. The average mRNA level was found to be lower among minor allele carriers (the risk allele) than average expression among homozygotes for the major allele. Metal levels were analyzed by flamed atomic absorption spectrometer system. Highly statistically significant associations were detected between the SNP and Cd, Zn, Cu and Pb levels. The results of Spearman's rank correlation showed that the expressions of MT2A and Cu, Pb and Ni concentrations were negatively correlated. On the basis of the results obtained in this study, we suggest that SNP polymorphism may affect the MT2A gene expression in prostate and this is associated with some metal accumulation. - Highlights: • MT2A gene expression and metal content in prostate cancer tissues • Association between SNP (rs28366003) and expression of MT2A • Significant associations between the SNP and Cd, Zn, Cu and Pb levels • Negative correlation between MT2A gene expression and Cu, Pb and Ni levels.

  9. 鸭副黏病毒和鸭圆环病毒二重荧光定量PCR检测方法的建立%Establishment of a Duplex Real-time PCR Assay for Detection of Duck Paramyxovirus and Duck Circovirus

    Institute of Scientific and Technical Information of China (English)

    许宗丽; 谢芝勋; 谢丽基; 刘加波; 谢志勤; 庞耀珊; 邓显文; 范晴

    2013-01-01

    根据鸭副黏病毒(DPMV)和鸭圆环病毒(DuCV)保守基因序列,设计了2对针对鸭副黏病毒和鸭圆环病毒的特异性引物和2条不同荧光基团标记的TaqMan探针,建立了鸭副黏病毒和鸭圆环病毒的二重荧光定量PCR检测方法.该方法敏感性好,对鸭副黏病毒和鸭圆环病毒的检测敏感性分别达到1 60和140个拷贝数;该方法特异性强,对鸭肝炎病毒、番鸭细小病毒、鸭瘟病毒和H9型禽流感病毒等病原体的检测全为阴性;应用该方法对118份临床病料进行检测,结果检出鸭副黏病毒和鸭圆环病毒阳性感染率分别为0.85%和8.47%,无混合感染.本试验建立的二重荧光定量PCR具有快速、特异、敏感和重复性好等优点,适用于鸭副黏病毒和鸭圆环病毒的快速诊断和监测.%A duplex Real-time polymerase chain reaction(drRT-PCR) assay was developed and optimized to simultaneously detect duck paramyxovirus and duck circovirus in one reaction. Two sets of specific primers for duck paramyxovirus and duck circovirus, along with two TaqMan probes specific for each virus were used in the assay. This drRT-PCR assay was found to be specific and be able to detect and differentiate duck paramyxovirus and duck circovirus, and no positive results were observed, when nucleic acid from duck hepatitis virus, Muscovy duck parvovirus, duck plague virus and H9 subtype avian influenza virus and so on were used as drRT-PCR templates. The sensitivity of this drRT-PCR assay was 160 and 140 copies for duck paramyxovirus and duck circovirus, respectivly. The drRT-PCR assay was applied to 118 clinical samples detection. Among these samples, duck paramyxovirus positive rate was 0. 85%, duck circovirus positive tate was 8. 47%, no co-infection. This drRT-PCR assay was a quick, sensitive and specific test for detecting duck paramyxovirus and duck circovirus,and would be useful for the control of these viruses in ducks.

  10. Detection of BRAF Mutations Using a Fully Automated Platform and Comparison with High Resolution Melting, Real-Time Allele Specific Amplification, Immunohistochemistry and Next Generation Sequencing Assays, for Patients with Metastatic Melanoma.

    Directory of Open Access Journals (Sweden)

    Alexandre Harlé

    Full Text Available Metastatic melanoma is a severe disease with one of the highest mortality rate in skin diseases. Overall survival has significantly improved with immunotherapy and targeted therapies. Kinase inhibitors targeting BRAF V600 showed promising results. BRAF genotyping is mandatory for the prescription of anti-BRAF therapies.Fifty-nine formalin-fixed paraffin-embedded melanoma samples were assessed using High-Resolution-Melting (HRM PCR, Real-time allele-specific amplification (RT-ASA PCR, Next generation sequencing (NGS, immunohistochemistry (IHC and the fully-automated molecular diagnostics platform IdyllaTM. Sensitivity, specificity, positive predictive value and negative predictive value were calculated using NGS as the reference standard to compare the different assays.BRAF mutations were found in 28(47.5%, 29(49.2%, 31(52.5%, 29(49.2% and 27(45.8% samples with HRM, RT-ASA, NGS, IdyllaTM and IHC respectively. Twenty-six (81.2% samples were found bearing a c.1799T>A (p.Val600Glu mutation, three (9.4% with a c.1798_1799delinsAA (p.Val600Lys mutation and one with c.1789_1790delinsTC (p.Leu597Ser mutation. Two samples were found bearing complex mutations.HRM appears the less sensitive assay for the detection of BRAF V600 mutations. The RT-ASA, IdyllaTM and IHC assays are suitable for routine molecular diagnostics aiming at the prescription of anti-BRAF therapies. IdyllaTM assay is fully-automated and requires less than 2 minutes for samples preparation and is the fastest of the tested assays.

  11. Corrosion behavior of duplex coatings

    Directory of Open Access Journals (Sweden)

    K. Raghu Ram Mohan Reddy

    2016-07-01

    Full Text Available The titanium alloys are used in defense, aerospace, automobile, chemical plants and biomedical applications due to their very high strength and lightweight properties. However, corrosion is a life-limiting factor when Ti alloys are exposed to different chemical environments at high temperatures. In the present paper, duplex NiCrAlY/WC–Co coating is coated onto Ti6Al4V substrate to investigate the corrosion behavior of both coated samples and the substrate. The duplex coating was performed with NiCrAlY as the intermediate coat of 200 μm thickness deposited by HVOF process and WC–Co ceramic top coat with varying thicknesses of 250 μm, 350 μm and 450 μm deposited by DS process. Potentiodynamic polarization tests were employed to investigate the corrosion performance of duplex coated samples and substrate in Ringer’s solution at 37 °C and pH value was set to 5.7. Finally the results reveal that 350 μm thick coated samples showed highest corrosion resistance compared to 250 μm thick samples as well as bare substrate. However, the 450 μm thick coated sample showed poor corrosion resistance compared to the substrate. The scale formed on the samples upon corrosion was characterized by using SEM analysis to understand the degree of corrosion behavior.

  12. 鸭Ⅰ型肝炎病毒和番鸭细小病毒二重荧光定量RT-PCR方法的建立%Development of duplex real-time RT-PCR assay for detection of duck hepatitis virus type Ⅰ and muscovy duck parvovirus

    Institute of Scientific and Technical Information of China (English)

    谢丽基; 谢芝勋; 邓显文; 谢志勤; 庞耀珊; 范晴; 刘加波

    2013-01-01

    A duplex real-time reverse polymerase chain reaction (drRT-PCR) assay was developed and optimized to simultaneously detect duck hepatitis virus type Ⅰ and muscovy duck parvovirus in one reaction.Two sets of specific oligonucleotide primers for duck hepatitis virus type Ⅰ and muscovy duck parvovirus,along with two TaqMan probes specific for each virus were used in the assay.This drRT-PCR assay was found to be specific and to be able to detect and differentiate duck hepatitis virus type Ⅰ and muscovy duck parvovirus,and no positive results were observed when nucleic acid from duck circovirus,gosling plague virus,duck parvovirus,duck plague virus and avian influenza virus were used as drRT-PCR templates.The sensitivity of this drRT-PCR assay was 200 template copies for duck hepatitis virus type Ⅰ and muscovy duck parvovirus.This drRT-PCR assay is a quick,sensitive,and specific test for detection of duck hepatitis virus type Ⅰ and muscovy duck parvovirus,and will be useful for the control of these viruses in ducks.%根据基因库中鸭Ⅰ型肝炎病毒和番鸭细小病毒的基因序列,设计2对特异性引物和2条用不同荧光基团标记的TaqMan探针.对反应条件和试剂浓度进行优化,建立能够同时检测鸭Ⅰ型肝炎病毒和番鸭细小病毒的二重荧光定量RT-PCR方法.该方法敏感性好,对鸭Ⅰ型肝炎病毒和番鸭细小病毒的检测敏感性均达到200个模板拷贝数;该方法特异性强,对鸭圆环病毒、鹅细小病毒、鸭副黏病毒、鸭瘟病毒和禽流感病毒等病原体的检测全为阴性.本研究建立的鸭Ⅰ型肝炎病毒和番鸭细小病毒的二重荧光定量RT-PCR方法,具有快速、敏感、特异、定量和重复性好等优点,可用于临床上鸭Ⅰ型肝炎病毒和番鸭细小病毒感染的检测.

  13. The Plasmodium falciparum merozoite surface protein-1 19 KD antibody response in the Peruvian Amazon predominantly targets the non-allele specific, shared sites of this antigen

    Directory of Open Access Journals (Sweden)

    Silva Claudia

    2010-01-01

    Full Text Available Abstract Background Plasmodium falciparum re-emerged in Iquitos, Peru in 1994 and is now hypoendemic (P. falciparum infections can be followed using this population dynamic. Previous work demonstrated a strong association between this population's antibody response to PfMSP1-19KD and protection against febrile illness and parasitaemia. Therefore, some selection for PfMSP1-19KD allelic diversity would be expected if the protection is to allele-specific sites of PfMSP1-19KD. Here, the potential for allele-specific polymorphisms in this population is investigated, and the allele-specificity of antibody responses to PfMSP1-19KD are determined. Methods The 42KD region in PfMSP1 was genotyped from 160 individual infections collected between 2003 and 2007. Additionally, the polymorphic block 2 region of Pfmsp1 (Pfmsp1-B2 was genotyped in 781 infection-months to provide a baseline for population-level diversity. To test whether PfMSP1-19KD genetic diversity had any impact on antibody responses, ELISAs testing IgG antibody response were performed on individuals using all four allele-types of PfMSP1-19KD. An antibody depletion ELISA was used to test the ability of antibodies to cross-react between allele-types. Results Despite increased diversity in Pfmsp1-B2, limited diversity within Pfmsp1-42KD was observed. All 160 infections genotyped were Mad20-like at the Pfmsp1-33KD locus. In the Pfmsp1-19KD locus, 159 (99.4% were the Q-KSNG-F haplotype and 1 (0.6% was the E-KSNG-L haplotype. Antibody responses in 105 individuals showed that Q-KNG and Q-TSR alleles generated the strongest immune responses, while Q-KNG and E-KNG responses were more concordant with each other than with those from Q-TSR and E-TSR, and vice versa. The immuno-depletion ELISAs showed all samples responded to the antigenic sites shared amongst all allelic forms of PfMSP1-19KD. Conclusions A non-allele specific antibody response in PfMSP1-19KD may explain why other allelic forms have not

  14. Screening of Modified RNA duplexes

    DEFF Research Database (Denmark)

    Schyth, Brian Dall; Bramsen, Jesper Bertram; Kjems, Jørgen

    Because of sequence specific gene targeting activity siRNAs are regarded as promising active compounds in gene medicine. But one serious problem with delivering siRNAs as treatment is the now well-established non-specific activities of some RNA duplexes. Cellular reactions towards double stranded...... RNAs include the 2´-5´ oligoadenylate synthetase system, the protein kinase R, RIG-I and Toll-like receptor activated pathways all resulting in antiviral defence mechanism. We have previously shown that antiviral innate immune reactions against double stranded RNAs could be detected in vivo as partial...... protection against a fish pathogenic virus. This protection corresponded with an interferon response in the fish. Here we use this fish model to screen siRNAs containing various chemical modifications of the RNA backbone for their antiviral activity, the overall aim being identification of an siRNA form...

  15. A Generalized Linear Model for Decomposing Cis-regulatory, Parent-of-Origin, and Maternal Effects on Allele-Specific Gene Expression

    Directory of Open Access Journals (Sweden)

    Yasuaki Takada

    2017-07-01

    Full Text Available Joint quantification of genetic and epigenetic effects on gene expression is important for understanding the establishment of complex gene regulation systems in living organisms. In particular, genomic imprinting and maternal effects play important roles in the developmental process of mammals and flowering plants. However, the influence of these effects on gene expression are difficult to quantify because they act simultaneously with cis-regulatory mutations. Here we propose a simple method to decompose cis-regulatory (i.e., allelic genotype, genomic imprinting [i.e., parent-of-origin (PO], and maternal [i.e., maternal genotype (MG] effects on allele-specific gene expression using RNA-seq data obtained from reciprocal crosses. We evaluated the efficiency of method using a simulated dataset and applied the method to whole-body Drosophila and mouse trophoblast stem cell (TSC and liver RNA-seq data. Consistent with previous studies, we found little evidence of PO and MG effects in adult Drosophila samples. In contrast, we identified dozens and hundreds of mouse genes with significant PO and MG effects, respectively. Interestingly, a similar number of genes with significant PO effect were detect in mouse TSCs and livers, whereas more genes with significant MG effect were observed in livers. Further application of this method will clarify how these three effects influence gene expression levels in different tissues and developmental stages, and provide novel insight into the evolution of gene expression regulation.

  16. Electromobility Shift Assay Reveals Evidence in Favor of Allele-Specific Binding of RUNX1 to the 5' Hypersensitive Site 4-Locus Control Region.

    Science.gov (United States)

    Dehghani, Hossein; Ghobakhloo, Sepideh; Neishabury, Maryam

    2016-08-01

    In our previous studies on the Iranian β-thalassemia (β-thal) patients, we identified an association between the severity of the β-thal phenotype and the polymorphic palindromic site at the 5' hypersensitive site 4-locus control region (5'HS4-LCR) of the β-globin gene cluster. Furthermore, a linkage disequilibrium was observed between this region and XmnI-HBG2 in the patient population. Based on this data, it was suggested that the well-recognized phenotype-ameliorating role assigned to positive XmnI could be associated with its linked elements in the LCR. To investigate the functional significance of polymorphisms at the 5'HS4-LCR, we studied its influence on binding of transcription factors. Web-based predictions of transcription factor binding revealed a binding site for runt-related transcription factor 1 (RUNX1), when the allele at the center of the palindrome (TGGGG(A/G)CCCCA) was A but not when it was G. Furthermore, electromobility shift assay (EMSA) presented evidence in support of allele-specific binding of RUNX1 to 5'HS4. Considering that RUNX1 is a well-known regulator of hematopoiesis, these preliminary data suggest the importance of further studies to confirm this interaction and consequently investigate its functional and phenotypical relevance. These studies could help us to understand the molecular mechanism behind the phenotype modifying role of the 5'HS4-LCR polymorphic palindromic region (rs16912979), which has been observed in previous studies.

  17. Direct Detection of Erythromycin-Resistant Bordetella pertussis in Clinical Specimens by PCR.

    Science.gov (United States)

    Wang, Zengguo; Han, Ruijun; Liu, Ying; Du, Quanli; Liu, Jifeng; Ma, Chaofeng; Li, Hengxin; He, Qiushui; Yan, Yongping

    2015-11-01

    Resistance of Bordetella pertussis to erythromycin has been increasingly reported. We developed an allele-specific PCR method for rapid detection of erythromycin-resistant B. pertussis directly from nasopharyngeal (NP) swab samples submitted for diagnostic PCR. Based on the proven association of erythromycin resistance with the A2047G mutation in the 23S rRNA of B. pertussis, four primers, two of which were designed to be specific for either the wild-type or the mutant allele, were used in two different versions of the allele-specific PCR assay. The methods were verified with results obtained by PCR-based sequencing of 16 recent B. pertussis isolates and 100 NP swab samples submitted for diagnostic PCR. The detection limits of the two PCR assays ranged from 10 to 100 fg per reaction for both erythromycin-susceptible and -resistant B. pertussis. Two amplified fragments of each PCR, of 286 and 112 bp, respectively, were obtained from a mutant allele of the isolates and/or NP swab samples containing B. pertussis DNAs. For the wild-type allele, only a 286-bp fragment was visible when the allele-specific PCR assay 1 was performed. No amplification was found when a number of non-Bordetella bacterial pathogens and NP swab samples that did not contain the DNAs of B. pertussis were examined. This assay can serve as an alternative for PCR-based sequencing, especially for local laboratories in resource-poor countries.

  18. Genome-wide detection of allele specific copy number variation associated with insulin resistance in African Americans from the HyperGEN study.

    Directory of Open Access Journals (Sweden)

    Marguerite R Irvin

    Full Text Available African Americans have been understudied in genome wide association studies of diabetes and related traits. In the current study, we examined the joint association of single nucleotide polymorphisms (SNPs and copy number variants (CNVs with fasting insulin and an index of insulin resistance (HOMA-IR in the HyperGEN study, a family based study with proband ascertainment for hypertension. This analysis is restricted to 1,040 African Americans without diabetes. We generated allele specific CNV genotypes at 872,243 autosomal loci using Birdsuite, a freely available multi-stage program. Joint tests of association for SNPs and CNVs were performed using linear mixed models adjusting for covariates and familial relationships. Our results highlight SNPs associated with fasting insulin and HOMA-IR (rs6576507 and rs8026527, 3.7*10(-7≤P≤1.1*10(-5 near ATPase, class V, type 10A (ATP10A, and the L Type voltage dependent calcium channel (CACNA1D, rs1401492, P≤5.2*10(-6. ATP10A belongs to a family of aminophospholipid-transporting ATPases and has been associated with type 2 diabetes in mice. CACNA1D has been linked to pancreatic beta cell generation in mice. The two most significant copy variable markers (rs10277702 and rs361367; P<2.0*10(-4 were in the beta variable region of the T-cell receptor gene (TCRVB. Human and mouse TCR has been shown to mimic insulin and its receptor and could contribute to insulin resistance. Our findings differ from genome wide association studies of fasting insulin and other diabetes related traits in European populations, highlighting the continued need to investigate unique genetic influences for understudied populations such as African Americans.

  19. SAAS-CNV: A Joint Segmentation Approach on Aggregated and Allele Specific Signals for the Identification of Somatic Copy Number Alterations with Next-Generation Sequencing Data.

    Directory of Open Access Journals (Sweden)

    Zhongyang Zhang

    2015-11-01

    Full Text Available Cancer genomes exhibit profound somatic copy number alterations (SCNAs. Studying tumor SCNAs using massively parallel sequencing provides unprecedented resolution and meanwhile gives rise to new challenges in data analysis, complicated by tumor aneuploidy and heterogeneity as well as normal cell contamination. While the majority of read depth based methods utilize total sequencing depth alone for SCNA inference, the allele specific signals are undervalued. We proposed a joint segmentation and inference approach using both signals to meet some of the challenges. Our method consists of four major steps: 1 extracting read depth supporting reference and alternative alleles at each SNP/Indel locus and comparing the total read depth and alternative allele proportion between tumor and matched normal sample; 2 performing joint segmentation on the two signal dimensions; 3 correcting the copy number baseline from which the SCNA state is determined; 4 calling SCNA state for each segment based on both signal dimensions. The method is applicable to whole exome/genome sequencing (WES/WGS as well as SNP array data in a tumor-control study. We applied the method to a dataset containing no SCNAs to test the specificity, created by pairing sequencing replicates of a single HapMap sample as normal/tumor pairs, as well as a large-scale WGS dataset consisting of 88 liver tumors along with adjacent normal tissues. Compared with representative methods, our method demonstrated improved accuracy, scalability to large cancer studies, capability in handling both sequencing and SNP array data, and the potential to improve the estimation of tumor ploidy and purity.

  20. SAAS-CNV: A Joint Segmentation Approach on Aggregated and Allele Specific Signals for the Identification of Somatic Copy Number Alterations with Next-Generation Sequencing Data.

    Science.gov (United States)

    Zhang, Zhongyang; Hao, Ke

    2015-11-01

    Cancer genomes exhibit profound somatic copy number alterations (SCNAs). Studying tumor SCNAs using massively parallel sequencing provides unprecedented resolution and meanwhile gives rise to new challenges in data analysis, complicated by tumor aneuploidy and heterogeneity as well as normal cell contamination. While the majority of read depth based methods utilize total sequencing depth alone for SCNA inference, the allele specific signals are undervalued. We proposed a joint segmentation and inference approach using both signals to meet some of the challenges. Our method consists of four major steps: 1) extracting read depth supporting reference and alternative alleles at each SNP/Indel locus and comparing the total read depth and alternative allele proportion between tumor and matched normal sample; 2) performing joint segmentation on the two signal dimensions; 3) correcting the copy number baseline from which the SCNA state is determined; 4) calling SCNA state for each segment based on both signal dimensions. The method is applicable to whole exome/genome sequencing (WES/WGS) as well as SNP array data in a tumor-control study. We applied the method to a dataset containing no SCNAs to test the specificity, created by pairing sequencing replicates of a single HapMap sample as normal/tumor pairs, as well as a large-scale WGS dataset consisting of 88 liver tumors along with adjacent normal tissues. Compared with representative methods, our method demonstrated improved accuracy, scalability to large cancer studies, capability in handling both sequencing and SNP array data, and the potential to improve the estimation of tumor ploidy and purity.

  1. Allele-specific analysis of cell fusion-mediated pluripotent reprograming reveals distinct and predictive susceptibilities of human X-linked genes to reactivation.

    Science.gov (United States)

    Cantone, Irene; Dharmalingam, Gopuraja; Chan, Yi-Wah; Kohler, Anne-Celine; Lenhard, Boris; Merkenschlager, Matthias; Fisher, Amanda G

    2017-01-25

    Inactivation of one X chromosome is established early in female mammalian development and can be reversed in vivo and in vitro when pluripotency factors are re-expressed. The extent of reactivation along the inactive X chromosome (Xi) and the determinants of locus susceptibility are, however, poorly understood. Here we use cell fusion-mediated pluripotent reprograming to study human Xi reactivation and allele-specific single nucleotide polymorphisms (SNPs) to identify reactivated loci. We show that a subset of human Xi genes is rapidly reactivated upon re-expression of the pluripotency network. These genes lie within the most evolutionary recent segments of the human X chromosome that are depleted of LINE1 and enriched for SINE elements, predicted to impair XIST spreading. Interestingly, this cadre of genes displays stochastic Xi expression in human fibroblasts ahead of reprograming. This stochastic variability is evident between clones, by RNA-sequencing, and at the single-cell level, by RNA-FISH, and is not attributable to differences in repressive histone H3K9me3 or H3K27me3 levels. Treatment with the DNA demethylating agent 5-deoxy-azacytidine does not increase Xi expression ahead of reprograming, but instead reveals a second cadre of genes that only become susceptible to reactivation upon induction of pluripotency. Collectively, these data not only underscore the multiple pathways that contribute to maintaining silencing along the human Xi chromosome but also suggest that transcriptional stochasticity among human cells could be useful for predicting and engineering epigenetic strategies to achieve locus-specific or domain-specific human Xi gene reactivation.

  2. Practical, real-time, full duplex wireless

    KAUST Repository

    Jain, Mayank

    2011-01-01

    This paper presents a full duplex radio design using signal inversion and adaptive cancellation. Signal inversion uses a simple design based on a balanced/unbalanced (Balun) transformer. This new design, unlike prior work, supports wideband and high power systems. In theory, this new design has no limitation on bandwidth or power. In practice, we find that the signal inversion technique alone can cancel at least 45dB across a 40MHz bandwidth. Further, combining signal inversion cancellation with cancellation in the digital domain can reduce self-interference by up to 73dB for a 10MHz OFDM signal. This paper also presents a full duplex medium access control (MAC) design and evaluates it using a testbed of 5 prototype full duplex nodes. Full duplex reduces packet losses due to hidden terminals by up to 88%. Full duplex also mitigates unfair channel allocation in AP-based networks, increasing fairness from 0.85 to 0.98 while improving downlink throughput by 110% and uplink throughput by 15%. These experimental results show that a re- design of the wireless network stack to exploit full duplex capability can result in significant improvements in network performance. © 2011 ACM.

  3. 非洲马瘟病毒VP7和NS2双重荧光RT-PCR检测技术的建立与应用%Development and Application of Duplex Real-time RT-PCR Assay for the Detection of VP7 and NS2 of African Horse Sickness Virus

    Institute of Scientific and Technical Information of China (English)

    高志强; 张鹤晓; 乔彩霞; 蒲静; 张伟; 谷强; 刘环; 张利峰; 马贵平

    2013-01-01

      Nucleic acid sequences of representative strains of different genotypes of African horse sickness viruses(AHSV)were aligned with the DNAMAN software. The two highly conservative NS2 and VP7 regions were then subjected to design primers and probes. The artificially synthesized nucleic acid fragments including amplification regions were used to prepare double strand RNA (dsRNA)by in vitro transcription in two directions(T7 and SP6). A duplex real-time TaqMan RT-PCR assay was developed to detect and quantify AHSV by optimization of reaction conditions using prepared dsRNA. The developed assay was used to detect a set of extracted pathogen RNA/DNA including those of AHSV,Eastern and Western equine encephalomyelitis virus(EEEV and WEEV), equine arteritis virus(EAV),equine influenza virus subtype H3N8(EIV H3N8),Salmonella abortus equi,Streptococcus equi subsp. zooepidimicus resulting in positive only for AHSV RNA,but negative for other virus nucleic acids,suggesting that the developed assay was specific and reliable. The detection limit of the real-time RT-PCR was 1.0×102copies per reaction,10 times more sensitive than that of the conventional RT–PCR based on gel electrophoresis. Also the double gene design could ensure the reliability, and efficiently reduce false negative results. By testing 248 clinical samples,it is confirmed that this assay was rapid,sensitive and repeatable,meeting the requirements for rapid diagnosis of AHSV.%  利用DNAMAN软件对非洲马瘟病毒不同基因型代表株的序列进行分析,选择其高度保守的VP7和NS2基因设计合成引物和探针。人工分别合成包含有扩增区域的VP7和NS2核苷酸片段进行双向(T7和SP6)体外转录制备双链RNA(dsRNA)。使用制备的dsRNA在对荧光定量RT-PCR的反应条件优化的基础上,建立了适用于非洲马瘟病毒检测的双重通用荧光定量RT-PCR检测技术。应用建立的方法对非洲马瘟病毒核酸,马流感病毒核酸

  4. Technical aspects of typing for HLA-DP alleles using allele-specific DNA in vitro amplification and sequence-specific oligonucleotide probes. Detection of single base mismatches

    DEFF Research Database (Denmark)

    Fugger, L; Morling, N; Ryder, L P

    1990-01-01

    The polymerase chain reaction (PCR) is an effective method for in vitro DNA amplification which combined with probing with synthetic oligonucleotides can be used for, e.g., HLA-typing. We have studied the technical aspects of HLA-DP typing with the technique. DNA from mononuclear nucleated cells ...... mismatches may be detected in the PCR and typing for HLA-DP gene variants, which differ for only one base, may be performed.......The polymerase chain reaction (PCR) is an effective method for in vitro DNA amplification which combined with probing with synthetic oligonucleotides can be used for, e.g., HLA-typing. We have studied the technical aspects of HLA-DP typing with the technique. DNA from mononuclear nucleated cells...... was extracted with either a simple salting out method or phenol/chloroform. Both DNAs could be readily used for PCR. The MgC2 concentration of the PCR buffer and the annealing temperature of the thermal cycle of the PCR were the two most important variables. The MgCl2 concentration and the temperature must...

  5. Thermodynamics of Oligonucleotide Duplex Melting

    Science.gov (United States)

    Schreiber-Gosche, Sherrie; Edwards, Robert A.

    2009-01-01

    Melting temperatures of oligonucleotides are useful for a number of molecular biology applications, such as the polymerase chain reaction (PCR). Although melting temperatures are often calculated with simplistic empirical equations, application of thermodynamics provides more accurate melting temperatures and an opportunity for students to apply…

  6. Full Duplex Communications in 5G Small Cells

    DEFF Research Database (Denmark)

    Mahmood, Nurul Huda; Gatnau, Marta; Berardinelli, Gilberto

    2017-01-01

    Full duplex communication promises system performance improvement over conventional half duplex communication by allowing simultaneous transmission and reception. However, such concurrent communication results in strong self interference and an increase in the overall network interference, and ca...

  7. Full Duplex Emulation via Spatial Separation of Half Duplex Nodes in a Planar Cellular Network

    DEFF Research Database (Denmark)

    Thomsen, Henning; Kim, Dong Min; Popovski, Petar;

    2016-01-01

    A Full Duplex Base Station (FD-BS) can be used to serve simultaneously two Half-Duplex (HD) Mobile Stations (MSs), one working in the uplink and one in the downlink, respectively. The same functionality can be realized by having two interconnected and spatially separated Half Duplex Base Stations...... (HD-BSs), which is a scheme termed CoMPflex (CoMP for In-Band Wireless Full Duplex). A FD-BS can be seen as a special case of CoMPflex with separation distance zero. In this paper we study the performance of CoMPflex in a two-dimensional cellular scenario using stochastic geometry and compare...... of communication reliability. Following the trend of wireless network densification, CoMPflex can be regarded as a method with a great potential to effectively use the dense HD deployments....

  8. Duplex ultrasound for identifying renal artery stenosis

    DEFF Research Database (Denmark)

    Zachrisson, Karin; Herlitz, Hans; Lönn, Lars

    2017-01-01

    Background Renal artery duplex ultrasound (RADUS) is an established method for diagnosis of renal artery stenosis (RAS), but there is no consensus regarding optimal RADUS criteria. Purpose To define optimal cutoff values for RADUS parameters when screening for RAS using intra-arterial trans...

  9. Ultra-short silicon MMI duplexer

    Science.gov (United States)

    Yi, Huaxiang; Huang, Yawen; Wang, Xingjun; Zhou, Zhiping

    2012-11-01

    The fiber-to-the-home (FTTH) systems are growing fast these days, where two different wavelengths are used for upstream and downstream traffic, typically 1310nm and 1490nm. The duplexers are the key elements to separate these wavelengths into different path in central offices (CO) and optical network unit (ONU) in passive optical network (PON). Multimode interference (MMI) has some benefits to be a duplexer including large fabrication tolerance, low-temperature dependence, and low-polarization dependence, but its size is too large to integrate in conventional case. Based on the silicon photonics platform, ultra-short silicon MMI duplexer was demonstrated to separate the 1310nm and 1490nm lights. By studying the theory of self-image phenomena in MMI, the first order images are adopted in order to keep the device short. A cascaded MMI structure was investigated to implement the wavelength splitting, where both the light of 1310nm and 1490nm was input from the same port, and the 1490nm light was coupling cross the first MMI and output at the cross-port in the device while the 1310nm light was coupling through the first and second MMI and output at the bar-port in the device. The experiment was carried on with the SOI wafer of 340nm top silicon. The cascaded MMI was investigated to fold the length of the duplexer as short as 117μm with the extinct ratio over 10dB.

  10. On the Secrecy Degrees of Freedom with Full Duplex Communication

    DEFF Research Database (Denmark)

    Mahmood, Nurul Huda; Mogensen, Preben Elgaard

    2017-01-01

    and the delay reduction of full duplex communication are somewhat limited in realistic network settings, leading researchers to study other possible applications of full duplex communication which can provide significantly higher gains over half duplex communication. Physical layer security is an example...... of such an application. The potential of full duplex nodes in improving the physical layer security of a communication link is investigated in this contribution. We specifically derive the information theoretic secrecy degrees of freedom measure for a pair of nodes communicating in full duplex mode. Moreover, closed...

  11. On the Ergodic Secrecy Capacity with Full Duplex Communication

    DEFF Research Database (Denmark)

    Mahmood, Nurul Huda; Shafique Ansari, Imran; Mogensen, Preben Elgaard

    2017-01-01

    Full duplex communication promises performance gains in terms of the throughput and the delay. Generally, it has been shown that the throughput and delay gains of full duplex communication are somewhat limited in realistic network settings, leading researchers to study other possible applications...... capacity for a pair of nodes with full duplex communication. The ergodic secrecy rate with full duplex communication is found to grow linearly with the log of the direct channel SNR as opposed to the flattened out secrecy rate with conventional half duplex communication, irrespective of the eavesdropper...

  12. Determination of allele frequency in pooled DNA: comparison of three PCR-based methods.

    Science.gov (United States)

    Wilkening, Stefan; Hemminki, Kari; Thirumaran, Ranjit Kumar; Bermejo, Justo Lorenzo; Bonn, Stefan; Försti, Asta; Kumar, Rajiv

    2005-12-01

    Determination of allele frequency in pooled DNA samples is a powerful and efficient tool for large-scale association studies. In this study, we tested and compared three PCR-based methods for accuracy, reproducibility, cost, and convenience. The methods compared were: (i) real-time PCR with allele-specific primers, (ii) real-time PCR with allele-specific TaqMan probes, and (iii) quantitative sequencing. Allele frequencies of three single nucleotide polymorphisms in three different genes were estimated from pooled DNA. The pools were made of genomic DNA samples from 96 cases with basal cell carcinoma of the skin and 96 healthy controls with known genotypes. In this study, the allele frequency estimation made by real-time PCR with allele-specific primers had the smallest median deviation (MD) from the real allele frequency with 1.12% (absolute percentage points) and was also the cheapest method. However; this method required the most time for optimization and showed the highest variation between replicates (SD = 6.47%). Quantitative sequencing, the simplest method, was found to have intermediate accuracies (MD = 1.44%, SD = 4.2%). Real-time PCR with TaqMan probes, a convenient but very expensive method, had an MD of 1.47% and the lowest variation between replicates (SD = 3.18%).

  13. Protection of aluminium by duplex coatings

    Energy Technology Data Exchange (ETDEWEB)

    Musil, J. [Ceska Akademie Ved, Prague (Czech Republic). Fyzikalni Ustav; Vlcek, J. [West Bohemia Univ., Plzen (Czech Republic). Dept. of Phys.; Jezek, V. [West Bohemia Univ., Plzen (Czech Republic). Dept. of Phys.; Benda, M. [West Bohemia Univ., Plzen (Czech Republic). Dept. of Phys.

    1995-11-01

    The paper reports on a new way of producing duplex coatings consisting of two steps. First, the substrate is coated by a physically vapour-deposited coating. Then, this precoated substrate is plasma nitrided or vacuum heat treated. This method was tested in the protection of substrates made of aluminium with a sputtered Ti coating about 5 {mu}m thick. The as-deposited and then plasma-nitrided or vacuum-heat-treated (Ti coating)/(Al substrate) couple was characterized by elemental depth profiles measured by glow discharge optical spectroscopy. It was shown that both the plasma nitriding and vacuum heat treatment process can stimulate a strong interdiffusion between Ti and the substrate elements. It results not only in the formation of a very broad interfacial region with a dramatic redistribution of the substrate elements in the Ti film but also in a formation of intermetallic Ti-Al compounds. This new duplex coating technique is described in detail. (orig.)

  14. Renal duplex Doppler ultrasound findings in diabetics

    Energy Technology Data Exchange (ETDEWEB)

    Shim, Hyang Yee; Kim, Young Geun; Kook, Cheol Keu; Yoon, Chong Hyun; Lee, Shin Hyung; Lee, Chang Joon [National Medical Center, Seoul (Korea, Republic of)

    1993-12-15

    The correlation between clinical-laboratory findings and renal duplex Doppler ultrasound findings was studied in 45 patients with diabetes mellitus to see the role of duplex Doppler ultrasound in the detection of diabetic nephropathy. The resistive indices in patients with elevated serum creatinine, BUN, proteinuria, and systolic blood pressure levels were statistically significantly higher than those in patients with normal levels (p<0.05). Also resistive indics in patients with retinopathy were higher than that in patients without retinopathy (p<0.05). But the ultrasound morphologic changes of kidney such as renal length, cortical eye-catching, and corticomedullarycontrast were not well correlated with clinical-laboratory data and resistive index. The resistive index of the kidney in conjunction with clinical-laboratory data in diabetics may be helpful in the evaluation of diabetic nephropathy

  15. Duplex Stainless Steels-An overview

    Directory of Open Access Journals (Sweden)

    Dr. Sunil D.Kahar

    2017-04-01

    Full Text Available Stainless steel is one of the most important materials in the engineering world. The material‟s wide applications in chemical, petrochemical, off-shore, and power generation plants prove that it is one of the most reliable materials. The Newest fast growing family of stainless steels is duplex alloys. The ferritic-austenitic grades have a ferrite matrix intermix with austenite and in other words island of austenite in a continuous matrix of highly alloyed ferrite commonly called „Duplex‟ stainless steel. Duplex stainless steel covers ferritic/austenitic Fe-Cr-Ni alloy with between 30% to 70 % Ferrite .Due to high level of Cr, Mo, and N steels shows high pitting & stress corrosion cracking resistance in chloride-containing environments. Hence it is frequently used in oilrefinery heat exchangers & typical applications where there is a risk for SCC and localized corrosion as a result of chloride-containing process streams, cooling waters or deposits. Modern duplex stainless steels have generally good Weldability. Due to a balanced composition, where nitrogen plays an important role, austenite formation in the heat affected zone (HAZ and weld metal is rapid. Under normal welding conditions a sufficient amount of austenite is formed to maintain good resistance to localized corrosion where as too rapid cooling may result in excessive amounts of ferrite, reducing the toughness. Therefore, welding with low heat input in thick walled materials should be avoided. Welding methods, such as resistance welding, laser welding and electron beam welding, which cause extremely rapid cooling should also be avoided or used with extreme caution. Too slow cooling can in the higher alloyed duplex grades cause formation of inter-metallic phases detrimental to corrosion resistance and toughness.

  16. Full duplex communication using visible light

    CERN Document Server

    Yang, Yongchao; Li, Yuanhang; Gao, Xumin; Yuan, Jialei; Zhu, Hongbo; Wang, Yongjin

    2016-01-01

    In this work, we propose, fabricate and characterize a full duplex communication system using visible light on a single chip. Both the suspended p-n junction InGaN/GaN multiple quantum well (MQW) devices and the suspended waveguides are obtained on a GaN-on-silicon platform by wafer-level processing. Two suspended p-n junction InGaN/GaN MQW devices that can both emit and detect light simultaneously are connected using suspended waveguides to form an in-plane visible light communication (VLC) system. The light that is emitted from one suspended p-n junction InGaN/GaN MQW device can induce a current in the device located at the other end of the waveguide via in-plane light coupling, thus leading to full duplex communication using visible light. This proof-of-concept in-plane VLC system paves the way towards the implementation of a full duplex communications system operating at the same frequency using visible light on a single chip.

  17. Joint duplex mode selection, channel allocation, and power control for full-duplex cognitive femtocell networks

    Directory of Open Access Journals (Sweden)

    Mingjie Feng

    2015-02-01

    Full Text Available In this paper, we aim to maximize the sum rate of a full-duplex cognitive femtocell network (FDCFN as well as guaranteeing the quality of service (QoS of users in the form of a required signal to interference plus noise ratios (SINR. We first consider the case of a pair of channels, and develop optimum-achieving power control solutions. Then, for the case of multiple channels, we formulate joint duplex model selection, power control, and channel allocation as a mixed integer nonlinear problem (MINLP, and propose an iterative framework to solve it. The proposed iterative framework consists of a duplex mode selection scheme, a near-optimal distributed power control algorithm, and a greedy channel allocation algorithm. We prove the convergence of the proposed iterative framework as well as a lower bound for the greedy channel allocation algorithm. Numerical results show that the proposed schemes effectively improve the sum rate of FDCFNs.

  18. Simplex and duplex event-specific analytical methods for functional biotech maize.

    Science.gov (United States)

    Lee, Seong-Hun; Kim, Su-Jeong; Yi, Bu-Young

    2009-08-26

    Analytical methods are very important in the control of genetically modified organism (GMO) labeling systems or living modified organism (LMO) management for biotech crops. Event-specific primers and probes were developed for qualitative and quantitative analysis for biotech maize event 3272 and LY 038 on the basis of the 3' flanking regions, respectively. The qualitative primers confirmed the specificity by a single PCR product and sensitivity to 0.05% as a limit of detection (LOD). Simplex and duplex quantitative methods were also developed using TaqMan real-time PCR. One synthetic plasmid was constructed from two taxon-specific DNA sequences of maize and two event-specific 3' flanking DNA sequences of event 3272 and LY 038 as reference molecules. In-house validation of the quantitative methods was performed using six levels of mixing samples, from 0.1 to 10.0%. As a result, the biases from the true value and the relative deviations were all within the range of +/-30%. Limits of quantitation (LOQs) of the quantitative methods were all 0.1% for simplex real-time PCRs of event 3272 and LY 038 and 0.5% for duplex real-time PCR of LY 038. This study reports that event-specific analytical methods were applicable for qualitative and quantitative analysis for biotech maize event 3272 and LY 038.

  19. q-Ising model on a duplex and a partially duplex clique

    CERN Document Server

    Chmiel, Anna; Sznajd-Weron, Katarzyna

    2016-01-01

    We analyze a modified kinetic Ising model, so called $q$- neighbor Ising model, with Metropolis dynamics,[Phys. Rev. E {\\bf92} 052105] on a duplex clique and a partially duplex clique. In the $q$-Ising model each spin interacts only with $q$ spins randomly chosen from the whole neighborhood. In the case of a duplex clique the change of a spin is allowed only if both levels simultaneously induce this change. Due to the mean-field like nature of the model we are able to derive the analytic form of transition probabilities and solve the corresponding master equation. The existence of the second level changes dramatically the character of the phase transition. In the case of the monoplex clique, the $q$-neighbor Ising model exhibits continuous phase transition for $q=3$, discontinuous phase transition for $q \\ge 4$ and for $q=1$ and $q=2$ the phase transition is not observed. On the other hand, in the case of the duplex clique continuous phase transitions are observed for all values of $q$, even for $q=1$ and $q=...

  20. Duplex tube steam reformer development program

    Energy Technology Data Exchange (ETDEWEB)

    Lewe, C K; Nieto, J M; Papadopoulos, A

    1978-09-01

    Work done in partial fulfillment of Task 7 of the Duplex Steam Reformer Development Program is described. The DSR concept acts as a double barrier between a process heat high temperature reactor plant (PNP) and a closed loop chemical heat pipe (CHP) for the long distance transport of chemical energy to a remote industrial user. The current state of the DSR design is described as well as related systems and equipment. The PNP concept presented is based upon work currently underway in the Federal Republic of Germany.

  1. Fundamentals of multiplexing with digital PCR.

    Science.gov (United States)

    Whale, Alexandra S; Huggett, Jim F; Tzonev, Svilen

    2016-12-01

    Over the past decade numerous publications have demonstrated how digital PCR (dPCR) enables precise and sensitive quantification of nucleic acids in a wide range of applications in both healthcare and environmental analysis. This has occurred in parallel with the advances in partitioning fluidics that enable a reaction to be subdivided into an increasing number of partitions. As the majority of dPCR systems are based on detection in two discrete optical channels, most research to date has focused on quantification of one or two targets within a single reaction. Here we describe 'higher order multiplexing' that is the unique ability of dPCR to precisely measure more than two targets in the same reaction. Using examples, we describe the different types of duplex and multiplex reactions that can be achieved. We also describe essential experimental considerations to ensure accurate quantification of multiple targets.

  2. RF Self-Interference cancellation for Full-Duplex

    NARCIS (Netherlands)

    van Liempd, B.; Debaillie, B.; Craninckx, J.; Lavin, C.; Palacios, C.; Malotaux, S.; Long, J.R.; van den Broek, Dirk-Jan; Klumperink, Eric A.M.

    2014-01-01

    This paper proposes two RF self-interference cancellation techniques. Their small form-factor enables full-duplex communication links for small-to-medium size portable devices and hence promotes the adoption of full-duplex in mass-market applications and next-generation standards, e.g. IEEE802.11 an

  3. Scintigraphic features of duplex kidneys on DMSA renal cortical scans.

    Science.gov (United States)

    Kwatra, Neha; Shalaby-Rana, Eglal; Majd, Massoud

    2013-09-01

    The spectrum of manifestations of duplex kidneys on (99m)Tc-dimercaptosuccinic acid (DMSA) renal cortical scans and correlating findings on other imaging modalities are presented. Relevant embryology of the duplex systems and technical aspects of DMSA scintigraphy are reviewed.

  4. On the Ergodic Secrecy Capacity with Full Duplex Communication

    DEFF Research Database (Denmark)

    Mahmood, Nurul Huda; Shafique Ansari, Imran; Mogensen, Preben Elgaard;

    2017-01-01

    Full duplex communication promises performance gains in terms of the throughput and the delay. Generally, it has been shown that the throughput and delay gains of full duplex communication are somewhat limited in realistic network settings, leading researchers to study other possible applications...

  5. Structural Properties of G,T-Parallel Duplexes

    Directory of Open Access Journals (Sweden)

    Anna Aviñó

    2010-01-01

    Full Text Available The structure of G,T-parallel-stranded duplexes of DNA carrying similar amounts of adenine and guanine residues is studied by means of molecular dynamics (MD simulations and UV- and CD spectroscopies. In addition the impact of the substitution of adenine by 8-aminoadenine and guanine by 8-aminoguanine is analyzed. The presence of 8-aminoadenine and 8-aminoguanine stabilizes the parallel duplex structure. Binding of these oligonucleotides to their target polypyrimidine sequences to form the corresponding G,T-parallel triplex was not observed. Instead, when unmodified parallel-stranded duplexes were mixed with their polypyrimidine target, an interstrand Watson-Crick duplex was formed. As predicted by theoretical calculations parallel-stranded duplexes carrying 8-aminopurines did not bind to their target. The preference for the parallel-duplex over the Watson-Crick antiparallel duplex is attributed to the strong stabilization of the parallel duplex produced by the 8-aminopurines. Theoretical studies show that the isomorphism of the triads is crucial for the stability of the parallel triplex.

  6. Spermine Condenses DNA, but Not RNA Duplexes

    Energy Technology Data Exchange (ETDEWEB)

    Katz, Andrea M.; Tolokh, Igor S.; Pabit, Suzette A.; Baker, Nathan; Onufriev, Alexey V.; Pollack, Lois

    2017-01-01

    Interactions between the polyamine spermine and nucleic acids drive important cellular processes. Spermine condenses DNA, and some RNAs such as poly(rA):poly(rU). A large fraction of the spermine present in cells is bound to RNA, but apparently does not condense it. Here, we study the effect of spermine binding to short duplex RNA and DNA and compare our findings with predictions of molecular dynamics simulations. When small numbers of spermine are introduced, RNA with a designed sequence, containing a mixture of 14 GC pairs and 11 AU pairs, resists condensation relative to DNA of an equivalent sequence or to 25 base pair poly(rA):poly(rU) RNA. Comparison of wide-angle x-ray scattering profiles with simulation suggests that spermine is sequestered deep within the major groove of mixed sequence RNA, preventing condensation by limiting opportunities to bridge to other molecules as well as stabilizing the RNA by locking it into a particular conformation. In contrast, for DNA, simulations suggest that spermine binds external to the duplex, offering opportunities for intermolecular interaction. The goal of this study is to explain how RNA can remain soluble, and available for interaction with other molecules in the cell, despite the presence of spermine at concentrations high enough to precipitate DNA.

  7. Duplex scanning using sparse data sequences

    DEFF Research Database (Denmark)

    Møllenbach, S. K.; Jensen, Jørgen Arendt

    2008-01-01

    The velocity distribution in vessels can be displayed using duplex scanning where B-mode acquisitions are interspaced with the velocity data. This gives an image for orientation, but lowers the maximum detectable velocity by a factor of two. Other pulse sequences either omits the B-mode image...... or leaves gaps in the velocity data, which makes it difficult to output audio data. The near full velocity range can be maintained and B-mode images shown by using a sparse data sequence with velocity and B-mode samples intermixed. The B-mode samples are placed in a (sparse) periodical pattern, which makes...... is scaled by the factor A/T. The approach has been investigated using in vivo RF data from the Hepatic vein, Carotid artery and Aorta from a 33 year old healthy male. A B-K Medical 3535 ultrasound scanner has been used in Duplex mode with a BK 8556, 3.2 MHz linear array probe. The sampling frequency...

  8. DNA Duplex Engineering for Enantioselective Fluorescent Sensor.

    Science.gov (United States)

    Hu, Yuehua; Lin, Fan; Wu, Tao; Zhou, Yufeng; Li, Qiusha; Shao, Yong; Xu, Zhiai

    2017-02-21

    The rapid identification of biomacromolecule structure that has a specific association with chiral enantiomers especially from natural sources will be helpful in developing enantioselective sensor and in speeding up drug exploitation. Herein, owing to its existence also in living cells, apurinic/apyrimidinic site (AP site) was first engineered into ds-DNA duplex to explore its competence in enantiomer selectivity. An AP site-specific fluorophore was utilized as an enantioselective discrimination probe to develop a straightforward chiral sensor using natural tetrahydropalmatine (L- and D-THP) as enantiomer representatives. We found that only L-THP can efficiently replace the prebound fluorophore to cause a significant fluorescence increase due to its specific binding with the AP site (two orders magnitude higher in affinity than binding with D-THP). The AP site binding specificity of L-THP over D-THP was assessed via intrinsic fluorescence, isothermal titration calorimetry, and DNA stability. The enantioselective performance can be easily tuned by the sequences near the AP site and the number of AP sites. A single AP site provides a perfect binding pocket to differentiate the chiral atom-induced structure discrepancy. We expect that our work will inspire interest in engineering local structures into a ds-DNA duplex for developing novel enantioselective sensors.

  9. Transgene detection by digital droplet PCR.

    Directory of Open Access Journals (Sweden)

    Dirk A Moser

    Full Text Available Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA included the term 'gene doping' in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR protocol for Insulin-Like Growth Factor 1 (IGF1 detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 33-day period. A duplex ddPCR protocol for simultaneous detection of Insulin-Like Growth Factor 1 (IGF1 and Erythropoietin (EPO transgenic elements was created. A new DNA extraction procedure with target-orientated usage of restriction enzymes including on-column DNA-digestion was established. In vivo data revealed that IGF1 transgenic elements could be reliably detected for a 33-day period in DNA extracted from whole blood. In vitro data indicated feasibility of IGF1 and EPO detection by duplex ddPCR with high reliability and sensitivity. On-column DNA-digestion allowed for significantly improved target detection in downstream PCR-based approaches. As ddPCR provides absolute quantification, it ensures excellent day-to-day reproducibility. Therefore, we expect this technique to be used in diagnosing and monitoring of viral and bacterial infection, in detecting mutated DNA sequences as well as profiling for the presence of foreign genetic material in elite athletes in the future.

  10. A novel duplex real-time reverse transcriptase-polymerase chain reaction assay for the detection of hepatitis C viral RNA with armored RNA as internal control

    Directory of Open Access Journals (Sweden)

    Meng Shuang

    2010-06-01

    Full Text Available Abstract Background The hepatitis C virus (HCV genome is extremely heterogeneous. Several HCV infections can not be detected using currently available commercial assays, probably because of mismatches between the template and primers/probes. By aligning the HCV sequences, we developed a duplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR assay using 2 sets of primers/probes and a specific armored RNA as internal control. The 2 detection probes were labelled with the same fluorophore, namely, 6-carboxyfluorescein (FAM, at the 5' end; these probes could mutually combine, improving the power of the test. Results The limit of detection of the duplex primer/probe assay was 38.99 IU/ml. The sensitivity of the assay improved significantly, while the specificity was not affected. All HCV genotypes in the HCV RNA Genotype Panel for Nucleic Acid Amplification Techniques could be detected. In the testing of 109 serum samples, the performance of the duplex real-time RT-PCR assay was identical to that of the COBAS AmpliPrep (CAP/COBAS TaqMan (CTM assay and superior to 2 commercial HCV assay kits. Conclusions The duplex real-time RT-PCR assay is an efficient and effective viral assay. It is comparable with the CAP/CTM assay with regard to the power of the test and is appropriate for blood-donor screening and laboratory diagnosis of HCV infection.

  11. Kinetics and thermodynamics of DNA, RNA, and hybrid duplex formation.

    Science.gov (United States)

    Rauzan, Brittany; McMichael, Elizabeth; Cave, Rachel; Sevcik, Lesley R; Ostrosky, Kara; Whitman, Elisabeth; Stegemann, Rachel; Sinclair, Audra L; Serra, Martin J; Deckert, Alice A

    2013-02-05

    The rates of duplex formation for two octamers of DNA (5' d-CACGGCTC/5' d-GAGCCGTG and 5' d-CACAGCAC/5' d-GTGCTGTG), the homologous RNA, and both sets of hybrids in 1 M NaCl buffer have been measured using stopped-flow spectroscopy. In addition, the thermodynamic parameters, ΔH° and ΔS°, have been determined for the same sequences under the same buffer conditions using optical melting techniques. These data reveal a linear free energy relationship between the free energy of activation for denaturation and the change in free energy for formation of the duplexes. This relationship indicates that these duplex formation reactions occur through a common unstructured transition state that is more similar to the single strands in solution than to the ensuing duplex. In addition, these data confirm that the greater stability of RNA duplexes relative to that of homologous DNA and hybrid duplexes is controlled by the denaturation rate and not the duplex formation rate.

  12. Buffer management in wireless full-duplex systems

    KAUST Repository

    Bouacida, Nader

    2015-10-19

    Wireless full-duplex radios can simultaneously transmit and receive using the same frequency. In theory, this can double the throughput. In fact, there is only little work addressing aspects other than throughput gains in full-duplex systems. Over-buffering in today\\'s networks or the so-called “bufferbloat” phenomenon creates excessive end-to-end delays resulting in network performance degradation. Our analysis shows that full-duplex systems may suffer from high latency caused by bloated buffers. In this paper, we address the problem of buffer management in full-duplex networks by using Wireless Queue Management (WQM), which is an active queue management technique for wireless networks. Our solution is based on Relay Full-Duplex MAC (RFD-MAC), an asynchronous media access control protocol designed for relay full-duplexing. We compare the performance of WQM in full-duplex environment to Drop Tail mechanism over various scenarios. Our solution reduces the end-to-end delay by two orders of magnitude while achieving similar throughput in most of the cases.

  13. Duplex 2209 Weld Overlay by ESSC Process

    Directory of Open Access Journals (Sweden)

    Er. Manoj Kumar

    2017-03-01

    Full Text Available In the modern world of industrialization the wear is eating metal assets worth millions of dollars per year. The wear is in the form of corrosion, erosion, abrasion etc. which occur in the process industries like oil & gas, refineries, cement plants, steel plants, shipping and offshore working structures. The equipments like pressure vessels, heat exchangers, hydro processing reactors which very often work at elevated temperatures face corrosion in the internal diameter. Duplex 2209 weld overlay on ferrous material is developed for high corrosion resistance properties and having high productivity by Electroslag strip cladding process due to its less dilution ~10% as compared to SMAW , GTAW or FCAW process. Because of Low Dilution ~10% undiluted chemistry can be achieved with single layer as compared to other weld overlay processes. The facility was developed inhouse to carry out weld overlay by ESSC and Testing

  14. MICROSCOPIC CORROSION STUDIES OF DUPLEX STAINLESS STEELS

    Institute of Scientific and Technical Information of China (English)

    C.Leygraf; J.Pan; M.Femenia

    2004-01-01

    Electrochemical scanning tunneling microscopy and scanning electrochemical microscopy have been used for in situ monitoring of localized corrosion processes of different Duplex stainless steels (DSS) in acidic chloride solutions. The techniques allow imaging of local dissolution events with micrometer resolution, as opposed to conventional electrochemical techniques, which only give an overall view of the corrosion behavior. In addition, combined scanning Kelvin probe force microscopy and magnetic force microscopy were used for mapping the Volta potential variation over the surface of DSSs. A significant difference in Volta potential between the austenite and ferrite phases suggests galvanic interaction between the phases. A compositional gradient appears within 2 micrometers across the phase boundary, as seen with scanning Auger microscopy (SAM). In all, the studies suggest that higher alloyed DSS exhibit a more homogeneous dissolution behavior than lower alloyed DSS, due to higher and more similar corrosion resistance of the two phases, and enhanced resistance of the ferrite/austenite phase boundary regions.

  15. Rietveld and impedance analysis of cold and hot rolled duplex and lean duplex steels for application in paper and pulp industry

    Energy Technology Data Exchange (ETDEWEB)

    Esteves, Luiza; Lins, Vanessa de Freitas Cunha, E-mail: luizaeq@yahoo.com.br [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Departamento de Engenharia Quimica; Paiva, Paulo Renato Perdigao [Centro Federal de Educacao Tecnologica de Minas Gerais (CEFET), Belo Horizonte, MG (Brazil); Viana, Adolfo Kalergis do Nascimento [APERAM South America, Timoteo, MG (Brazil)

    2017-01-15

    In this study, X-Ray Diffraction (XRD) and Rietveld Refinement were performed to identify and quantify the ferrite and austenite phase of cold and hot rolled duplex stainless steels (UNS S31803) and lean duplex stainless steels (UNS S32304). Electrochemical impedance spectroscopy (EIS) was applied to evaluate the chemical behavior of duplex and lean duplex stainless steels in white, green, and black liquors of paper and pulp industry. Rietveld analysis results showed a higher austenite content than the standard limit for duplex steels in the hot rolled condition. The hot rolling condition plays a major role in improving corrosion resistance in white liquor mainly for the lean duplex steel. (author)

  16. Duplex scanning on admission prevents unnecessary carotid endarterectomies.

    Science.gov (United States)

    Dalainas, I; Nano, G; Casana, R; Bianchi, P; Stegher, S; Malacrida, G; Tealdi, D G

    2006-06-01

    This retrospective single Institution study, aims to evaluate the performance of duplex scanning on admission of patients with carotid artery disease to avoid unnecessary carotid endarterectomies. From 1 January 1997 until 31 Decem-ber 2004, 1 504 patients were admitted to our Institution to undergo carotid endarterectomy. A duplex scan on admission was performed in all of them. A total of 1 369 from these patients (91%) underwent surgery, while 135 (9%) were dismissed because there was no indication for surgical treatment. They were put in conservative treatment and periodic duplex control appointments. In 9% of the patients, unnecessary carotid endarterectomy was avoided.

  17. Investigation of the Hot Plasticity of Duplex Stainless Steel

    Institute of Scientific and Technical Information of China (English)

    LIN Gang; ZHANG Zhi-xia; SONG Hong-wei; TONG Jun; ZHOU Can-dong

    2008-01-01

    Hot plasticity of a nitrogen alloyed 25Cr-7Ni-4 Mo duplex stainless steel was investigated.The results indicate that thc main factors affecting the hot plasticity of duplex stainless steel are listed as follows:coalescent force of phase interface,microstructure,and the phase ratio and difference between the mechanicsl propertms of ferrite and austenite.The heat treatment and sulphur contents have a notable effect on the hot plasticity.The reasonable heat treatrnents and the irlcreased interfacial coalescent force will effectively enhance the hot plasticity of duplex stainless steel.

  18. Analysing Self Interference Cancellation in Full Duplex Radios

    DEFF Research Database (Denmark)

    Mahmood, Nurul Huda; Shafique Ansari, Imran; Berardinelli, Gilberto;

    2016-01-01

    Full duplex communication promises a theoretical $100\\%$ throughput gain by doubling the number of simultaneous transmissions. Such compelling gains are conditioned on perfect cancellation of the self interference power resulting from simultaneous transmission and reception. Generally, self...... cancellation. In this study, we derive an analytical model for the residual self interference power, and demonstrate various applications of the derived model in analysing the performance of a Full Duplex radio. In general, full duplex communication is found to provide only modest throughput gains over half...

  19. Development of a new duplex real-time polymerase chain reaction assay for detection of dicer in G. gallus.

    Science.gov (United States)

    Ji, Xiaolin; Wang, Qi; Gao, Yulong; Wang, Yongqiang; Qin, Liting; Qi, Xiaole; Gao, Honglei; Wang, Xiaomei

    2013-05-01

    Recently, there has been a growing body of evidence showing that cellular microRNAs (miRNAs) are involved in virus-host interactions. Numerous studies have focused on analyses of the expression profiles of cellular miRNAs, but the expression patterns of Dicer, which is responsible for the generation of miRNAs, have only rarely been explored in Gallus gallus. We developed a duplex realtime reverse transcriptase polymerase chain reaction (RTPCR) assay for the relative quantification of the mRNAs of Dicer and beta-actin in G. gallus. To apply this method, the expression of Dicer in avian cells after infection with avian leukosis virus subgroup J (ALV-J) was detected using our established duplex real-time RT-PCR. The duplex realtime RT-PCR assay is sufficiently sensitive, specific, accurate, reproducible, and cost-effective for the detection of Dicer in G. gallus. Furthermore, this study, for the first time, demonstrated that ALV-J can induce differential expression of Dicer mRNA in the ALV-J-infected cells.

  20. Multi-primer target PCR for rapid identification of bovine DRB3 alleles.

    Science.gov (United States)

    Ledwidge, S A; Mallard, B A; Gibson, J P; Jansen, G B; Jiang, Z H

    2001-08-01

    Multi-primer target polymerase chain reaction (MPT-PCR) is a rapid method for the identification of specific BoLA-DRB3 alleles. In a single PCR reaction, the presence of two alleles associated with increased risk, DRB3.2*23 (DRB3*2701-2703, 2705-2707) and decreased risk, DRB3.2*16 (DRB3*1501, 1502), of mastitis in Canadian Holstein can be detected. Two outer primers amplify exon 2 of DRB3. Simultaneously, two inner, allele-specific primers amplify individual alleles. Initially, 40 cows previously typed by PCR-restriction fragment length polymorphism (PCR-RFLP) were genotyped using the multi-primer approach. An additional 30 cows were first genotyped by multi-primer target PCR, then by PCR-RFLP. All animals were correctly identified and there were no false positives. This technique can readily be modified to identify other BoLA alleles of interest.

  1. Establishment and application of the duplex TaqMan real-time RT-PCR with internal control for bovine viral diarrhea virus detection%牛病毒性腹泻病毒内标双重TaqMan荧光RT-PCR方法的建立及初步应用

    Institute of Scientific and Technical Information of China (English)

    季新成; 史茜; 郭春娟; 王科珂; 冉多良

    2014-01-01

    为建立牛病毒性腹泻病毒(BVDV)内标双重TaqMan荧光RT-PCR方法,本研究根据BVDV 5'-UTR区序列设计引物和荧光标记探针,通过重叠延伸PCR (SOE-PCR)扩增获得内标模板,经体外转录得到可以作为内标物的cRNA.经反应条件的优化,建立了BVDV内标双重TaqMan荧光RT-PCR检测体系.结果表明:当cRNA浓度为103拷贝/μL时,二者均有较好的扩增曲线;该方法对Ⅰ型BVDV的检测灵敏度为0.25 TCID50,对Ⅱ型BVDV的检测灵敏度为2.5 TCID50;利用该方法对猪瘟病毒等其他相关核酸的检测结果均为阴性;重复性检测Ct值变异系数为0.54~4.73.对506份临床样品检测,结果有23份样品为阳性,阳性样品检出数量和编号与不含内标的单一荧光RT-PCR方法完全一致,表明该方法可以用来对BVDV进行准确、快速检测,并且可以对检测结果进行质量监控.

  2. RNase H-dependent PCR (rhPCR: improved specificity and single nucleotide polymorphism detection using blocked cleavable primers

    Directory of Open Access Journals (Sweden)

    Powers Kristy M

    2011-08-01

    Full Text Available Abstract Background The polymerase chain reaction (PCR is commonly used to detect the presence of nucleic acid sequences both in research and diagnostic settings. While high specificity is often achieved, biological requirements sometimes necessitate that primers are placed in suboptimal locations which lead to problems with the formation of primer dimers and/or misamplification of homologous sequences. Results Pyrococcus abyssi (P.a. RNase H2 was used to enable PCR to be performed using blocked primers containing a single ribonucleotide residue which are activated via cleavage by the enzyme (rhPCR. Cleavage occurs 5'-to the RNA base following primer hybridization to the target DNA. The requirement of the primer to first hybridize with the target sequence to gain activity eliminates the formation of primer-dimers and greatly reduces misamplification of closely related sequences. Mismatches near the scissile linkage decrease the efficiency of cleavage by RNase H2, further increasing the specificity of the assay. When applied to the detection of single nucleotide polymorphisms (SNPs, rhPCR was found to be far more sensitive than standard allele-specific PCR. In general, the best discrimination occurs when the mismatch is placed at the RNA:DNA base pair. Conclusion rhPCR eliminates the formation of primer dimers and markedly improves the specificity of PCR with respect to off-target amplification. These advantages of the assay should find utility in challenging qPCR applications such as genotyping, high level multiplex assays and rare allele detection.

  3. Magnetic resonance urography in duplex kidney with ectopic ureteral insertion

    Science.gov (United States)

    Thambidorai, Conjeevaram Rajendrarao; Anuar, Zulfiqar

    2011-01-01

    This is a report on the use of magnetic resonance urography (MRU) in a 6-year-old girl who presented with urinary incontinence. She had a left duplex kidney with poorly functioning upper moiety and ectopic insertion of the dilated upper pole ureter. MRU has been shown to be superior to conventional imaging techniques in delineating poorly functioning moieties of duplex kidneys and ectopic ureters. PMID:21897576

  4. Magnetic resonance urography in duplex kidney with ectopic ureteral insertion

    Directory of Open Access Journals (Sweden)

    Conjeevaram Rajendrarao Thambidorai

    2011-01-01

    Full Text Available This is a report on the use of magnetic resonance urography (MRU in a 6-year-old girl who presented with urinary incontinence. She had a left duplex kidney with poorly functioning upper moiety and ectopic insertion of the dilated upper pole ureter. MRU has been shown to be superior to conventional imaging techniques in delineating poorly functioning moieties of duplex kidneys and ectopic ureters.

  5. Properties of duplex stainless steels made by powder metallurgy

    OpenAIRE

    Rosso, M.; M. Actis Grande; Z. Brytan; L.A. Dobrzański

    2007-01-01

    Purpose: of this paper was to examine the mechanical properties of duplex stainless steels.Design/methodology/approach: In presented study duplex stainless steels were obtained through powder metallurgy starting from austenitic, martensitic base powders by controlled addition of alloying elements, such as Cr, Ni, Mo and Cu. In the studies behind the preparation of mixes, Schaeffler’s diagram was taken into consideration. Prepared mixes have been sintered in a vacuu...

  6. Phase Transformations in Cast Duplex Stainless Steels

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Yoon-Jun [Iowa State Univ., Ames, IA (United States)

    2004-01-01

    Duplex stainless steels (DSS) constitute both ferrite and austenite as a matrix. Such a microstructure confers a high corrosion resistance with favorable mechanical properties. However, intermetallic phases such as σ and χ can also form during casting or high-temperature processing and can degrade the properties of the DSS. This research was initiated to develop time-temperature-transformation (TTT) and continuous-cooling-transformation (CCT) diagrams of two types of cast duplex stainless steels, CD3MN (Fe-22Cr-5Ni-Mo-N) and CD3MWCuN (Fe-25Cr-7Ni-Mo-W-Cu-N), in order to understand the time and temperature ranges for intermetallic phase formation. The alloys were heat treated isothermally or under controlled cooling conditions and then characterized using conventional metallographic methods that included tint etching, and also using electron microscopy (SEM, TEM) and wavelength dispersive spectroscopy (WDS). The kinetics of intermetallic-phase (σ + χ) formation were analyzed using the Johnson-Mehl-Avrami (MA) equation in the case of isothermal transformations and a modified form of this equation in the case of continuous cooling transformations. The rate of intermetallic-phase formation was found to be much faster in CD3MWCuN than CD3MN due mainly to differences in the major alloying contents such as Cr, Ni and Mo. To examine in more detail the effects of these elements of the phase stabilities; a series of eight steel castings was designed with the Cr, Ni and Mo contents systematically varied with respect to the nominal composition of CD3MN. The effects of varying the contents of alloying additions on the formation of intermetallic phases were also studied computationally using the commercial thermodynamic software package, Thermo-Calc. In general, σ was stabilized with increasing Cr addition and χ by increasing Mo addition. However, a delicate balance among Ni and other minor elements such as N and Si also exists. Phase equilibria in DSS can be affected by

  7. Phase Transformations in Cast Duplex Stainless Steels

    Energy Technology Data Exchange (ETDEWEB)

    Yoon-Jun Kim

    2004-12-19

    Duplex stainless steels (DSS) constitute both ferrite and austenite as a matrix. Such a microstructure confers a high corrosion resistance with favorable mechanical properties. However, intermetallic phases such as {sigma} and {chi} can also form during casting or high-temperature processing and can degrade the properties of the DSS. This research was initiated to develop time-temperature-transformation (TTT) and continuous-cooling-transformation (CCT) diagrams of two types of cast duplex stainless steels, CD3MN (Fe-22Cr-5Ni-Mo-N) and CD3MWCuN (Fe-25Cr-7Ni-Mo-W-Cu-N), in order to understand the time and temperature ranges for intermetallic phase formation. The alloys were heat treated isothermally or under controlled cooling conditions and then characterized using conventional metallographic methods that included tint etching, and also using electron microscopy (SEM, TEM) and wavelength dispersive spectroscopy (WDS). The kinetics of intermetallic-phase ({sigma} + {chi}) formation were analyzed using the Johnson-Mehl-Avrami (MA) equation in the case of isothermal transformations and a modified form of this equation in the case of continuous cooling transformations. The rate of intermetallic-phase formation was found to be much faster in CD3MWCuN than CD3MN due mainly to differences in the major alloying contents such as Cr, Ni and Mo. To examine in more detail the effects of these elements of the phase stabilities; a series of eight steel castings was designed with the Cr, Ni and Mo contents systematically varied with respect to the nominal composition of CD3MN. The effects of varying the contents of alloying additions on the formation of intermetallic phases were also studied computationally using the commercial thermodynamic software package, Thermo-Calc. In general, {sigma} was stabilized with increasing Cr addition and {chi} by increasing Mo addition. However, a delicate balance among Ni and other minor elements such as N and Si also exists. Phase equilibria in

  8. Real-time PCR assays for hepatitis B virus DNA quantification may require two different targets.

    Science.gov (United States)

    Liu, Chao; Chang, Le; Jia, Tingting; Guo, Fei; Zhang, Lu; Ji, Huimin; Zhao, Junpeng; Wang, Lunan

    2017-05-12

    Quantification Hepatitis B virus (HBV) DNA plays a critical role in the management of chronic HBV infections. However, HBV is a DNA virus with high levels of genetic variation, and drug-resistant mutations have emerged with the use of antiviral drugs. If a mutation caused a sequence mismatched in the primer or probe of a commercial DNA quantification kit, this would lead to an underestimation of the viral load of the sample. The aim of this study was to determine whether commercial kits, which use only one pair of primers and a single probe, accurately quantify the HBV DNA levels and to develop an improved duplex real-time PCR assay. We developed a new duplex real-time PCR assay that used two pairs of primers and two probes based on the conserved S and C regions of the HBV genome. We performed HBV DNA quantitative detection of HBV samples and compared the results of our duplex real-time PCR assays with the COBAS TaqMan HBV Test version 2 and Daan real-time PCR assays. The target region of the discordant sample was amplified, sequenced, and validated using plasmid. The results of the duplex real-time PCR were in good accordance with the commercial COBAS TaqMan HBV Test version 2 and Daan real-time PCR assays. We showed that two samples from Chinese HBV infections underestimated viral loads when quantified by the Roche kit because of a mismatch between the viral sequence and the reverse primer of the Roche kit. The HBV DNA levels of six samples were undervalued by duplex real-time PCR assays of the C region because of mutations in the primer of C region. We developed a new duplex real-time PCR assay, and the results of this assay were similar to the results of commercial kits. The HBV DNA level could be undervalued when using the COBAS TaqMan HBV Test version 2 for Chinese HBV infections owing to a mismatch with the primer/probe. A duplex real-time PCR assay based on the S and C regions could solve this problem to some extent.

  9. Wavelength-tunable duplex integrated light source

    Science.gov (United States)

    Okamoto, Hiroshi; Yasaka, Hiroshi; Oe, Kunishige

    1996-04-01

    A monolithically integrated opto-electronic device is proposed as a fast wavelength-switching light source. This tunable duplex integrated light source comprises two wavelength-tunable distributed Bragg reflector (DBR) laser diodes (LDs), two MQW-electro-absorption optical switches, a Y-shaped waveguide coupler, a MQW-electro-absorption modulator, and two thermal drift compensators (TDCs). The wavelength-switching time of the optical switches was estimated to be 60 ps including a 50-ps rise time for the electrical-pulse generator. The wavelength of a 10-Gbit/s NRZ-modulated optical signal can be switched without bit loss. The function of the TDCs is to keep the device-chip temperature constant. Thermal-transient- induced wavelength drift with a millisecond-order time constant, which has been reported for DBR-LDs, and thermal crosstalk between the tuning regions of the integrated LDs, which causes wavelength fluctuation, are effectively suppressed by thermal-drift-compensation operation using the TDCs.

  10. Ferrite Quantification Methodologies for Duplex Stainless Steel

    Directory of Open Access Journals (Sweden)

    Arnaldo Forgas Júnior

    2016-07-01

    Full Text Available In order to quantify ferrite content, three techniques, XRD, ferritoscope and optical metallography, were applied to a duplex stainless steel UNS S31803 solution-treated for 30 min at 1,000, 1,100 and 1,200 °C, and then compared to equilibrium of phases predicted by ThermoCalc® simulation. As expected, the microstructure is composed only by austenite and ferrite phases, and ferrite content increases as the solution treatment temperature increases. The microstructure presents preferred grains orientation along the rolling directions even for a sample solution treated for 30 min at 1,200 °C. For all solution treatment temperatures, the ferrite volume fractions obtained by XRD measurements were higher than those achieved by the other two techniques and ThermoCalc® simulation, probably due to texturing effect of previous rolling process. Values obtained by quantitative metallography look more assertive as it is a direct measurement method but the ferritoscope technique should be considered mainly for in loco measurement.

  11. Corrosion behaviour of sintered duplex stainless steels

    Energy Technology Data Exchange (ETDEWEB)

    Utrilla, M. Victoria; Urena, Alejandro; Otero, Enrique; Munez, Claudio Jose [Escuela Superior de Ciencias Experimentales y Tecnologia, Universidad Rey Juan Carlos, C/ Tulipan s/n, 28933 Mostoles, Madrid (Spain)

    2004-07-01

    Duplex austenite-ferrite stainless steels were prepared by mixing austenitic (316L) and ferritic (434L) atomized powders. Although different 316L/434L ratios were prepared, present work centred its study on 50% ferrite - 50% austenite sintered steel. The powders were mixed and pressed at 700 MPa and sintered at 1250 deg. C for 30 min in vacuum. The cooling rate was 5 deg. C/min. Solution treatment was carried out to homogenize the microstructure at 1100 deg. C during 20 min. A microstructural study of the material in solution was performed, evaluating the microstructure, proportion and shape of porosity, and ferrite percentage. This last was measured by two methods, quantitative metallography and Fischer ferrito-metry. The materials were heat treated in the range of 700 to 1000 deg. C, for 10, 30 and 60 min and water quenched, to study the microstructural changes and the influence on the intergranular corrosion resistance. The method used to evaluate the sensitization to the intergranular corrosion was the electrochemical potentio-kinetic reactivation procedure (EPR). The test solution was 0.5 M H{sub 2}SO{sub 4} + 0,01 M KSCN at 30 deg. C. The criterion used to evaluate the sensitization was the ratio between the maximum reactivation density (Ir) and the maximum activation density (Ia). The results of the electrochemical tests were discussed in relation with the microstructures observed at the different heat treatments. (authors)

  12. Droplet digital PCR for simultaneous quantification of general and human-associated fecal indicators for water quality assessment.

    Science.gov (United States)

    Cao, Yiping; Raith, Meredith R; Griffith, John F

    2015-03-01

    Despite wide application to beach water monitoring and microbial source identification, results produced by quantitative PCR (qPCR) methods are subject to bias introduced by reliance on quantitative standards. Digital PCR technology provides direct, standards-free quantification and may potentially alleviate or greatly reduce other qPCR limitations such as difficulty in multiplexing and susceptibility to PCR inhibition. This study examined the efficacy of employing a duplex droplet digital PCR (ddPCR) assay that simultaneously quantifies Enterococcus spp. and the human fecal-associated HF183 marker for water quality assessment. Duplex ddPCR performance was evaluated side-by-side with qPCR and simplex ddPCR using reference material and 131 fecal and water samples. Results for fecal and water samples were highly correlated between ddPCR and simplex qPCR (coefficients > 0.93, p competition and resulted in non-detection or underestimation of the target with low concentration relative to the other, while results produced by simplex and duplex ddPCR were consistent and often indistinguishable from one another. ddPCR showed greater tolerance for inhibition, with no discernable effect on quantification at inhibitor concentrations one to two orders of magnitude higher than that tolerated by qPCR. Overall, ddPCR also exhibited improved precision, higher run-to-run repeatability, similar diagnostic sensitivity and specificity on the HF183 marker, but a lower upper limit of quantification than qPCR. Digital PCR has the potential to become a reliable and economical alternative to qPCR for recreational water monitoring and fecal source identification. Findings from this study may also be of interest to other aspects of water research such as detection of pathogens and antibiotic resistance genes.

  13. PCR thermocycler

    Science.gov (United States)

    Benett, William J.; Richards, James B.

    2003-01-01

    A sleeve-type silicon polymerase chain reaction (PCR) chamber or thermocycler having improved thermal performance. The silicon sleeve reaction chamber is improved in thermal performance by etched features therein that reduce thermal mass and increase the surface area of the sleeve for cooling. This improved thermal performance of the thermocycler enables an increase in speed and efficiency of the reaction chamber. The improvement is accomplished by providing grooves in the faces of the sleeve and a series of grooves on the interior surfaces that connect with grooves on the faces of the sleeve. The grooves can be anisotropically etched in the silicon sleeve simultaneously with formation of the chamber.

  14. High frequency of SLC22A12 variants causing renal hypouricemia 1 in the Czech and Slovak Roma population; simple and rapid detection method by allele-specific polymerase chain reaction.

    Science.gov (United States)

    Gabrikova, Dana; Bernasovska, Jarmila; Sokolova, Jitka; Stiburkova, Blanka

    2015-10-01

    Renal hypouricemia is a rare heterogeneous inherited disorder characterized by impaired tubular uric acid transport with severe complications, such as acute kidney injury. Type 1 and 2 are caused by loss-of-function mutations in the SLC22A12 and SLC2A9 gene, respectively. A cohort of 881 randomly chosen ethnic Roma from two regions in Eastern Slovakia and two regions in the Czech Republic participated. Genomic DNA was isolated from buccal swabs and/or from blood samples. The c.1245_1253del and c.1400C>T genotypes were determined using polymerase chain reaction with allele-specific primers in a multiplex arrangement and/or direct sequencing of exon 7 and 9. Allele frequencies and genotypes were tested for Hardy-Weinberg equilibrium using the Chi-square test. 25 subjects were heterozygous and three were homozygous for the c.1245_1253del, while 92 subjects were heterozygous and two were homozygous for the c.1400C>T. Moreover, two participants were compound heterozygotes. Frequencies of the c.1245_1253del and c.1400C>T variants were 1.87 and 5.56 %, respectively. Our finding confirms an uneven geographical and ethnic distribution of SLC22A12 mutant variants. We found that the c.1245_1253del and c.1400C>T variants were present in the Czech and Slovak Roma population at unexpectedly high frequencies. Renal hypouricemia should be kept in mind during differential diagnostic on Roma patients with low serum uric acid concentrations.

  15. Defined presentation of carbohydrates on a duplex DNA scaffold.

    Science.gov (United States)

    Schlegel, Mark K; Hütter, Julia; Eriksson, Magdalena; Lepenies, Bernd; Seeberger, Peter H

    2011-12-16

    A new method for the spatially defined alignment of carbohydrates on a duplex DNA scaffold is presented. The use of an N-hydroxysuccinimide (NHS)-ester phosphoramidite along with carbohydrates containing an alkylamine linker allows for on-column labeling during solid-phase oligonucleotide synthesis. This modification method during solid-phase synthesis only requires the use of minimal amounts of complex carbohydrates. The covalently attached carbohydrates are presented in the major groove of the B-form duplex DNA as potential substrates for murine type II C-type lectin receptors mMGL1 and mMGL2. CD spectroscopy and thermal melting revealed only minimal disturbance of the overall helical structure. Surface plasmon resonance and cellular uptake studies with bone-marrow-derived dendritic cells were used to assess the capability of these carbohydrate-modified duplexes to bind to mMGL receptors.

  16. Sarcomatoid carcinoma of the renal pelvis in duplex kidney

    Institute of Scientific and Technical Information of China (English)

    CHEN Ge-ming; CHEN Shan-wen; XIA Dan; LI Jun; YAN Sheng; JIN Bai-ye

    2011-01-01

    Sarcomatoid transitional cell carcinoma of the renal pelvis is a rare neoplasm with only 14 well-illustrated examples reported previously. Duplex kidney is the most common congenital abnormality of the urinary tract, with an incidence of around 2%. Neoplasia of the renal pelvis in duplex kidney is rare. We reported a case whose sarcomatoid carcinoma originated from the upper portion of the duplicated renal pelvis with hydronephrosis, and total nephroureterectomy with bladder cuff excision surgery of both renal units was carried out. Because of the rare nature of renal pelvic sarcomatoid carcinoma and its apparent lack of response to adjuvant therapy, it is essential to do early diagnosis and early radical surgery to improve survival. It is important to stress the need for frequent and diligent monitoring or treating complex duplex kidney with hydronephrosis of either moiety in case of a risk of having neoplasias.

  17. KRAS mutation analysis by PCR: a comparison of two methods.

    Directory of Open Access Journals (Sweden)

    Louise Bolton

    Full Text Available KRAS mutation assays are important companion diagnostic tests to guide anti-EGFR antibody treatment of metastatic colorectal cancer. Direct comparison of newer diagnostic methods with existing methods is an important part of validation of any new technique. In this this study, we have compared the Therascreen (Qiagen ARMS assay with Competitive Allele-Specific TaqMan PCR (castPCR, Life Technologies to determine equivalence for KRAS mutation analysis.DNA was extracted by Maxwell (Promega from 99 colorectal cancers. The ARMS-based Therascreen and a customized castPCR assay were performed according to the manufacturer's instructions. All assays were performed on either an Applied Biosystems 7500 Fast Dx or a ViiA7 real-time PCR machine (both from Life Technologies. The data were collected and discrepant results re-tested with newly extracted DNA from the same blocks in both assay types.Of the 99 tumors included, Therascreen showed 62 tumors to be wild-type (WT for KRAS, while 37 had KRAS mutations on initial testing. CastPCR showed 61 tumors to be wild-type (WT for KRAS, while 38 had KRAS mutations. Thirteen tumors showed BRAF mutation in castPCR and in one of these there was also a KRAS mutation. The custom castPCR plate included several other KRAS mutations and BRAF V600E, not included in Therascreen, explaining the higher number of mutations detected by castPCR. Re-testing of discrepant results was required in three tumors, all of which then achieved concordance for KRAS. CastPCR assay Ct values were on average 2 cycles lower than Therascreen.There was excellent correlation between the two methods. Although castPCR assay shows lower Ct values than Therascreen, this is unlikely to be clinically significant.

  18. Comparison of simulated and measured quantities of a duplex reactor

    Energy Technology Data Exchange (ETDEWEB)

    Koskela, M.; Kajava, M. [ABB Marine, Helsinki (Finland)

    1997-12-31

    The purpose of this article is to illustrate the use of an analog simulator as a design tool when designing new power electric equipment. The purpose of simulation is to predict the functionality of electrical equipment to be constructed. Duplex reactor is an electromagnetic device designed to reduce voltage harmonics and short circuit currents in the ship electrical network. In this report a comparison between simulated and measured electrical quantities of a duplex reactor has been made. The purpose of the measurements was to show the correct functioning of the reactor. The simulation results and the measured waveforms corresponds well to each other. (orig.) 4 refs.

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

    Science.gov (United States)

    O'Halloran, Damien M

    2016-02-08

    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.

  20. A semi-nested real-time PCR method to detect low chimerism percentage in small quantity of hematopoietic stem cell transplant DNA samples.

    Science.gov (United States)

    Aloisio, Michelangelo; Bortot, Barbara; Gandin, Ilaria; Severini, Giovanni Maria; Athanasakis, Emmanouil

    2017-02-01

    Chimerism status evaluation of post-allogeneic hematopoietic stem cell transplantation samples is essential to predict post-transplant relapse. The most commonly used technique capable of detecting small increments of chimerism is quantitative real-time PCR. Although this method is already used in several laboratories, previously described protocols often lack sensitivity and the amount of the DNA required for each chimerism analysis is too high. In the present study, we compared a novel semi-nested allele-specific real-time PCR (sNAS-qPCR) protocol with our in-house standard allele-specific real-time PCR (gAS-qPCR) protocol. We selected two genetic markers and analyzed technical parameters (slope, y-intercept, R2, and standard deviation) useful to determine the performances of the two protocols. The sNAS-qPCR protocol showed better sensitivity and precision. Moreover, the sNAS-qPCR protocol requires, as input, only 10 ng of DNA, which is at least 10-fold less than the gAS-qPCR protocols described in the literature. Finally, the proposed sNAS-qPCR protocol could prove very useful for performing chimerism analysis with a small amount of DNA, as in the case of blood cell subsets.

  1. Breaking the Transmitter-Receiver Isolation Barrier in Mobile Handsets with Spatial Duplexing

    DEFF Research Database (Denmark)

    Alrabadi, Osama; Tatomirescu, Alexandru; Knudsen, Mikael;

    2013-01-01

    In full-duplex radio communication systems like eUTRAN, CDMA-2000, the radio transmitter (Tx) is active at the same time as the radio receiver (Rx). The Tx and the Rx will be using separate dedicated frequency bands and the Tx-Rx isolation is ensured by duplex filters. However, agile duplexers req...

  2. On the Potential of Full Duplex Performance in 5G Ultra-Dense Small Cell Networks

    DEFF Research Database (Denmark)

    Gatnau, Marta; Fleischer, Marko; Berardinelli, Gilberto;

    2016-01-01

    Full duplex allows a device to transmit and receive simultaneously in the same frequency band, theoretically doubling the throughput compared to traditional half duplex systems. However, several limitations restrict the promised full duplex gain: non-ideal self-interference cancellation, increase...

  3. Development of a genome-wide multiple duplex-SSR protocol and its applications for the identification of selfed progeny in switchgrass

    Directory of Open Access Journals (Sweden)

    Liu Linglong

    2012-10-01

    Full Text Available Abstract Background Switchgrass (Panicum virgatum is a herbaceous crop for the cellulosic biofuel feedstock development in the USA and Europe. As switchgrass is a naturally outcrossing species, accurate identification of selfed progeny is important to producing inbreds, which can be used in the production of heterotic hybrids. Development of a technically reliable, time-saving and easily used marker system is needed to quantify and characterize breeding origin of progeny plants of targeted parents. Results Genome-wide screening of 915 mapped microsatellite (simple sequence repeat, SSR markers was conducted, and 842 (92.0% produced clear and scorable bands on a pooled DNA sample of eight switchgrass varieties. A total of 166 primer pairs were selected on the basis of their relatively even distribution in switchgrass genome and PCR amplification quality on 16 tetraploid genotypes. Mean polymorphic information content value for the 166 markers was 0.810 ranging from 0.116 to 0.959. From them, a core set of 48 loci, which had been mapped on 17 linkage groups, was further tested and optimized to develop 24 sets of duplex markers. Most of (up to 87.5% targeted, but non-allelic amplicons within each duplex were separated by more than 10-bp. Using the established duplex PCR protocol, selfing ratio (i.e., selfed/all progeny x100% was identified as 0% for a randomly selected open-pollinated ‘Kanlow’ genotype grown in the field, 15.4% for 22 field-grown plants of bagged inflorescences, and 77.3% for a selected plant grown in a growth chamber. Conclusions The study developed a duplex SSR-based PCR protocol consisting of 48 markers, providing ample choices of non-tightly-linked loci in switchgrass whole genome, and representing a powerful, time-saving and easily used method for the identification of selfed progeny in switchgrass. The protocol should be a valuable tool in switchgrass breeding efforts.

  4. Effect of Radiofrequency Radiation on DNA Duplex Stability and Replication.

    Science.gov (United States)

    1983-08-01

    R.W., J.B. Dodgson, I.F. Nes, and R.D. Wells. Duplex regions in single-stranded OX174 DNA are cleaved by a restriction endonuclease from Haemophilus ... aegyptius . J Biol Chem 252:7300-7306 (1977). *i Brown, R.F., S.V. Marshall, and C.W. Hughes. Effect of radiofrequency radi- ation (RFR) on excision

  5. Analysis of full-duplex relay networks with opportunistic scheduling

    Directory of Open Access Journals (Sweden)

    Lei Shao

    2015-05-01

    Full Text Available This Letter addresses a two-hop decode-and-forward relay system with full-duplex relaying and opportunistic scheduling. Exact expressions for outage probability, average capacity and symbol error rate are presented in an independent identically distributed Rayleigh fading environment. Numerical and simulated results show the validity of the analytical results.

  6. Reliability Capacity of Half-Duplex Channels with Strict Deadlines

    DEFF Research Database (Denmark)

    Costa, Rui; Roetter, Daniel Enrique Lucani; Vinhoza, Tiago

    2015-01-01

    A fundamental characterization of a half-duplex wireless system with packet losses under traffic with hard deadlines is instrumental to understanding and developing efficient, coding aware policies for real-time applications. We set forth the concept of reliability capacity with a limited number...

  7. Full-duplex wireless communications systems self-interference cancellation

    CERN Document Server

    Le-Ngoc, Tho

    2017-01-01

    This book introduces the development of self-interference (SI)-cancellation techniques for full-duplex wireless communication systems. The authors rely on estimation theory and signal processing to develop SI-cancellation algorithms by generating an estimate of the received SI and subtracting it from the received signal. The authors also cover two new SI-cancellation methods using the new concept of active signal injection (ASI) for full-duplex MIMO-OFDM systems. The ASI approach adds an appropriate cancelling signal to each transmitted signal such that the combined signals from transmit antennas attenuate the SI at the receive antennas. The authors illustrate that the SI-pre-cancelling signal does not affect the data-bearing signal. This book is for researchers and professionals working in wireless communications and engineers willing to understand the challenges of deploying full-duplex and practical solutions to implement a full-duplex system. Advanced-level students in electrical engineering and computer ...

  8. Capacity of the Degraded Half-Duplex Relay Channel

    CERN Document Server

    Vijayakumaran, Saravanan; Lok, Tat M

    2007-01-01

    A discrete memoryless half-duplex relay channel is constructed from a broadcast channel from the source to the relay and destination and a multiple access channel from the source and relay to the destination. When the relay listens, the channel operates in the broadcast mode. The channel switches to the multiple access mode when the relay transmits. If the broadcast component channel is physically degraded, the half-duplex relay channel will also be referred to as physically degraded. The capacity of this degraded half-duplex relay channel is examined. It is shown that the block Markov coding suggested in the seminal paper by Cover and El Gamal can be modified to achieve capacity for the degraded half-duplex relay channel. In the code construction, the listen-transmit schedule of the relay is made to depend on the message to be sent and hence the schedule carries information itself. If the schedule is restricted to be deterministic, it is shown that the capacity can be achieved by a simple management of infor...

  9. Full-duplex MIMO system based on antenna cancellation technique

    DEFF Research Database (Denmark)

    Foroozanfard, Ehsan; Franek, Ondrej; Tatomirescu, Alexandru

    2014-01-01

    The performance of an antenna cancellation technique for a multiple-input– multiple-output (MIMO) full-duplex system that is based on null-steering beamforming and antenna polarization diversity is investigated. A practical implementation of a symmetric antenna topology comprising three dual-pola...

  10. Micromechanics of base pair unzipping in the DNA duplex.

    Science.gov (United States)

    Volkov, Sergey N; Paramonova, Ekaterina V; Yakubovich, Alexander V; Solov'yov, Andrey V

    2012-01-25

    All-atom molecular dynamics (MD) simulations of DNA duplex unzipping in a water environment were performed. The investigated DNA double helix consists of a Drew-Dickerson dodecamer sequence and a hairpin (AAG) attached to the end of the double-helix chain. The considered system is used to examine the process of DNA strand separation under the action of an external force. This process occurs in vivo and now is being intensively investigated in experiments with single molecules. The DNA dodecamer duplex is consequently unzipped pair by pair by means of the steered MD. The unzipping trajectories turn out to be similar for the duplex parts with G·C content and rather distinct for the parts with A·T content. It is shown that during the unzipping each pair experiences two types of motion: relatively quick rotation together with all the duplex and slower motion in the frame of the unzipping fork. In the course of opening, the complementary pair passes through several distinct states: (i) the closed state in the double helix, (ii) the metastable preopened state in the unzipping fork and (iii) the unbound state. The performed simulations show that water molecules participate in the stabilization of the metastable states of the preopened base pairs in the DNA unzipping fork.

  11. Increased Y-chromosome detection by SRY duplexing

    DEFF Research Database (Denmark)

    Hansen, Morten Høgh; Clausen, Frederik Banch; Dziegiel, Morten Hanefeld

    2012-01-01

    Determining fetal sex noninvasively is dependent of a robust assay. We designed a novel SRY assay and combined it with a SRY assay from literature forming a duplex assay with the same fluorescent dye to increase detection of Y-chromosome at low cell-free fetal DNA or chimeric DNA concentrations....

  12. Free energy estimation of short DNA duplex hybridizations

    Directory of Open Access Journals (Sweden)

    Leger Serge

    2010-02-01

    Full Text Available Abstract Background Estimation of DNA duplex hybridization free energy is widely used for predicting cross-hybridizations in DNA computing and microarray experiments. A number of software programs based on different methods and parametrizations are available for the theoretical estimation of duplex free energies. However, significant differences in free energy values are sometimes observed among estimations obtained with various methods, thus being difficult to decide what value is the accurate one. Results We present in this study a quantitative comparison of the similarities and differences among four published DNA/DNA duplex free energy calculation methods and an extended Nearest-Neighbour Model for perfect matches based on triplet interactions. The comparison was performed on a benchmark data set with 695 pairs of short oligos that we collected and manually curated from 29 publications. Sequence lengths range from 4 to 30 nucleotides and span a large GC-content percentage range. For perfect matches, we propose an extension of the Nearest-Neighbour Model that matches or exceeds the performance of the existing ones, both in terms of correlations and root mean squared errors. The proposed model was trained on experimental data with temperature, sodium and sequence concentration characteristics that span a wide range of values, thus conferring the model a higher power of generalization when used for free energy estimations of DNA duplexes under non-standard experimental conditions. Conclusions Based on our preliminary results, we conclude that no statistically significant differences exist among free energy approximations obtained with 4 publicly available and widely used programs, when benchmarked against a collection of 695 pairs of short oligos collected and curated by the authors of this work based on 29 publications. The extended Nearest-Neighbour Model based on triplet interactions presented in this work is capable of performing accurate

  13. Amplificação gênica alelo-específica na caracterização das hemoglobinas S, C e D e as interações entre elas e talassemias beta Allele-specific genic amplification in the characterization of hemoglobins S, C, D and interactions among them and with beta thalassemia

    Directory of Open Access Journals (Sweden)

    Luciane Cristina Bertholo

    2006-08-01

    possible interactions, based on the allele-specific genic amplification (PCR-AE with the use in parallel two primers that differ at their 3’ extremities and are complementary to the normal or mutated sequences. RESULTS AND DISCUSSION: The results make evident the validation of this methodology in the characterization of these mutations, once this procedure is easy to execute,to reproduce, as well as it is possible to be applied to a significative number of samples.

  14. DMT of Multi-hop Cooperative Networks - Part II: Half-Duplex Networks with Full-Duplex Performance

    CERN Document Server

    Sreeram, K; Kumar, P Vijay

    2008-01-01

    We consider single-source single-sink (ss-ss) multi-hop relay networks, with slow-fading links and single-antenna half-duplex relay nodes. In a companion paper, we established some basic results which laid the foundation for the results presented here. In the present paper, we consider two families of networks of half-duplex networks. KPP networks may be viewed as the union of K node-disjoint parallel relaying paths. Generalizations of these networks include KPP(I) networks, which permit interference between paths and KPP(D) networks, which possess a direct link between source and sink. We characterize the DMT of these families of networks completely and show that they can achieve the cut-set bound, thus proving that full-duplex performance can be obtained even in the presence of the half-duplex constraint. We then consider layered networks, and prove that a linear DMT between maximum diversity and maximum multiplexing gain is achievable. All protocols in this paper are explicit and use only amplify-and-forwa...

  15. Comprehensive and Rapid Real-Time PCR Analysis of 21 Foodborne Outbreaks

    Directory of Open Access Journals (Sweden)

    Hiroshi Fukushima

    2009-01-01

    Full Text Available A set of four duplex SYBR Green I PCR (SG-PCR assay combined with DNA extraction using QIAamp DNA Stool Mini kit was evaluated for the detection of foodborne bacteria from 21 foodborne outbreaks. The causative pathogens were detected in almost all cases in 2 hours or less. The first run was for the detection of 8 main foodborne pathogens in 5 stool specimens within 2 hours and the second run was for the detection of other unusual suspect pathogens within a further 45 minutes. After 2 to 4 days, the causative agents were isolated and identified. The results proved that for comprehensive and rapid molecular diagnosis in foodborne outbreaks, Duplex SG-PCR assay is not only very useful, but is also economically viable for one-step differentiation of causative pathogens in fecal specimens obtained from symptomatic patients. This then allows for effective diagnosis and management of foodborne outbreaks.

  16. Full-Duplex Communications in Large-Scale Cellular Networks

    KAUST Repository

    AlAmmouri, Ahmad

    2016-04-01

    In-band full-duplex (FD) communications have been optimistically promoted to improve the spectrum utilization and efficiency. However, the penetration of FD communications to the cellular networks domain is challenging due to the imposed uplink/downlink interference. This thesis presents a tractable framework, based on stochastic geometry, to study FD communications in multi-tier cellular networks. Particularly, we assess the FD communications effect on the network performance and quantify the associated gains. The study proves the vulnerability of the uplink to the downlink interference and shows that the improved FD rate gains harvested in the downlink (up to 97%) comes at the expense of a significant degradation in the uplink rate (up to 94%). Therefore, we propose a novel fine-grained duplexing scheme, denoted as α-duplex scheme, which allows a partial overlap between the uplink and the downlink frequency bands. We derive the required conditions to harvest rate gains from the α-duplex scheme and show its superiority to both the FD and half-duplex (HD) schemes. In particular, we show that the α-duplex scheme provides a simultaneous improvement of 28% for the downlink rate and 56% for the uplink rate. We also show that the amount of the overlap can be optimized based on the network design objective. Moreover, backward compatibility is an essential ingredient for the success of new technologies. In the context of in-band FD communication, FD base stations (BSs) should support HD users\\' equipment (UEs) without sacrificing the foreseen FD gains. The results show that FD-UEs are not necessarily required to harvest rate gains from FD-BSs. In particular, the results show that adding FD-UEs to FD-BSs offers a maximum of 5% rate gain over FD-BSs and HD-UEs case, which is a marginal gain compared to the burden required to implement FD transceivers at the UEs\\' side. To this end, we shed light on practical scenarios where HD-UEs operation with FD-BSs outperforms the

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

    Science.gov (United States)

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

    2016-07-07

    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). 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. 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. The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and

  18. Rapid detection of SNP (c.309T>G in the MDM2 gene by the Duplex SmartAmp method.

    Directory of Open Access Journals (Sweden)

    Yasuaki Enokida

    Full Text Available BACKGROUND: Genetic polymorphisms in the human MDM2 gene are suggested to be a tumor susceptibility marker and a prognostic factor for cancer. It has been reported that a single nucleotide polymorphism (SNP c.309T>G in the MDM2 gene attenuates the tumor suppressor activity of p53 and accelerates tumor formation in humans. METHODOLOGY: In this study, to detect the SNP c.309T>G in the MDM2 gene, we have developed a new SNP detection method, named "Duplex SmartAmp," which enabled us to simultaneously detect both 309T and 309G alleles in one tube. To develop this new method, we introduced new primers i.e., nBP and oBPs, as well as two different fluorescent dyes that separately detect those genetic polymorphisms. RESULTS AND CONCLUSIONS: By the Duplex SmartAmp method, the genetic polymorphisms of the MDM2 gene were detected directly from a small amount of genomic DNA or blood samples. We used 96 genomic DNA and 24 blood samples to validate the Duplex SmartAmp by comparison with results of the conventional PCR-RFLP method; consequently, the Duplex SmartAmp results agreed totally with those of the PCR-RFLP method. Thus, the new SNP detection method is considered useful for detecting the SNP c.309T>G in the MDM2 gene so as to judge cancer susceptibility against some cellular stress in the clinical setting, and also to handle a large number of samples and enable rapid clinical diagnosis.

  19. Comments on process of duplex coatings on aluminum alloys

    Institute of Scientific and Technical Information of China (English)

    Samir H.A.; QIAN Han-cheng(钱翰城); XIA Bo-cai(夏伯才); WU Shi-ming(吴仕明)

    2004-01-01

    Despite the great achievements made in improvement of wear resistance properties of aluminum alloys,their applications in heavy surface load-bearing are limited. Single coating is insufficient to produce the desired combination of surface properties. These problems can be solved through the duplex coatings. The aim of the present study is to overview the research advances on processes of duplex coatings on aluminum alloys combined with micro plasma oxidation process and with other modern processes such as physical vapour deposition and plasma assisted chemical vapour deposition and also to evaluate the performance of micro plasma oxidation coatings in improving the load-bearing, friction and wear resistance properties of aluminum alloys in comparison with other coatings. Wherein, a more detailed presentation of the processes and their performances and disadvantages are given as well.

  20. Herpes Zoster Duplex Unilateralis: Two Cases and Brief Literature Review

    Science.gov (United States)

    Son, Jee Hee; Chung, Bo Young; Kim, Hye One; Cho, Hee Jin

    2016-01-01

    Cases involving dermatomal herpes zoster in two or more locations are rare, especially in immunocompetent patients. When two noncontiguous dermatomes are involved, if affected unilaterally, it is called herpes zoster duplex unilateralis; if bilaterally, bilateralis. Here, we report two cases of herpes zoster duplex unilateralis. A 66-year-old man presented with painful erythematous grouped vesicles on his left scalp, forehead, trunk, and back (left [Lt.] V1, Lt. T8). Histologic findings were consistent with herpetic infection. A 33-year-old woman presented with painful erythematous grouped vesicles and crust on her left forehead and neck (Lt. V1, Lt. C5). Both patients were treated with oral administration of famcyclovir 750 mg/day for seven days. PMID:27904277

  1. Listen and talk full-duplex cognitive radio networks

    CERN Document Server

    Liao, Yun; Han, Zhu

    2016-01-01

    This brief focuses on the use of full-duplex radio in cognitive radio networks, presenting a novel spectrum sharing protocol that allows the secondary users to simultaneously sense and access the vacant spectrum. This protocol, called “Listen-and-talk” (LAT), is evaluated by both mathematical analysis and computer simulations in comparison with other existing protocols, including the listen-before-talk protocol. In addition to LAT-based signal processing and resource allocation, the brief discusses techniques such as spectrum sensing and dynamic spectrum access. The brief proposes LAT as a suitable access scheme for cognitive radio networks, which can support the quality-of-service requirements of these high priority applications. Fundamental theories and key techniques of cognitive radio networks are also covered. Listen and Talk: Full-duplex Cognitive Radio Networks is designed for researchers, developers, and professionals involved in cognitive radio networks. Advanced-level students studying signal pr...

  2. Smectic phase in suspensions of gapped DNA duplexes

    Science.gov (United States)

    Salamonczyk, Miroslaw; Zhang, Jing; Portale, Giuseppe; Zhu, Chenhui; Kentzinger, Emmanuel; Gleeson, James T.; Jakli, Antal; de Michele, Cristiano; Dhont, Jan K. G.; Sprunt, Samuel; Stiakakis, Emmanuel

    2016-11-01

    Smectic ordering in aqueous solutions of monodisperse stiff double-stranded DNA fragments is known not to occur, despite the fact that these systems exhibit both chiral nematic and columnar mesophases. Here, we show, unambiguously, that a smectic-A type of phase is formed by increasing the DNA's flexibility through the introduction of an unpaired single-stranded DNA spacer in the middle of each duplex. This is unusual for a lyotropic system, where flexibility typically destabilizes the smectic phase. We also report on simulations suggesting that the gapped duplexes (resembling chain-sticks) attain a folded conformation in the smectic layers, and argue that this layer structure, which we designate as smectic-fA phase, is thermodynamically stabilized by both entropic and energetic contributions to the system's free energy. Our results demonstrate that DNA as a building block offers an exquisitely tunable means to engineer a potentially rich assortment of lyotropic liquid crystals.

  3. Conserved guanine-guanine stacking in tetraplex and duplex DNA.

    Science.gov (United States)

    Kypr, J; Fialová, M; Chládková, J; Tůmová, M; Vorlícková, M

    2001-12-01

    Using a series of suitably chosen oligonucleotides, we demonstrate that the DNA duplex of d(CCCCGGGG) provides an almost identical CD spectrum as the parallel-stranded tetraplex of d(GGGG). The CD spectra are very sensitive to base stacking in DNA so that the above observation indicates that guanine-guanine stacking is essentially the same within the duplex of d(CCCCGGGG) and the tetraplex of d(GGGG). A very similar CD spectrum is also provided by the A-form of d(CCCCGGGG) induced by trifluoroethanol. These results reveal that guanine-guanine stacking is a structural invariant conserved in various nucleic acid conformers. The structural invariance is likely to cohere with evolution of the genetic molecules and be important for fundamental functions, e.g. initiation of transcription.

  4. A 2-order MIMO Full-Duplex Antenna System

    DEFF Research Database (Denmark)

    Tsakalaki, Elpiniki; Foroozanfard, Ehsan; De Carvalho, Elisabeth

    2014-01-01

    The paper presents an antenna system with combined full-duplex and 2-order multiple-input-multiple-output (MIMO) functionalities, i.e., a system capable of spatially multiplexing and spatially demultiplexing 2 datastreams in the same frequency and in the same time. By exploiting symmetries....... On the other hand, the 2 MIMO ports (either at the Tx or at the Rx) are sufficiently decoupled thanks to polarization diversity. The proposed antenna system exhibits a remarkable level of fullduplex isolation over a wide bandwidth while maintaining low coupling between its MIMO ports and can serve...... as a concrete implementation of an antenna system equipped with both MIMO as well as full-duplex capabilities....

  5. Sex determination of porcine embryos using a new developed duplex polymerase chain reaction procedure based on the amplification of repetitive sequences.

    Science.gov (United States)

    Torner, Eva; Bussalleu, Eva; Briz, M Dolors; Gutiérrez-Adán, Alfonso; Bonet, Sergi

    2013-01-01

    Polymerase chain reaction (PCR)-based assays have become increasingly prevalent for sexing embryos. The aim of the present study was to develop a suitable duplex PCR procedure based on the amplification of porcine repetitive sequences for sexing porcine tissues, embryos and single cells. Primers were designed targeting the X12696 Y chromosome-specific repeat sequence (SUSYa and SUSYb; sex-related primer sets), the multicopy porcine-specific mitochondrial 12S rRNA gene (SUS12S; control primer set) and the X51555 1 chromosome repeat sequence (SUS1; control primer set). The specificity of the primer sets was established and the technique was optimised by testing combinations of two specific primer sets (SUSYa/SUS12S; SUSYb/SUS12S), different primer concentrations, two sources of DNA polymerase, different melting temperatures and different numbers of amplification cycles using genomic DNA from porcine ovarian and testicular tissue. The optimised SUSYa/SUS12S- and SUSYb/SUS12S-based duplex PCR procedures were applied to porcine in vitro-produced (IVP) blastocysts, cell-stage embryos and oocytes. The SUSYb/SUS12S primer-based procedure successfully sexed porcine single cells and IVP cell-stage embryos (100% efficiency), as well as blastocysts (96.6% accuracy; 96.7% efficiency). This is the first report to demonstrate the applicability of these repetitive sequences for this purpose. In conclusion, the SUSYb/SUS12S primer-based duplex PCR procedure is highly reliable and sensitive for sexing porcine IVP embryos.

  6. Reverse-transcription PCR (RT-PCR).

    Science.gov (United States)

    Bachman, Julia

    2013-01-01

    RT-PCR is commonly used to test for genetic diseases and to characterize gene expression in various tissue types, cell types, and over developmental time courses. This serves as a form of expression profiling, but typically as a candidate approach. RT-PCR is also commonly used to clone cDNAs for further use with other molecular biology techniques (e.g., see Oligo(dT)-primed RT-PCR isolation of polyadenylated RNA degradation intermediates and Circularized RT-PCR (cRT-PCR): analysis of RNA 5' ends, 3' ends, and poly(A) tails).

  7. Implementation of real-time duplex synthetic aperture ultrasonography

    DEFF Research Database (Denmark)

    Hemmsen, Martin Christian; Larsen, Lee; Kjeldsen, Thomas;

    2015-01-01

    This paper presents a real-time duplex synthetic aperture imaging system, implemented on a commercially available tablet. This includes real-time wireless reception of ultrasound signals and GPU processing for B-mode and Color Flow Imaging (CFM). The objective of the work is to investigate the im...... and that the required bandwidth between the probe and processing unit is within the current Wi-Fi standards....

  8. Effect of heat treatment on corrosion behavior of duplex coatings

    Directory of Open Access Journals (Sweden)

    K. Raghu Ram Mohan Reddy

    2017-01-01

    Full Text Available In the present paper, duplex WC-Co/NiCrAlY coating is coated onto Ti6Al4V substrate and vacuum heat treatment is employed to investigate the corrosion behavior of heat treated samples as well as Ti6Al4V substrate for comparison. In this duplex coating system, High Velocity Oxy Fuel (HVOF process is used to deposit NiCrAlY interlayer with a constant thickness of 200 μm and WC-Co ceramic top layer with varying thickness of 250 μm, 350 μm and 450 μm deposited by Detonation Spray (DS process. Different heat treatment temperatures (600–1150 °C were employed for the coated samples to study the microstructure and the effect on corrosion resistance of the duplex coatings. Potentiodynamic polarization tests were carried to investigate the corrosion performance of duplex coated heat treated samples and the substrate in Ringer’s solution at 37 °C and prepared the pH to 5.7. The microstructure upon corrosion after heat treatment was characterized by SEM analysis to understand the corrosion behavior. The results disclosed that at all heat treatment temperatures, all the coated samples exhibited better corrosion resistance than the base substrate. However, during 950 °C and 1150 °C heat treatment temperatures, it was observed highest corrosion potential than 600 °C and 800 °C. The 350 μm thickness, coated sample exhibited highest corrosion resistance compared to other two coated samples and the substrate at all heat treatment temperatures.

  9. Evolution of microstresses in plastically deformed duplex steel

    Energy Technology Data Exchange (ETDEWEB)

    Baczmanski, A.; Wierzbanowski, K. [Akademia Gorniczo-Hutnicza, Krakow (Poland). WFTJ; Braham, C. [LMMM, URA-CNRS 1219, Ecole Nationale Superieure d' Arts et Metiers, Paris (France); Lodini, A. [IFTS, Univ. de Reims Charleville-Mezieres (France)

    2000-07-01

    The X-ray diffraction method has been applied to determine the internal stresses in two phases austeno-ferritic steel during uniaxial tensile test. The elasto-plastic deformation model was used to predict internal stresses and critical resolved shear stress for the both phases. The model calculations were successfully compared with the information obtained from the shift and broadening of diffraction peak. Finally, the parameters characterising elasto-plastic deformation for duplex steel were determined. (orig.)

  10. Tensile properties of duplex UNS S32205 and lean duplex UNS S32304 steels and the influence of short duration 475 ºC aging

    Directory of Open Access Journals (Sweden)

    Sérgio Souto Maior Tavares

    2012-12-01

    Full Text Available Duplex stainless steels are high strength and corrosion resistant steels extensively used in the petrochemical and chemical industries. The aging at 475 ºC for long periods of time provokes embrittlement and deterioration of corrosion resistance. However, short duration aging at 475 ºC may be used as heat treatment to improve mechanical resistance with small decrease in the other properties. In this work the flow stress curves of lean duplex UNS S32304 and duplex UNS S32205 steels were modeled with Hollomon's equation and work hardening exponents (n were determined. The analyses were conducted in specimens annealed and heat treated at 475 ºC for short periods of time. The aging at 475 ºC for 4 hours, 8 hours and 12 hours promoted significant hardening with small decrease of ductility. The work hardening exponents of both steels were compared, being higher in the duplex steel than in the lean duplex grade.

  11. Duplex Schemes in Multiple Antenna Two-Hop Relaying

    Directory of Open Access Journals (Sweden)

    Anja Klein

    2008-04-01

    Full Text Available A novel scheme for two-hop relaying defined as space division duplex (SDD relaying is proposed. In SDD relaying, multiple antenna beamforming techniques are applied at the intermediate relay station (RS in order to separate downlink and uplink signals of a bi-directional two-hop communication between two nodes, namely, S1 and S2. For conventional amplify-and-forward two-hop relaying, there appears a loss in spectral efficiency due to the fact that the RS cannot receive and transmit simultaneously on the same channel resource. In SDD relaying, this loss in spectral efficiency is circumvented by giving up the strict separation of downlink and uplink signals by either time division duplex or frequency division duplex. Two novel concepts for the derivation of the linear beamforming filters at the RS are proposed; they can be designed either by a three-step or a one-step concept. In SDD relaying, receive signals at S1 are interfered by transmit signals of S1, and receive signals at S2 are interfered by transmit signals of S2. An efficient method in order to combat this kind of interference is proposed in this paper. Furthermore, it is shown how the overall spectral efficiency of SDD relaying can be improved if the channels from S1 and S2 to the RS have different qualities.

  12. On full duplex Gaussian relay channels with self-interference

    KAUST Repository

    Behboodi, Arash

    2016-08-15

    Self interference (SI) in full duplex (FD) systems is the interference caused by the transmission stream on the reception stream. Being one of the main restrictive factors for performance of practical full duplex systems, however, not too much is known about its effect on the fundamental limits of relaying systems. In this work, we consider the full duplex three-node relay channel with SI where SI is modeled as an additive Gaussian noise whose variance is dependent on instantaneous input power. The classical achievable rates and upper bounds for the single three-node relay channel no longer apply due to the structure of SI. Achievable rates for Decode-and-Forward (DF) and Compress-and-Forward (CF) and upper bounds on the capacity are derived assuming Gaussian inputs and SI. The deterministic model is also introduced and its capacity is characterized. The optimal joint source-relay distributions is discussed. Numerical results are provided comparing the achievable rates and upper bound. © 2016 IEEE.

  13. Single-Step qPCR and dPCR Detection of Diverse CRISPR-Cas9 Gene Editing Events In Vivo

    Directory of Open Access Journals (Sweden)

    Micol Falabella

    2017-10-01

    Full Text Available Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-CRISPR-associated protein 9 (Cas9-based technology is currently the most flexible means to create targeted mutations by recombination or indel mutations by nonhomologous end joining. During mouse transgenesis, recombinant and indel alleles are often pursued simultaneously. Multiple alleles can be formed in each animal to create significant genetic complexity that complicates the CRISPR-Cas9 approach and analysis. Currently, there are no rapid methods to measure the extent of on-site editing with broad mutation sensitivity. In this study, we demonstrate the allelic diversity arising from targeted CRISPR editing in founder mice. Using this DNA sample collection, we validated specific quantitative and digital PCR methods (qPCR and dPCR, respectively for measuring the frequency of on-target editing in founder mice. We found that locked nucleic acid (LNA probes combined with an internal reference probe (Drop-Off Assay provide accurate measurements of editing rates. The Drop-Off LNA Assay also detected on-target CRISPR-Cas9 gene editing in blastocysts with a sensitivity comparable to PCR-clone sequencing. Lastly, we demonstrate that the allele-specific LNA probes used in qPCR competitor assays can accurately detect recombinant mutations in founder mice. In summary, we show that LNA-based qPCR and dPCR assays provide a rapid method for quantifying the extent of on-target genome editing in vivo, testing RNA guides, and detecting recombinant mutations.

  14. EFFECT OF INTERMETALLIC PHASES ON CORROSION BEHAVIOR AND MECHANICAL PROPERTIES OF DUPLEX STAINLESS STEEL AND SUPER-DUPLEX STAINLESS STEEL

    Directory of Open Access Journals (Sweden)

    Prabhu Paulraj

    2015-08-01

    Full Text Available Duplex Stainless Steels (DSS and Super Duplex Stainless Steel (SDSS have excellent integration of mechanical and corrosion properties. However, the formation of intermetallic phases is a major problem in their usage. The mechanical and corrosion properties are deteriorated due to the presence of intermetallic phases. These phases are induced during welding, prolonged exposure to high temperatures, and improper heat treatments. The main emphasis of this review article is on intermetallic phases and their effects on corrosion and mechanical properties. First the effect of various alloying elements on DSS and SDSS has been discussed followed by formation of various intermetallic phases. The intermetallic phases affect impact toughness and corrosion resistance significantly. Their deleterious effect on weldments has also been reviewed.

  15. Characterization of microstructure and texture across dissimilar super duplex/austenitic stainless steel weldment joint by super duplex filler metal

    Energy Technology Data Exchange (ETDEWEB)

    Eghlimi, Abbas, E-mail: a.eghlimi@ma.iut.ac.ir [Department of Materials Engineering, Isfahan University of Technology, Isfahan 84156-83111 (Iran, Islamic Republic of); Shamanian, Morteza [Department of Materials Engineering, Isfahan University of Technology, Isfahan 84156-83111 (Iran, Islamic Republic of); Eskandarian, Masoomeh [Department of Materials Engineering, Shiraz University, Shiraz 71348-51154 (Iran, Islamic Republic of); Zabolian, Azam [Department of Natural Resources, Isfahan University of Technology, Isfahan 84156-83111 (Iran, Islamic Republic of); Szpunar, Jerzy A. [Department of Mechanical Engineering, University of Saskatchewan, Saskatoon SK S7N 5A9 (Canada)

    2015-08-15

    In the present paper, microstructural changes across an as-welded dissimilar austenitic/duplex stainless steel couple welded by a super duplex stainless steel filler metal using gas tungsten arc welding process is characterized with optical microscopy and electron back-scattered diffraction techniques. Accordingly, variations of microstructure, texture, and grain boundary character distribution of base metals, heat affected zones, and weld metal were investigated. The results showed that the weld metal, which was composed of Widmanstätten austenite side-plates and allotriomorphic grain boundary austenite morphologies, had the weakest texture and was dominated by low angle boundaries. The welding process increased the ferrite content but decreased the texture intensity at the heat affected zone of the super duplex stainless steel base metal. In addition, through partial ferritization, it changed the morphology of elongated grains of the rolled microstructure to twinned partially transformed austenite plateaus scattered between ferrite textured colonies. However, the texture of the austenitic stainless steel heat affected zone was strengthened via encouraging recrystallization and formation of annealing twins. At both interfaces, an increase in the special character coincident site lattice boundaries of the primary phase as well as a strong texture with <100> orientation, mainly of Goss component, was observed. - Graphical abstract: Display Omitted - Highlights: • Weld metal showed local orientation at microscale but random texture at macroscale. • Intensification of <100> orientated grains was observed adjacent to the fusion lines. • The austenite texture was weaker than that of the ferrite in all duplex regions. • Welding caused twinned partially transformed austenites to form at SDSS HAZ. • At both interfaces, the ratio of special CSL boundaries of the primary phase increased.

  16. Extensive Recombination Due to Heteroduplexes Generates Large Amounts of Artificial Gene Fragments during PCR

    Science.gov (United States)

    Liu, Jia; Song, Hongshuo; Liu, Donglai; Zuo, Tao; Lu, Fengmin; Zhuang, Hui; Gao, Feng

    2014-01-01

    Artificial recombinants can be generated during PCR when more than two genetically distinct templates coexist in a single PCR reaction. These recombinant amplicons can lead to the false interpretation of genetic diversity and incorrect identification of biological phenotypes that do not exist in vivo. We investigated how recombination between 2 or 35 genetically distinct HIV-1 genomes was affected by different PCR conditions using the parallel allele-specific sequencing (PASS) assay and the next generation sequencing method. In a standard PCR condition, about 40% of amplicons in a PCR reaction were recombinants. The high recombination frequency could be significantly reduced if the number of amplicons in a PCR reaction was below a threshold of 1013–1014 using low thermal cycles, fewer input templates, and longer extension time. Heteroduplexes (each DNA strand from a distinct template) were present at a large proportion in the PCR products when more thermal cycles, more templates, and shorter extension time were used. Importantly, the majority of recombinants were identified in heteroduplexes, indicating that the recombinants were mainly generated through heteroduplexes. Since prematurely terminated extension fragments can form heteroduplexes by annealing to different templates during PCR amplification, recombination has a better chance to occur with samples containing different genomes when the number of amplicons accumulate over the threshold. New technologies are warranted to accurately characterize complex quasispecies gene populations. PMID:25211143

  17. Extensive recombination due to heteroduplexes generates large amounts of artificial gene fragments during PCR.

    Directory of Open Access Journals (Sweden)

    Jia Liu

    Full Text Available Artificial recombinants can be generated during PCR when more than two genetically distinct templates coexist in a single PCR reaction. These recombinant amplicons can lead to the false interpretation of genetic diversity and incorrect identification of biological phenotypes that do not exist in vivo. We investigated how recombination between 2 or 35 genetically distinct HIV-1 genomes was affected by different PCR conditions using the parallel allele-specific sequencing (PASS assay and the next generation sequencing method. In a standard PCR condition, about 40% of amplicons in a PCR reaction were recombinants. The high recombination frequency could be significantly reduced if the number of amplicons in a PCR reaction was below a threshold of 10(13-10(14 using low thermal cycles, fewer input templates, and longer extension time. Heteroduplexes (each DNA strand from a distinct template were present at a large proportion in the PCR products when more thermal cycles, more templates, and shorter extension time were used. Importantly, the majority of recombinants were identified in heteroduplexes, indicating that the recombinants were mainly generated through heteroduplexes. Since prematurely terminated extension fragments can form heteroduplexes by annealing to different templates during PCR amplification, recombination has a better chance to occur with samples containing different genomes when the number of amplicons accumulate over the threshold. New technologies are warranted to accurately characterize complex quasispecies gene populations.

  18. Residual stresses and fatigue in a duplex stainless steel

    Energy Technology Data Exchange (ETDEWEB)

    Johansson, Johan

    1999-05-01

    Duplex stainless steels, consisting of approximately equal amounts of austenite and ferrite, often combine the best features of austenitic and ferritic stainless steels. They generally have good mechanical properties, including high strength and ductility, and the corrosion resistance is often better than conventional austenitic grades. This has lead to a growing use of duplex stainless steels as a material in mechanically loaded constructions. However, detailed knowledge regarding its mechanical properties and deformation mechanisms are still lacking. In this thesis special emphasis has been placed on the residual stresses and their influence on mechanical behaviour of duplex stainless steels. Due to the difference in coefficient of thermal expansion between the two phases, tensile microstresses are found in the austenitic phase and balancing compressive microstresses in the ferritic phase. The first part of this thesis is a literature survey, which will give an introduction to duplex stainless steels and review the fatigue properties of duplex stainless steels and the influence of residual stresses in two-phase material. The second part concerns the evolution of the residual stress state during uniaxial loading. Initial residual stresses were found to be almost two times higher in the transverse direction compared to the rolling direction. During loading the absolute value of the microstresses increased in the macroscopic elastic regime but started to decrease with increasing load in the macroscopic plastic regime. A significant increase of the microstresses was also found to occur during unloading. Finite element simulations also show stress variation within one phase and a strong influence of both the elastic and plastic anisotropy of the individual phases on the simulated stress state. In the third part, the load sharing between the phases during cyclic loading is studied. X-ray diffraction stress analysis and transmission electron microscopy show that even if

  19. Effect of ultrafine grain on tensile behaviour and corrosion resistance of the duplex stainless steel.

    Science.gov (United States)

    Jinlong, Lv; Tongxiang, Liang; Chen, Wang; Limin, Dong

    2016-05-01

    The ultrafine grained 2205 duplex stainless steel was obtained by cold rolling and annealing. The tensile properties were investigated at room temperature. Comparing with coarse grained stainless steel, ultrafine grained sample showed higher strength and plasticity. In addition, grain size changed deformation orientation. The strain induced α'-martensite was observed in coarse grained 2205 duplex stainless steel with large strain. However, the grain refinement inhibited the transformation of α'-martensite;nevertheless, more deformation twins improved the strength and plasticity of ultrafine grained 2205 duplex stainless steel. In addition, the grain refinement improved corrosion resistance of the 2205 duplex stainless steel in sodium chloride solution.

  20. Analyzing the Potential of Full Duplex in 5G Ultra-Dense Small Cell Networks

    DEFF Research Database (Denmark)

    Gatnau, Marta; Berardinelli, Gilberto; Mahmood, Nurul Huda;

    2016-01-01

    Full duplex technology has become an attractive solution for future 5th Generation (5G) systems for accommodating the exponentially growing mobile traffic demand. Full duplex allows a node to transmit and receive simultaneously in the same frequency band, thus, theoretically, doubling the system......-interference cancellation are demonstrated using our own developed test bed. Secondly, a detailed evaluation of full duplex communication in 5G ultra-dense small cell networks via system level simulations is provided. The results are presented in terms of throughput and delay. Two types of full duplex are studied: when...

  1. Simultaneous detection of ricin and abrin DNA by real-time PCR (qPCR).

    Science.gov (United States)

    Felder, Eva; Mossbrugger, Ilona; Lange, Mirko; Wölfel, Roman

    2012-09-01

    Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR) assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5'-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.

  2. Simultaneous Detection of Ricin and Abrin DNA by Real-Time PCR (qPCR

    Directory of Open Access Journals (Sweden)

    Roman Wölfel

    2012-08-01

    Full Text Available Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5′-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.

  3. Sex Determination Using PCR

    Science.gov (United States)

    Kima, Peter E.; Rasche, Madeline E.

    2004-01-01

    PCR has revolutionized many aspects of biochemistry and molecular biology research. In the following exercise, students learn PCR by isolating their own DNA, amplifying specific segments of the X and Y chromosomes, and estimating the sizes of the PCR products using agarose gel electrophoresis. Based on the pattern of PCR products, students can…

  4. A new quantitative RT-PCR method for sensitive detection of dengue virus in serum samples.

    Science.gov (United States)

    Sadon, Nadine; Delers, Anne; Jarman, Richard G; Klungthong, Chonticha; Nisalak, Ananda; Gibbons, Robert V; Vassilev, Ventzislav

    2008-10-01

    In order to detect and identify dengue serotypes in serum samples, we developed a single-step quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) assay (referred to as Q-PCR). Sets of primers were selected from the capsid region of the viral genome. Dengue serotypes 1/3 and 2/4 were detected in two separate duplex amplification reactions using specific primers and fluorogenic TaqMan probes. Results obtained with this Q-PCR and the classical nested RT-PCR (N-PCR) assays were compared using a panel of 97 representative human sera collected from patients in Bangkok, Thailand. It is shown that the Q-PCR is a rapid, sensitive and reproducible tool for the detection and quantitation of the four dengue serotypes in clinical samples, and therefore of great interest for diagnostic use or for large cohort studies.

  5. Quantitative duplex TaqMan real-time polymerase chain reaction for the assessment of the etiologic agent of epizootic bovine abortion.

    Science.gov (United States)

    Brooks, Roxann S; Blanchard, Myra T; Anderson, Mark L; Hall, Mark R; Stott, Jeffery L

    2011-11-01

    Epizootic bovine abortion (EBA), also commonly known as "foothill abortion," is a late-term abortion primarily in beef cattle with significant economic impacts in California, Nevada, and Oregon. The causative agent is a novel deltaproteobacterium (aoEBA) closely related to the order Myxococcales and vectored by the soft-shelled tick Ornithodoros coriaceus. Historically, diagnosis has relied upon the pathologic examination of the fetus and the presence of elevated fetal serum immunoglobulins. Identification of the etiologic agent, a unique deltaproteobacterium, permitted the development of a quantitative duplex real-time polymerase chain reaction (qPCR) using a unique 90-bp sequence of aoEBA 16S ribosomal RNA gene in conjunction with an 88-bp sequence of the bovine β-actin gene. Reaction efficiencies were 100.9% for the 16S aoEBA gene and 93.1% for the bovine β-actin gene. Application of the duplex TaqMan to a set of aoEBA-infected fetal bovine necropsy tissues demonstrated the assay to be robust in quantitatively identifying the aoEBA bacteria and establishing host-tissue pathogen load. Consistent with previously reported immunohistochemical data, organized lymphoid tissue generally carried the heaviest bacterial load as compared to non-lymphoid tissue. The newly developed duplex TaqMan assay will facilitate diagnosis in difficult cases and provide an invaluable tool for delineating the pathogenesis of EBA.

  6. Force-Induced Rupture of a DNA Duplex: From Fundamentals to Force Sensors.

    Science.gov (United States)

    Mosayebi, Majid; Louis, Ard A; Doye, Jonathan P K; Ouldridge, Thomas E

    2015-12-22

    The rupture of double-stranded DNA under stress is a key process in biophysics and nanotechnology. In this article, we consider the shear-induced rupture of short DNA duplexes, a system that has been given new importance by recently designed force sensors and nanotechnological devices. We argue that rupture must be understood as an activated process, where the duplex state is metastable and the strands will separate in a finite time that depends on the duplex length and the force applied. Thus, the critical shearing force required to rupture a duplex depends strongly on the time scale of observation. We use simple models of DNA to show that this approach naturally captures the observed dependence of the force required to rupture a duplex within a given time on duplex length. In particular, this critical force is zero for the shortest duplexes, before rising sharply and then plateauing in the long length limit. The prevailing approach, based on identifying when the presence of each additional base pair within the duplex is thermodynamically unfavorable rather than allowing for metastability, does not predict a time-scale-dependent critical force and does not naturally incorporate a critical force of zero for the shortest duplexes. We demonstrate that our findings have important consequences for the behavior of a new force-sensing nanodevice, which operates in a mixed mode that interpolates between shearing and unzipping. At a fixed time scale and duplex length, the critical force exhibits a sigmoidal dependence on the fraction of the duplex that is subject to shearing.

  7. Numerical modeling and optimization of machining duplex stainless steels

    Directory of Open Access Journals (Sweden)

    Rastee D. Koyee

    2015-01-01

    Full Text Available The shortcomings of the machining analytical and empirical models in combination with the industry demands have to be fulfilled. A three-dimensional finite element modeling (FEM introduces an attractive alternative to bridge the gap between pure empirical and fundamental scientific quantities, and fulfill the industry needs. However, the challenging aspects which hinder the successful adoption of FEM in the machining sector of manufacturing industry have to be solved first. One of the greatest challenges is the identification of the correct set of machining simulation input parameters. This study presents a new methodology to inversely calculate the input parameters when simulating the machining of standard duplex EN 1.4462 and super duplex EN 1.4410 stainless steels. JMatPro software is first used to model elastic–viscoplastic and physical work material behavior. In order to effectively obtain an optimum set of inversely identified friction coefficients, thermal contact conductance, Cockcroft–Latham critical damage value, percentage reduction in flow stress, and Taylor–Quinney coefficient, Taguchi-VIKOR coupled with Firefly Algorithm Neural Network System is applied. The optimization procedure effectively minimizes the overall differences between the experimentally measured performances such as cutting forces, tool nose temperature and chip thickness, and the numerically obtained ones at any specified cutting condition. The optimum set of input parameter is verified and used for the next step of 3D-FEM application. In the next stage of the study, design of experiments, numerical simulations, and fuzzy rule modeling approaches are employed to optimize types of chip breaker, insert shapes, process conditions, cutting parameters, and tool orientation angles based on many important performances. Through this study, not only a new methodology in defining the optimal set of controllable parameters for turning simulations is introduced, but also

  8. Technology of Welding Joints Mixed with Duplex Steel

    OpenAIRE

    Słania J.; Krawczyk R.; Masłoń D.

    2016-01-01

    Results of the examinations of sample plates of mixed joints with the duplex steel were discussed. Examinations were taken on the sample plates of mixed joints of sheet plates type P355NL1 and X2CrNiMoN22-5-3 welded by the flux-cored wire DW-329A by the Kobelco company of the following category T 22 9 3 NL RC/M3 in the gas shroud M21 (Ar+18%CO2) (plate no.1), and nickel covered electrodes E Ni 6082 by the Böhler company (plate no. 2). Results of the side bend test of welded joint, transverse ...

  9. Protocols For Half-Duplex Multiple Relay Networks

    CERN Document Server

    Rost, P

    2007-01-01

    In this paper we present several strategies for multiple relay networks which are constrained by a half-duplex operation, i. e., each node either transmits or receives on a particular resource. Using the discrete memoryless multiple relay channel we present achievable rates for a multilevel partial decode-and-forward approach which generalizes previous results presented by Kramer and Khojastepour et al.. Furthermore, we derive a compress-and-forward approach using a regular encoding scheme which simplifies the encoding and decoding scheme and improves the achievable rates in general. Finally, we give achievable rates for a mixed strategy used in a four-terminal network with alternately transmitting relay nodes.

  10. Reliability Capacity of Half-Duplex Channels with Strict Deadlines

    DEFF Research Database (Denmark)

    Costa, Rui; Roetter, Daniel Enrique Lucani; Vinhoza, Tiago;

    2015-01-01

    A fundamental characterization of a half-duplex wireless system with packet losses under traffic with hard deadlines is instrumental to understanding and developing efficient, coding aware policies for real-time applications. We set forth the concept of reliability capacity with a limited number...... and in terms of its advantages towards the mean number of packets that can be transmitted reliably. Optimal schemes for leveraging feedback are presented and the results show that a judicious use of even a single feedback packet can have a significant impact on the mean performance....

  11. Corrosion resistance properties of sintered duplex stainless steel

    Directory of Open Access Journals (Sweden)

    L.A. Dobrzański

    2006-09-01

    Full Text Available Purpose: of this paper was to examine the corrosion resistance of duplex stainless steels using electrochemical methods in 1M NaCl solution. The influence of powder mixes preparation and cooling cycle after sintering on corrosion properties was evaluated.Design/methodology/approach: In presented study duplex stainless steels were obtained through powder metallurgy starting from austenitic, martensitic base powders by controlled addition of alloying elements, such as Cr, Ni, Mo and Cu. In the studies behind the preparation of mixes, Schaeffler’s diagram was taken into consideration. Prepared mixes have been compacted at 800 MPa and sintered in a vacuum furnace with argon backfilling at 1260°C for 1 h. After sintering two different cooling cycles were applied: rapid cooling with an average cooling rate of 245 °C/min and slow cooling of 5 °C/min in argon atmosphere. Produced duplex stainless steels have been studied by scanning and optical microscopy and EDS chemical analysis of microstructure components. Corrosion properties have been studied through electrochemical methods in 1M NaCl water solutionFindings: According to achieved results, it was affirmed that applied sintering method as well as powder mixes preparation allows for manufacturing the sintered duplex steels with good corrosion properties which depends on austenite/ferrite ratio in the microstructure and elements partitioning between phases. Corrosion resistance of sintered stainless steels is strictly connected with the density and the pore morphology present in the microstructure too. The highest resistance to pitting corrosion in 1M NaCl solution was achieved for composition with approximate balance of ferrite and austenite in the microstructure.Research limitations/implications: According to the powders characteristic, the applied fast cooling rate seems to be a good compromise for corrosion properties and microstructures, nevertheless further tests should be carried out in

  12. The Terahertz Controlled Duplex Isolator: Physical Grounds and Numerical Experiment

    Directory of Open Access Journals (Sweden)

    Konstantin Vytovtov

    2016-01-01

    Full Text Available Electromagnetic properties of an anisotropic stratified slab with an arbitrary orientation of the anisotropy axis under an oblique incidence of a plane harmonic wave are studied. The dependence of the eigenwave wavenumbers and the reflection coefficient on an anisotropy axis orientation and frequency is investigated. For the first time, the expression for the translation matrix is obtained in the compact analytical form. The controlled two-way dual-frequency (duplex isolator based on the above described slab is presented for the first time. It is based on the properties of the anisotropic structure described here but not on the Faraday effect.

  13. Duplex ultrasound scanning of peripheral arterial disease of the lower limb

    DEFF Research Database (Denmark)

    Eiberg, Jonas Peter; Rasmussen, John Bøje Grønvall; Hansen, Marc Allan

    2010-01-01

    To assess the reliability and applicability of duplex ultrasound scanning (DUS) of lower limb arteries, compared with digital subtraction angiography (DSA), in patients with peripheral arterial disease (PAD).......To assess the reliability and applicability of duplex ultrasound scanning (DUS) of lower limb arteries, compared with digital subtraction angiography (DSA), in patients with peripheral arterial disease (PAD)....

  14. RF Self-Interference reduction techniques for compact full duplex radios

    NARCIS (Netherlands)

    Deballie, B.; van den Broek, Dirk-Jan; Lavin, C.; van Liempd, B.; Klumperink, Eric A.M.; Palacios, C.; Craninckx, J.; Nauta, Bram

    2015-01-01

    This paper describes three RF self-interference reduction techniques for full-duplex wireless links, which specifically target integration in compact radios. Concretely, a self-interference cancelling front-end, a dual-polarized antenna, and an electrical balance duplexer are proposed. Each techniqu

  15. Importance of diastolic velocities in the detection of celiac and mesenteric artery disease by duplex ultrasound

    DEFF Research Database (Denmark)

    Perko, M J; Just, S; Schroeder, T V

    1997-01-01

    To assess the predictive value of ultrasound duplex scanning in the detection of superior mesenteric artery (SMA) and celiac artery (CA) occlusive disease.......To assess the predictive value of ultrasound duplex scanning in the detection of superior mesenteric artery (SMA) and celiac artery (CA) occlusive disease....

  16. Accuracy of colour duplex sonography for the diagnosis of renal artery stenosis

    DEFF Research Database (Denmark)

    Saeed, Aso; Bergström, Göran; Zachrisson, Karin

    2009-01-01

    OBJECTIVE: To examine the diagnostic value of novel velocimetric colour duplex sonography indices in the screening of renal artery stenosis (RAS). METHODS: We performed a retrospective analysis of all consecutively studied patients at our centre with suspected RAS, and a colour duplex sonography ...

  17. Pushing the limits of Full-duplex: Design and Real-time Implementation

    CERN Document Server

    Sahai, Achaleshwar; Sabharwal, Ashutosh

    2011-01-01

    Recent work has shown the feasibility of single-channel full-duplex wireless physical layer, allowing nodes to send and receive in the same frequency band at the same time. In this report, we first design and implement a real-time 64-subcarrier 10 MHz full-duplex OFDM physical layer, FD-PHY. The proposed FD-PHY not only allows synchronous full-duplex transmissions but also selective asynchronous full-duplex modes. Further, we show that in over-the-air experiments using optimal antenna placement on actual devices, the self-interference can be suppressed upto 80dB, which is 10dB more than prior reported results. Then we propose a full-duplex MAC protocol, FD-MAC, which builds on IEEE 802.11 with three new mechanisms -- shared random backoff, header snooping and virtual backoffs. The new mechanisms allow FD-MAC to discover and exploit full-duplex opportunities in a distributed manner. Our over-the-air tests show over 70% throughput gains from using full-duplex over half-duplex in realistically used cases.

  18. Deformation in thrust-ramp anticlines and duplexes: implications for geometry and porosity

    Energy Technology Data Exchange (ETDEWEB)

    Groshong, R.H. Jr.; Usdansky, S.I.

    1986-05-01

    A computerized kinematic model of thrust-ramp anticline geometry allows workers to predict the zones of greatest deformation in ramp anticlines and fault duplexes. The model assumes a constant cross-section area, symmetrical fold hinges, and slip in the hanging wall parallel to the ramp and forelimb. Assuming that the collapse of original porosity or the generation of secondary fracture porosity is proportional to deformation, the model can be used to predict porosity changes. Deformation in a single ramp anticline is greatest in the forelimb and backlimb, and may be absent in the crest. A duplex structure results from comparatively closely spaced thrusts that have a common upper detachment horizon. Relatively wide spacing between the duplex faults yields a bumpy roofed duplex as in the central Appalachians. Forelimbs may be deformed twice and should show greater porosity modification. Relatively close spacing between ramp-and-flat thrusts can produce a listric-fault, snakehead anticline geometry because younger faults deform the preexisting thrust slices. The resulting geometry is here called a snakehead duplex and appears to be fairly common, as in the Jumpingpound field in the Canadian Rockies. Each thrust slice within the duplex is deformed six times or more, providing the maximum opportunity for deformation-related porosity changes. Maximum fracture porosity should occur in thrusts having listric-fan or snakehead duplex geometry. Structures involving duplexes generally should be better than isolated ramp anticlines.

  19. Thermal Aging Phenomena in Cast Duplex Stainless Steels

    Energy Technology Data Exchange (ETDEWEB)

    Byun, T. S.; Yang, Y.; Overman, N. R.; Busby, J. T.

    2016-02-28

    Cast stainless steels (CASSs) have been extensively used for the large components of light water reactor (LWR) power plants such as primary coolant piping and pump casing. The thermal embrittlement of CASS components is one of the most serious concerns related to the extended-term operation of nuclear power plants. Many past researches have concluded that the formation of Cr–rich α'-phase by Spinodal decomposition of δ-ferrite phase is the primary mechanism for the thermal embrittlement. Cracking mechanism in the thermally-embrittled duplex stainless steels consists of the formation of cleavage at ferrite and its propagation via separation of ferrite-austenite interphase. This article intends to provide an introductory overview on the thermal aging phenomena in LWR relevant conditions. Firstly, the thermal aging effect on toughness is discussed in terms of the cause of embrittlement and influential parameters. An approximate analysis of thermal reaction using Arrhenius equation was carried out to scope the aging temperatures for the accelerated aging experiments to simulate the 60 and 80 years of services. Further, equilibrium precipitation calculation was performed for model CASS alloys using the CALPHAD program and the results are used to describe the precipitation behaviors in duplex stainless steels. These results are also to be used to guide an on-going research aiming to provide knowledge-based conclusive prediction for the integrity of the CASS components of LWR power plants during the service life extended up to and beyond 60 years.

  20. Fabrication of Duplex Coated U-Mo-Ti Atomized Powder

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Ji Min; Kim, Woo Jeong; Lee, Kyu Hong [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Ryu, Ho Jin [Korea Advanced Institue of Science and Technology, Daejeon (Korea, Republic of)

    2013-10-15

    The results of an annealing test showed that the coating layers inhibit the formation of interaction layers. The results of duplex coating showed that nitride coating layers inhibit the formation of other coating layers. High-density U-Mo alloys are regarded as promising candidates for advanced research reactor fuel as they have shown stable irradiation performance when compared to other uranium alloys and compounds. However, interaction layer formation between the U-Mo alloys and Al matrix degrades the irradiation performance of U-Mo dispersion fuel. Therefore, the addition of Ti in U-Mo alloys, the addition of Si in a Al matrix, and silicide or nitride coating on the surface of U-Mo particles have been proposed to inhibit the interaction layer growth. In this study, U-Mo-Ti alloy powder was produced using a centrifugal atomization method. In addition, nitride and silicide duplex coating layers were fabricated on the surface of the U-Mo-Ti particles. The coated powders were characterized by using X-ray diffraction, SEM, and EDX. Silicide and nitride single coating layers were fabricated on the surface of U-7wt%Mo-1wt%Ti alloys with a thickness of about 10.20 micrometers.

  1. Physical-Layer Security of a Buffer-Aided Full-Duplex Relaying System

    KAUST Repository

    El Shafie, Ahmed

    2016-07-07

    This letter proposes a novel hybrid half-/full-duplex relaying scheme to enhance the relay channel security. A source node (Alice) communicates with her destination node (Bob) in the presence of a buffer-aided full-duplex relay node (Rooney) and a potential eavesdropper (Eve). Rooney adopts two different relaying, namely randomize-and-forward and decode-andforward relaying strategies, to improve the security of the legitimate system. In the first relaying strategy, Rooney uses a codebook different from that used at Alice. In the second relaying strategy, Rooney and Alice use the same codebooks. In addition, Rooney switches between half-duplex and full-duplex modes to further enhance the security of the legitimate system. The numerical results demonstrate that our proposed scheme achieves a significant average secrecy end-to-end throughput improvement relative to the conventional bufferless full-duplex relaying scheme.

  2. Hole Transport in A-form DNA/RNA Hybrid Duplexes

    Science.gov (United States)

    Wong, Jiun Ru; Shao, Fangwei

    2017-01-01

    DNA/RNA hybrid duplexes are prevalent in many cellular functions and are an attractive target form for electrochemical biosensing and electric nanodevice. However the electronic conductivities of DNA/RNA hybrid duplex remain relatively unexplored and limited further technological applications. Here cyclopropyl-modified deoxyribose- and ribose-adenosines were developed to explore hole transport (HT) in both DNA duplex and DNA/RNA hybrids by probing the transient hole occupancies on adenine tracts. HT yields through both B-form and A-form double helixes displayed similar shallow distance dependence, although the HT yields of DNA/RNA hybrid duplexes were lower than those of DNA duplexes. The lack of oscillatory periods and direction dependence in HT through both helixes implied efficient hole propagation can be achieved via the hole delocalization and coherent HT over adenine tracts, regardless of the structural variations.

  3. Throughput Analysis of Full Duplex Communication with Asymmetric Traffic in Small Cell Systems

    DEFF Research Database (Denmark)

    Mahmood, Nurul Huda; Berardinelli, Gilberto; Mogensen, Preben Elgaard;

    2015-01-01

    in this contribution using network analysis tools from stochastic geometry. The analytical findings are further confirmed through computer-based Monte-Carlo simulations. Asymmetric downlink/uplink traffic pattern and the increased network interference stemming from full duplex transmissions are found to limit its......Full duplex communication promises a 100% throughput gain by enabling simultaneous transmission and reception. However, such simultaneous communication leads to a corresponding increase in the network interference. In addition, full duplex communication can only be exploited when traffic...... is available in both uplink and downlink directions; while, cellular network traffic tend to be downlink heavy in practice. The potential throughput gains of full duplex communication over conventional half duplex transmission in a small cell network with asymmetric traffic conditions are investigated...

  4. Sequence-selective targeting of duplex DNA by peptide nucleic acids

    DEFF Research Database (Denmark)

    Nielsen, Peter E

    2010-01-01

    Sequence-selective gene targeting constitutes an attractive drug-discovery approach for genetic therapy, with the aim of reducing or enhancing the activity of specific genes at the transcriptional level, or as part of a methodology for targeted gene repair. The pseudopeptide DNA mimic peptide...... nucleic acid (PNA) can recognize duplex DNA with high sequence specificity and affinity in triplex, duplex and double-duplex invasive modes or non-invasive triplex modes. Novel PNA modification has improved the affinity for DNA recognition via duplex invasion, double-duplex invasion and triplex...... recognition considerably. Such modifications have also resulted in new approaches to targeted gene repair and sequence-selective double-strand cleavage of genomic DNA....

  5. Tissue and allelic-specific expression of hsp70 gene in chickens: basal and heat-stress-induced mRNA level quantified with real-time reverse transcriptase polymerase chain reaction.

    Science.gov (United States)

    Zhen, F-S; Du, H-L; Xu, H-P; Luo, Q-B; Zhang, X-Q

    2006-08-01

    1. The 70 kDa heat shock proteins (hsp70) are a family of molecular chaperones, which promote protein folding and participate in many cellular functions. The objective of the current research was to investigate the relationship between tissue or allele and the expression of chicken hsp70 under normal growth conditions and during acute heat stress (44 degrees C for 4 h). 2. A total of 279 individuals were genotyped for two single nucleotide polymorphisms (A258G and C276G) in chicken hsp70 gene by single-strand conformation polymorphism (SSCP) analysis. 3. The mRNA abundance of chicken hsp70 genes was evaluated by real-time reverse transcriptase polymerase chain reaction (RT-PCR). The expression of hsp70 gene in the liver (9.83 +/- 0.84, 10(7)) was significantly higher than that in the muscle (4.42 +/- 0.36, 10(7)) under normal growth conditions. However, during acute heat stress, the expression of hsp70 gene in the brain (1.82 +/- 0.25, 10(9)) was the highest and was significantly different from those in the liver (1.08 +/- 0.16, 10(9)) and muscle (1.08 +/- 0.13, 10(9)). 4. The expression of hsp70 among different genotypes or haplotype combinations is quite different under normal and heat-stress conditions. The haplotype H3 (GC) is probably advantageous to improving heat resistance of chickens. 5. The results from the present study indicate that the expression of hsp70 in chickens is affected by heat stress, and is tissue- and allele-dependent.

  6. Enhanced H-bonding and pi-stacking in DNA: a potent duplex-stabilizing and mismatch sensing nucleobase analogue

    DEFF Research Database (Denmark)

    Lou, Chenguang; Dallmann, Andre; Marafini, Pietro;

    2014-01-01

    X-pyrene is a new nucleic acid duplex stabilizing cytosine analogue that combines enhanced pi-stacking, hydrogen bonding and electrostatic interactions to greatly increase the stability of bulged DNA duplexes and DNA/RNA hybrids. X-pyrene is highly selective for guanine as a partner and duplex...... analogue for use in a variety of biological applications....

  7. Water-evaporation reduction by duplex films: application to the human tear film.

    Science.gov (United States)

    Cerretani, Colin F; Ho, Nghia H; Radke, C J

    2013-09-01

    Water-evaporation reduction by duplex-oil films is especially important to understand the physiology of the human tear film. Secreted lipids, called meibum, form a duplex film that coats the aqueous tear film and purportedly reduces tear evaporation. Lipid-layer deficiency is correlated with the occurrence of dry-eye disease; however, in-vitro experiments fail to show water-evaporation reduction by tear-lipid duplex films. We review the available literature on water-evaporation reduction by duplex-oil films and outline the theoretical underpinnings of spreading and evaporation kinetics that govern behavior of these systems. A dissolution-diffusion model unifies the data reported in the literature and identifies dewetting of duplex films into lenses as a key challenge to obtaining significant evaporation reduction. We develop an improved apparatus for measuring evaporation reduction by duplex-oil films including simultaneous assessment of film coverage, stability, and temperature, all under controlled external mass transfer. New data reported in this study fit into the larger body of work conducted on water-evaporation reduction by duplex-oil films. Duplex-oil films of oxidized mineral oil/mucin (MOx/BSM), human meibum (HM), and bovine meibum (BM) reduce water evaporation by a dissolution-diffusion mechanism, as confirmed by agreement between measurement and theory. The water permeability of oxidized-mineral-oil duplex films agrees with those reported in the literature, after correction for the presence of mucin. We find that duplex-oil films of bovine and human meibum at physiologic temperature reduce water evaporation only 6-8% for a 100-nm film thickness pertinent to the human tear film. Comparison to in-vivo human tear-evaporation measurements is inconclusive because evaporation from a clean-water surface is not measured and because the mass-transfer resistance is not characterized.

  8. Reliable allele detection using SNP-based PCR primers containing Locked Nucleic Acid: application in genetic mapping

    Directory of Open Access Journals (Sweden)

    Trognitz Friederike

    2007-02-01

    Full Text Available Abstract Background The diploid, Solanum caripense, a wild relative of potato and tomato, possesses valuable resistance to potato late blight and we are interested in the genetic base of this resistance. Due to extremely low levels of genetic variation within the S. caripense genome it proved impossible to generate a dense genetic map and to assign individual Solanum chromosomes through the use of conventional chromosome-specific SSR, RFLP, AFLP, as well as gene- or locus-specific markers. The ease of detection of DNA polymorphisms depends on both frequency and form of sequence variation. The narrow genetic background of close relatives and inbreds complicates the detection of persisting, reduced polymorphism and is a challenge to the development of reliable molecular markers. Nonetheless, monomorphic DNA fragments representing not directly usable conventional markers can contain considerable variation at the level of single nucleotide polymorphisms (SNPs. This can be used for the design of allele-specific molecular markers. The reproducible detection of allele-specific markers based on SNPs has been a technical challenge. Results We present a fast and cost-effective protocol for the detection of allele-specific SNPs by applying Sequence Polymorphism-Derived (SPD markers. These markers proved highly efficient for fingerprinting of individuals possessing a homogeneous genetic background. SPD markers are obtained from within non-informative, conventional molecular marker fragments that are screened for SNPs to design allele-specific PCR primers. The method makes use of primers containing a single, 3'-terminal Locked Nucleic Acid (LNA base. We demonstrate the applicability of the technique by successful genetic mapping of allele-specific SNP markers derived from monomorphic Conserved Ortholog Set II (COSII markers mapped to Solanum chromosomes, in S. caripense. By using SPD markers it was possible for the first time to map the S. caripense alleles

  9. Half-Duplex and Full-Duplex AF and DF Relaying with Energy-Harvesting in Log-Normal Fading

    KAUST Repository

    Rabie, Khaled M.

    2017-08-15

    Energy-harvesting (EH) and wireless power transfer in cooperative relaying networks have recently attracted a considerable amount of research attention. Most of the existing work on this topic however focuses on Rayleigh fading channels, which represent outdoor environments. In contrast, this paper is dedicated to analyze the performance of dual-hop relaying systems with EH over indoor channels characterized by log-normal fading. Both half-duplex (HD) and full-duplex (FD) relaying mechanisms are studied in this work with decode-and-forward (DF) and amplify-and-forward (AF) relaying protocols. In addition, three EH schemes are investigated, namely, time switching relaying, power splitting relaying and ideal relaying receiver which serves as a lower bound. The system performance is evaluated in terms of the ergodic outage probability for which we derive accurate analytical expressions. Monte Carlo simulations are provided throughout to validate the accuracy of our analysis. Results reveal that, in both HD and FD scenarios, AF relaying performs only slightly worse than DF relaying which can make the former a more efficient solution when the processing energy cost at the DF relay is taken into account. It is also shown that FD relaying systems can generally outperform HD relaying schemes as long as the loop-back interference in FD is relatively small. Furthermore, increasing the variance of the log-normal channel has shown to deteriorate the performance in all the relaying and EH protocols considered.

  10. Detection of Toxoplasma gondii and Epstein-Barr virus in HIV patients with clinical symptoms of suspected central nervous system infection using duplex real-time polymerase chain reaction

    Science.gov (United States)

    Rahmawati, E.; Ibrahim, F.; Imran, D.; Sudarmono, P.

    2017-08-01

    Focal brain lesion is a neurological complication in HIV, which is marked as a space occupying lesion (SOL) and needs rapid and effective treatment. This lesion is mainly caused by encephalitis toxoplasma and primary central nervous system lymphoma related to the Epstein-Barr virus (EBV) infection, which is difficult to distinguish using CT scan or magnetic resonance imaging (MRI). The gold standard of diagnosing focal brain lesion has been brain biopsy, but this examination is an invasive procedure that causes complications. The objective of this study is to obtain the rapid laboratory diagnosis of Toxoplasma gondii (T. gondii) and EBV infection. In this experimental study, blood and cerebrospinal fluid were obtained from HIV patients who were admitted to the Neurology Department of Cipto Mangunkusumo Hospital. The samples were examined using duplex real-time polymerase chain reaction (PCR) to detect T. gondii and EBV. The first step was the optimization of duplex real-time PCR, including the annealing temperature, primer and probe concentration, elution volume, and template volume. Minimal DNA detection was used to measure minimal T. gondii and EBV. Cross reactions were determined for technical specificity using the bacteria and viruses Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa, Mycobacterium tuberculosis H37Rv, Candida spp, cytomegalovirus, herpes zoster virus, and varicella zoster virus. Duplex real-time PCR was applied optimally to patients. In the optimization of duplex real-time PCR, the annealing temperature of T. gondii and EBV were 58 °C, the concentration of primer forward and reverse for T. gondii and EBV were 0.2 μM, the concentration of probe for T. gondii and EBV were 0.4μM and 0.2 μM, respectively. Minimal DNA detection of T. gondii and EBV were 5.68 copy/ml and 1.31 copy/ml, respectively. There was no cross reaction between another bacteria and virus that were used as the primer and probe for T. gondii and EBV. The

  11. A duplex real-time reverse transcription polymerase chain reaction for the detection and quantitation of avian leukosis virus subgroups A and B.

    Science.gov (United States)

    Zhou, Gang; Cai, Wenbo; Liu, Xiaolei; Niu, Chengming; Gao, Caixia; Si, Changde; Zhang, Wei; Qu, Liandong; Han, Lingxia

    2011-05-01

    Avian leukosis is a disease that is spreading widely in the world causing large economic losses to the poultry industry. In this study, a duplex quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay was developed to detect and quantify avian leukosis virus subgroups A and B (ALVA/B). The assay was optimised to measure viral gp85 and chicken housekeeping (β-actin) genes. The result showed that the assay was specific for reference strains of ALVA/B subtype and no cross-reaction was detected with ALV subtypes E and J or with four other non-ALV viruses. The assay detected as few as 56 gp85 cDNA copies and was 100-fold more sensitive than a conventional RT-PCR. Seventy clinical blood samples were evaluated by both the qRT-PCR and the conventional RT-PCR assay, and the results show that 65 samples were positive by the qRT-PCR compared with 43 by the conventional RT-PCR. When this assay was used to quantify the viral load in ALV-inoculated embryos from three congenic chicken lines, the embryos from the B21 line showed the highest viral load, whereas the lowest load was found in the B5 line. This assay provides a powerful tool for quantitative detection of the ALVA/B and for the study of host genetic resistance to avian leukosis.

  12. CODEHOP PCR and CODEHOP PCR primer design.

    Science.gov (United States)

    Staheli, Jeannette P; Boyce, Richard; Kovarik, Dina; Rose, Timothy M

    2011-01-01

    While PCR primer design for the amplification of known sequences is usually quite straightforward, the design, and successful application of primers aimed at the detection of as yet unknown genes is often not. The search for genes that are presumed to be distantly related to a known gene sequence, such as homologous genes in different species, paralogs in the same genome, or novel pathogens in diverse hosts, often turns into the proverbial search for the needle in the haystack. PCR-based methods commonly used to address this issue involve the use of either consensus primers or degenerate primers, both of which have significant shortcomings regarding sensitivity and specificity. We have developed a novel primer design approach that diminishes these shortcomings and instead takes advantage of the strengths of both consensus and degenerate primer designs, by combining the two concepts into a Consensus-Degenerate Hybrid Oligonucleotide Primer (CODEHOP) approach. CODEHOP PCR primers contain a relatively short degenerate 3' core and a 5' nondegenerate clamp. The 3' degenerate core consists of a pool of primers containing all possible codons for a 3-4 aminoacid motif that is highly conserved in multiply aligned sequences from known members of a protein family. Each primer in the pool also contains a single 5' nondegenerate nucleotide sequence derived from a codon consensus across the aligned aminoacid sequences flanking the conserved motif. During the initial PCR amplification cycles, the degenerate core is responsible for specific binding to sequences encoding the conserved aminoacid motif. The longer consensus clamp region serves to stabilize the primer and allows the participation of all primers in the pool in the efficient amplification of products during later PCR cycles. We have developed an interactive web site and algorithm (iCODEHOP) for designing CODEHOP PCR primers from multiply aligned protein sequences, which is freely available online. Here, we describe the

  13. La PCR quantitative en temps réel : application à la quantification des OGM

    Directory of Open Access Journals (Sweden)

    Alary Rémi

    2002-11-01

    Full Text Available Suite à l’obligation d’étiquetage, au seuil de 1 %, des aliments contenant des OGM autorisés, il est nécessaire de disposer de méthodes fiables de quantification. Pour répondre à cette obligation, la technique de PCR quantitative en temps réel semble actuellement la mieux adaptée. Son principe, ses avantages et sa mise en oeuvre pour la détermination de la teneur en OGM de farines de soja sont présentés. Les PCR simplex et duplex sont comparées.

  14. l-Proline and RNA Duplex m-Value Temperature Dependence.

    Science.gov (United States)

    Schwinefus, Jeffrey J; Baka, Nadia L; Modi, Kalpit; Billmeyer, Kaylyn N; Lu, Shutian; Haase, Lucas R; Menssen, Ryan J

    2017-08-03

    The temperature dependence of l-proline interactions with the RNA dodecamer duplex surface exposed after unfolding was quantified using thermal and isothermal titration denaturation monitored by uv-absorbance. The m-value quantifying proline interactions with the RNA duplex surface area exposed after unfolding was measured using RNA duplexes with GC content ranging between 17 and 83%. The m-values from thermal denaturation decreased with increasing GC content signifying increasingly favorable proline interactions with the exposed RNA surface area. However, m-values from isothermal titration denaturation at 25.0 °C were independent of GC content and less negative than those from thermal denaturation. The m-value from isothermal titration denaturation for a 50% GC RNA duplex decreased (became more negative) as the temperature increased and was in nearly exact agreement with the m-value from thermal denaturation. Since RNA duplex transition temperatures increased with GC content, the more favorable proline interactions with the high GC content duplex surface area observed from thermal denaturation resulted from the temperature dependence of proline interactions rather than the RNA surface chemical composition. The enthalpy contribution to the m-value was positive and small (indicating a slight increase in duplex unfolding enthalpy with proline) while the entropic contribution to the m-value was positive and increased with temperature. Our results will facilitate proline's use as a probe of solvent accessible surface area changes during biochemical reactions at different reaction temperatures.

  15. Analysis of Structural Flexibility of Damaged DNA Using Thiol-Tethered Oligonucleotide Duplexes.

    Directory of Open Access Journals (Sweden)

    Masashi Fujita

    Full Text Available Bent structures are formed in DNA by the binding of small molecules or proteins. We developed a chemical method to detect bent DNA structures. Oligonucleotide duplexes in which two mercaptoalkyl groups were attached to the positions facing each other across the major groove were prepared. When the duplex contained the cisplatin adduct, which was proved to induce static helix bending, interstrand disulfide bond formation under an oxygen atmosphere was detected by HPLC analyses, but not in the non-adducted duplex, when the two thiol-tethered nucleosides were separated by six base pairs. When the insert was five and seven base pairs, the disulfide bond was formed and was not formed, respectively, regardless of the cisplatin adduct formation. The same reaction was observed in the duplexes containing an abasic site analog and the (6–4 photoproduct. Compared with the cisplatin case, the disulfide bond formation was slower in these duplexes, but the reaction rate was nearly independent of the linker length. These results indicate that dynamic structural changes of the abasic site- and (6–4 photoproduct-containing duplexes could be detected by our method. It is strongly suggested that the UV-damaged DNA-binding protein, which specifically binds these duplexes and functions at the first step of global-genome nucleotide excision repair, recognizes the easily bendable nature of damaged DNA.

  16. Preparation and property of duplex Ni-B-TiO2/Ni nano-composite coatings

    Science.gov (United States)

    Wang, Shu-Jen; Wang, Yuxin; Shu, Xin; Tay, Seeleng; Gao, Wei; Shakoor, R. A.; Kahraman, Ramazan

    2015-03-01

    The duplex Nickel-Boron-Titania/Nickel (Ni-B-TiO2/Ni) coatings were deposited on mild steel by using two baths with Ni as the inner layer. TiO2 nanoparticles were incorporated into the Ni-B coatings as the outer layer by using solid particle mixing method. The microstructure, morphology and corrosion resistance of the duplex Ni-B-TiO2/Ni nanocomposite coatings were systemically investigated. The results show that the duplex interface was uniform and the adhesion between two layers was very good. The microhardness of duplex Ni-B-TiO2/Ni coating was much higher than the Ni coating due to the outer layer of Ni-B-TiO2 coating. The corrosion resistance of the duplex Ni-B-TiO2/Ni coating was also significantly improved comparing with single Ni-B coating. The Ni-B-10 g/L TiO2/Ni coating was found to have the best corrosion resistance among these duplex coatings. This type of duplex Ni-B-TiO2/Ni coating, with high hardness and good corrosion resistance properties, should be able to find broad applications under adverse environmental conditions.

  17. Full Duplex Wireless Communications for Cognitive Radio Networks

    CERN Document Server

    Cheng, Wenchi; Zhang, Hailin

    2011-01-01

    As a key in cognitive radio networks (CRNs), dynamic spectrum access needs to be carefully designed to minimize the interference and delay to the \\emph{primary} (licensed) users. One of the main challenges in dynamic spectrum access is to determine when the \\emph{secondary} (unlicensed) users can use the spectrum. In particular, when the secondary user is using the spectrum, if the primary user becomes active to use the spectrum, it is usually hard for the secondary user to detect the primary user instantaneously, thus causing unexpected interference and delay to primary users. The secondary user cannot detect the presence of primary users instantaneously because the secondary user is unable to detect the spectrum at the same time while it is transmitting. To solve this problem, we propose the full duplex wireless communications scheme for CRNs. In particular, we employ the Antennas Cancellation (AC), the RF Interference Cancellation (RIC), and the Digital Interference Cancellation (DIC) techniques for second...

  18. Current status of duplex surface engineered Ti-based materials

    Institute of Scientific and Technical Information of China (English)

    T.Bell

    2004-01-01

    Industrial exploitation of the high specific strength and corrosion resistance of titanium were dominated historically by the technological advances which have been made in gas-turbine engine and aircraft components. Realization of the possible benefits in general engineering has been limited by the absence of any proven and reliable means of overcoming the poor wear resistance and galling tendency suffered by titanium alloys when in contact with other materials. This problem can only be addressed by optimizing and demonstrating industrially viable surface engineering processes for titanium in general engineering. The status of single and duplex surface engineering systems are reviewed. In addition, in order to fully realize the potential of advanced surface engineering of titanium components contact mechanics models are developed to enable the automotive engineers to design dynamically the loaded automotive engine and transmission components.

  19. Technology of Welding Joints Mixed with Duplex Steel

    Directory of Open Access Journals (Sweden)

    Słania J.

    2016-03-01

    Full Text Available Results of the examinations of sample plates of mixed joints with the duplex steel were discussed. Examinations were taken on the sample plates of mixed joints of sheet plates type P355NL1 and X2CrNiMoN22-5-3 welded by the flux-cored wire DW-329A by the Kobelco company of the following category T 22 9 3 NL RC/M3 in the gas shroud M21 (Ar+18%CO2 (plate no.1, and nickel covered electrodes E Ni 6082 by the Böhler company (plate no. 2. Results of the side bend test of welded joint, transverse tensile test, stretching of the weld metal, impact strength, micro and macroscopic metallographic examinations, and measurements of the delta ferrite content were presented.

  20. Duplex Stirling gas fired heating only heat pump

    Science.gov (United States)

    Beale, W.

    1982-03-01

    In phase I the Duplex Stirling Gas fired Heat Pump was analyzed with the aid of the Sunpower third order simulation and optimization code and found to show heating COP of 1.7 at the rating point of 8 C (17 F) with a heat delivery of 10 kW. Also it was found that a direct heat exchange between working fluid and source and sink air was consistent with high COP in the heat pump, thus eliminating the need for an intermediate heat transfer fluid and its associated pumps and controls. Further, it was demonstrated in the simulation that the heat engine power output could be matched to the heat pump load over the range of source temperatures without need of complex control systems. Cost estimates, along with COP figures from the analysis indicated a payback time of 2 to 4 years in completion with existing combustion heating systems in northern climates.

  1. The Modern Applications of Surface Duplex Treatment Technology

    Institute of Scientific and Technical Information of China (English)

    JerzySmolik; JanWalkowicz; AdamMazurkiewicz; JerzyTomaszewski

    2004-01-01

    The paper presents results of the research carried out by the authors in different fields of plasma surface technologies applications. Three groups of different surface engineering technologies are shown in the paper. The first one concerns the possibility of using the duplex treatment technology for creation of biocompatible diamond-like a-C:H films. The paper presents research results concerning influence of the process parameters of the a-C:H coatings creation by means of the RFPACVD method in the pure methane amlosphere on their phase structure and mechanical properties. In the second case authors present the concept of a new special multilayer thermal barrier coatings with the PAPVD diffusion barrier layers based on aluminium oxide. As the last one the special application of plasma techniques for creation of composite materials characterized by the muffling of mechanical vibration was presented.

  2. The Modern Applications of Surface Duplex Treatment Technology

    Institute of Scientific and Technical Information of China (English)

    Jerzy Smolik; Jan Walkowicz; Adam Mazurkiewicz; Jerzy Tomaszewski

    2004-01-01

    The paper presents results of the research carried out by the authors in different fields of plasma surface technologies applications. Three groups of different surface engineering technologies are shown in the paper. The first one concerns the possibility of using the duplex treatment technology for creation of biocompatible diamond-like a-C:H films.The paper presents research results concerning influence of the process parameters of the a-C:H coatings creation by means of the RF PACVD method in the pure methane atmosphere on their phase structure and mechanical properties. In the second case authors present the concept of a new special multilayer thermal barrier coatings with the PAPVD diffusion barrier layers based on aluminium oxide. As the last one the special application of plasma techniques for creation of composite materials characterized by the muffling of mechanical vibration was presented.

  3. Electromagnetic non-destructive technique for duplex stainless steel characterization

    Science.gov (United States)

    Rocha, João Vicente; Camerini, Cesar; Pereira, Gabriela

    2016-02-01

    Duplex stainless steel (DSS) is a two-phase (ferrite and austenite) material, which exhibits an attractive combination of mechanical properties and high corrosion resistance, being commonly employed for equipment of petrochemical plants, refining units and oil & gas platforms. The best properties of DSS are achieved when the phases are in equal proportions. However, exposition to high temperatures (e.g. welding process) may entail undesired consequences, such as deleterious phases precipitation (e.g. sigma, chi) and different proportion of the original phases, impairing dramatically the mechanical and corrosion properties of the material. A detailed study of the magnetic behavior of DSS microstructure with different ferrite austenite ratios and deleterious phases content was accomplished. The non destructive method evaluates the electromagnetic properties changes in the material and is capable to identify the presence of deleterious phases into DSS microstructure.

  4. Hybridization accompanying FRET event in labeled natural nucleoside-unnatural nucleoside containing chimeric DNA duplexes.

    Science.gov (United States)

    Bag, Subhendu Sekhar; Das, Suman K; Pradhan, Manoj Kumar; Jana, Subhashis

    2016-09-01

    Förster resonance energy transfer (FRET) is a highly efficient strategy in illuminating the structures, structural changes and dynamics of DNA, proteins and other biomolecules and thus is being widely utilized in studying such phenomena, in designing molecular/biomolecular probes for monitoring the hybridization event of two single stranded DNA to form duplex, in gene detection and in many other sensory applications in chemistry, biology and material sciences. Moreover, FRET can give information about the positional status of chromophores within the associated biomolecules with much more accuracy than other methods can yield. Toward this end, we want to report here the ability of fluorescent unnatural nucleoside, triazolylphenanthrene ((TPhen)BDo) to show FRET interaction upon hybridization with fluorescently labeled natural nucleosides, (Per)U or (OxoPy)U or (Per)U, forming two stable chimeric DNA duplexes. The pairing selectivity and the thermal duplex stability of the chimeric duplexes are higher than any of the duplexes with natural nucleoside formed. The hybridization results in a Förster resonance energy transfer (FRET) from donor triazolylphenanthrene of (TPhen)BDo to acceptor oxopyrene of (OxoPy)U and/or to perylene chromophore of (Per)U, respectively, in two chimeric DNA duplexes. Therefore, we have established the FRET process in two chimeric DNA duplexes wherein a fluorescently labeled natural nucleoside ((OxoPy)U or (Per)U) paired against an unnatural nucleoside ((TPhen)BDo) without sacrificing the duplex stability and B-DNA conformation. The hybridization accompanying FRET event in these classes of interacting fluorophores is new. Moreover, there is no report of such designed system of chimeric DNA duplex. Our observed phenomenon and the design can potentially be exploited in designing more of such efficient FRET pairs for useful application in the detection and analysis of biomolecular interactions and in material science application. Copyright

  5. Duplex Doppler ultrasound study of the temporomandibular joint.

    Science.gov (United States)

    Stagnitti, A; Marini, A; Impara, L; Drudi, F M; Lo Mele, L; Lillo Odoardi, G

    2012-06-01

    Sommario INTRODUZIONE: La fisiologia articolare dell’articolazione temporo-mandibolare (ATM) può essere esaminata sia dal punto di vista clinico che strumentale. La diagnostica per immagini ha da tempo contribuito con la risonanza magnetica (RM) e anche con la radiografia (Rx) e la tomografia computerizzata (TC) all’analisi della morfologia dei capi articolari e della cinetica condilare. L’esame duplex-ecodoppler è una metodica di largo impiego nello studio delle strutture in movimento in particolar modo a livello delle strutture del sistema vascolare. MATERIALI E METODI: È stata utilizzata un’apparecchiatura Toshiba APLIO SSA-770A, con l’uso di tecnica duplex-ecodoppler multi display, che consente la visualizzazione contemporanea dell’immagine ecografica e dei segnali Doppler utilizzando una sonda lineare del tipo phased array con cristalli trasduttori funzionanti ad una frequenza fondamentale di 6 MHz per gli spettri Doppler pulsati e 7.5 MHz per l’imaging ecografico. Sono stati esaminati nel Dipartimento di Scienze Radiologiche, Oncologiche e Anatomo-patologiche dell’Università “Sapienza” di Roma, 30 pazienti del reparto di Ortognatodonzia dell’Istituto di Odontoiatria della stessa Università. RISULTATI: Nei pazienti normali si è ottenuta un’alternanza regolare degli spettri Doppler, mentre nei soggetti con disfunzioni del complesso condilo-meniscale, si è persa la regolarità della sommatoria degli spettri di Fourier, con altezze incostanti in relazione a spostamenti irregolari del complesso condilo-meniscale. CONCLUSIONI: L’esame ecodoppler si è dimostrato, in tutti i pazienti, capace di discriminare quelli normali dai patologici e tra questi ultimi ha permesso di identificare gli aspetti più significativi delle patologie disfunzionali.

  6. DNA Duplexes with Hydrophobic Modifications Inhibit Fusion between HIV-1 and Cell Membranes

    OpenAIRE

    Xu, Liang; Cai, Lifeng; Chen, Xueliang; Jiang, Xifeng; Chong, Huihui; Zheng, Baohua; Wang, Kun; He, Junlin; Chen, Wei; ZHANG, Tao; Cheng, Maosheng; He, Yuxian; Liu, Keliang

    2013-01-01

    Discovery of new drugs for the treatment of AIDS typically possessing unique structures associated with novel mechanisms of action has been of great importance due to the quick drug-resistant mutations of HIV-1 strains. The work presented in this report describes a novel class of DNA duplex-based HIV-1 fusion inhibitors. Hydrophobic groups were introduced into a DNA duplex skeleton either at one end, at both ends, or in the middle. These modified DNA duplexes inhibited fusion between HIV-1 an...

  7. Duplex stainless steel surface bay laser cladding; Modificacion de las propiedades superficiales de aceros inoxidables Duplex mediante recubrimientos por laser

    Energy Technology Data Exchange (ETDEWEB)

    Amigo, V.; Pineda, Y.; Segovia, F.; Vicente, A.

    2004-07-01

    Laser cladding is one of the most promising techniques to restore damaged surfaces and achieve properties similar to those of the base metal. In this work, duplex stainless steels have been cladded by a nickel alloy under different processing conditions. The influence of the beam speed and defocusing variables ha been evaluated in the microstructure both of the cladding and heat affected zone, HAZ. These results have been correlated to mechanical properties by means of microhardness measurements from cladding area to base metal through the interface. This technique has shown to be very appropriate to obtain controlled mechanical properties as they are determined by the solidification microstructure, originated by the transfer of mass and heat in the system. (Author) 21 refs.

  8. Transcranial duplex doppler ultrasonography in dogs with hydrocephalus Ultrassonografia duplex Doppler transcraniana em cães com hidrocefalia

    Directory of Open Access Journals (Sweden)

    C.F. Carvalho

    2010-02-01

    Full Text Available Transcranial duplex Doppler ultrasound was performed in 32 conscious dogs presenting ventriculomegaly detected in B-mode, obtaining measures of lateral ventricles and resistance parameters of main cerebral arteries before and after 30 days of clinical treatment. The animals were distributed divided in two groups: group 1, dogs that presented remission or decrease of the neurological signs of the disease after clinical therapy; and group 2, dogs that displayed worsening of the neurological signs or clinical stability. The data were all presented in tables and were submitted to paired t test and to logistic regression models to evaluate the influence of the RI reduction on both groups. There was no significant influence of the variables in the B-mode. After the treatment, the mean resistive index (RI was significantly lower for the group that presented clinical improvement. There was no significant difference of the mean RI reduction for the same artery when the right and left sides were compared. It could be concluded that transcranial duplex Doppler ultrasound is a method of cerebral hemodynamic assessment able to monitor more precisely the treatment of hydrocephalus in dogs and verify the responses.Foi realizada a ultrassonografia duplex Doppler transcraniana em 32 cães, conscientes, que apresentavam ventriculomegalia previamente detectada ao exame modo-B, para obter as medidas dos ventrículos laterais e os parâmetros de resistência das artérias cerebrais antes e depois de 30 dias de tratamento clínico. Os animais foram distribuídos em dois grupos: 1 - formado por cães que apresentaram remissão ou diminuição dos sinais clínicos após tratamento; 2 - por cães que apresentaram piora dos sinais neurológicos ou estabilidade do quadro clínico. Todos os dados foram dispostos em tabelas e submetidos ao teste t pareado e a modelos de regressão logística para avaliar a influência da redução do índice de resistividade (IR. Não foi

  9. Quantitative analysis of food and feed samples with droplet digital PCR.

    Science.gov (United States)

    Morisset, Dany; Štebih, Dejan; Milavec, Mojca; Gruden, Kristina; Žel, Jana

    2013-01-01

    In this study, the applicability of droplet digital PCR (ddPCR) for routine analysis in food and feed samples was demonstrated with the quantification of genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of GMOs in products. However, its use is limited for detecting and quantifying very small numbers of DNA targets, as in some complex food and feed matrices. Using ddPCR duplex assay, we have measured the absolute numbers of MON810 transgene and hmg maize reference gene copies in DNA samples. Key performance parameters of the assay were determined. The ddPCR system is shown to offer precise absolute and relative quantification of targets, without the need for calibration curves. The sensitivity (five target DNA copies) of the ddPCR assay compares well with those of individual qPCR assays and of the chamber digital PCR (cdPCR) approach. It offers a dynamic range over four orders of magnitude, greater than that of cdPCR. Moreover, when compared to qPCR, the ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors. Finally, ddPCR throughput and cost are advantageous relative to those of qPCR for routine GMO quantification. It is thus concluded that ddPCR technology can be applied for routine quantification of GMOs, or any other domain where quantitative analysis of food and feed samples is needed.

  10. Quantitative analysis of food and feed samples with droplet digital PCR.

    Directory of Open Access Journals (Sweden)

    Dany Morisset

    Full Text Available In this study, the applicability of droplet digital PCR (ddPCR for routine analysis in food and feed samples was demonstrated with the quantification of genetically modified organisms (GMOs. Real-time quantitative polymerase chain reaction (qPCR is currently used for quantitative molecular analysis of the presence of GMOs in products. However, its use is limited for detecting and quantifying very small numbers of DNA targets, as in some complex food and feed matrices. Using ddPCR duplex assay, we have measured the absolute numbers of MON810 transgene and hmg maize reference gene copies in DNA samples. Key performance parameters of the assay were determined. The ddPCR system is shown to offer precise absolute and relative quantification of targets, without the need for calibration curves. The sensitivity (five target DNA copies of the ddPCR assay compares well with those of individual qPCR assays and of the chamber digital PCR (cdPCR approach. It offers a dynamic range over four orders of magnitude, greater than that of cdPCR. Moreover, when compared to qPCR, the ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors. Finally, ddPCR throughput and cost are advantageous relative to those of qPCR for routine GMO quantification. It is thus concluded that ddPCR technology can be applied for routine quantification of GMOs, or any other domain where quantitative analysis of food and feed samples is needed.

  11. Duplex real-time polymerase chain reaction reveals competition between Erwinia amylovora and E. pyrifoliae on pear blossoms.

    Science.gov (United States)

    Lehman, Susan M; Kim, Won-Sik; Castle, Alan J; Svircev, Antonet M

    2008-06-01

    Erwinia amylovora and E. pyrifoliae are the causative agents of fire blight and Asian pear blight, respectively. The pathogens are closely related, with overlapping host ranges. Data are unavailable on the current distribution of E. pyrifoliae and on the interaction between the two species when they are present together on the same host. In this study, a duplex real-time polymerase chain reaction (PCR) protocol was developed to monitor the population dynamics of E. amylovora and E. pyrifoliae on the surface of Bartlett pear blossoms. Bacterial cells washed from blossoms were used directly as the PCR template without DNA extraction. Primers and a probe based on the E. amylovora levansucrase gene detected all E. amylovora strains. All E. pyrifoliae strains, including the Japanese Erwinia strains previously described as E. amylovora, were detected with a primer and probe combination based on the E. pyrifoliae hrpW gene. Disease development and severity were not significantly different in blossoms inoculated with individual Erwinia species or with a mixture of the two species. However, E. amylovora grew to greater population sizes than did E. pyrifoliae in both single species inoculations and in mixtures, suggesting that E. amylovora has a greater competitive fitness on Bartlett pear blossoms than E. pyrifoliae.

  12. Two-temperature LATE-PCR endpoint genotyping

    Directory of Open Access Journals (Sweden)

    Reis Arthur H

    2006-12-01

    Full Text Available Abstract Background In conventional PCR, total amplicon yield becomes independent of starting template number as amplification reaches plateau and varies significantly among replicate reactions. This paper describes a strategy for reconfiguring PCR so that the signal intensity of a single fluorescent detection probe after PCR thermal cycling reflects genomic composition. The resulting method corrects for product yield variations among replicate amplification reactions, permits resolution of homozygous and heterozygous genotypes based on endpoint fluorescence signal intensities, and readily identifies imbalanced allele ratios equivalent to those arising from gene/chromosomal duplications. Furthermore, the use of only a single colored probe for genotyping enhances the multiplex detection capacity of the assay. Results Two-Temperature LATE-PCR endpoint genotyping combines Linear-After-The-Exponential (LATE-PCR (an advanced form of asymmetric PCR that efficiently generates single-stranded DNA and mismatch-tolerant probes capable of detecting allele-specific targets at high temperature and total single-stranded amplicons at a lower temperature in the same reaction. The method is demonstrated here for genotyping single-nucleotide alleles of the human HEXA gene responsible for Tay-Sachs disease and for genotyping SNP alleles near the human p53 tumor suppressor gene. In each case, the final probe signals were normalized against total single-stranded DNA generated in the same reaction. Normalization reduces the coefficient of variation among replicates from 17.22% to as little as 2.78% and permits endpoint genotyping with >99.7% accuracy. These assays are robust because they are consistent over a wide range of input DNA concentrations and give the same results regardless of how many cycles of linear amplification have elapsed. The method is also sufficiently powerful to distinguish between samples with a 1:1 ratio of two alleles from samples comprised of

  13. Two-temperature LATE-PCR endpoint genotyping

    Science.gov (United States)

    Sanchez, J Aquiles; Abramowitz, Jessica D; Salk, Jesse J; Reis, Arthur H; Rice, John E; Pierce, Kenneth E; Wangh, Lawrence J

    2006-01-01

    Background In conventional PCR, total amplicon yield becomes independent of starting template number as amplification reaches plateau and varies significantly among replicate reactions. This paper describes a strategy for reconfiguring PCR so that the signal intensity of a single fluorescent detection probe after PCR thermal cycling reflects genomic composition. The resulting method corrects for product yield variations among replicate amplification reactions, permits resolution of homozygous and heterozygous genotypes based on endpoint fluorescence signal intensities, and readily identifies imbalanced allele ratios equivalent to those arising from gene/chromosomal duplications. Furthermore, the use of only a single colored probe for genotyping enhances the multiplex detection capacity of the assay. Results Two-Temperature LATE-PCR endpoint genotyping combines Linear-After-The-Exponential (LATE)-PCR (an advanced form of asymmetric PCR that efficiently generates single-stranded DNA) and mismatch-tolerant probes capable of detecting allele-specific targets at high temperature and total single-stranded amplicons at a lower temperature in the same reaction. The method is demonstrated here for genotyping single-nucleotide alleles of the human HEXA gene responsible for Tay-Sachs disease and for genotyping SNP alleles near the human p53 tumor suppressor gene. In each case, the final probe signals were normalized against total single-stranded DNA generated in the same reaction. Normalization reduces the coefficient of variation among replicates from 17.22% to as little as 2.78% and permits endpoint genotyping with >99.7% accuracy. These assays are robust because they are consistent over a wide range of input DNA concentrations and give the same results regardless of how many cycles of linear amplification have elapsed. The method is also sufficiently powerful to distinguish between samples with a 1:1 ratio of two alleles from samples comprised of 2:1 and 1:2 ratios of the

  14. Structural basis for duplex RNA recognition and cleavage by Archaeoglobus fulgidus C3PO

    Science.gov (United States)

    Parizotto, Eneida A; Lowe, Edward D; Parker, James S

    2013-01-01

    Oligomeric complexes of Trax and Translin proteins, known as C3POs, participate in a variety of eukaryotic nucleic acid metabolism pathways including RNAi and tRNA processing. In RNAi in humans and Drosophila, C3PO activates pre-RISC by removing the passenger strand of the siRNA precursor duplex using nuclease activity present in Trax. It is not known how C3POs engage with nucleic acid substrates. Here we identify a single protein from Archaeoglobus fulgidus that assembles into an octamer with striking similarity to human C3PO. The structure in complex with duplex RNA reveals that the octamer entirely encapsulates a single thirteen base-pair RNA duplex inside a large inner cavity. Trax-like subunit catalytic sites target opposite strands of the duplex for cleavage, separated by seven base pairs. The structure provides insight into the mechanism of RNA recognition and cleavage by an archaeal C3PO-like complex. PMID:23353787

  15. Two-flux transfer matrix model for predicting the reflectance and transmittance of duplex halftone prints.

    Science.gov (United States)

    Mazauric, Serge; Hébert, Mathieu; Simonot, Lionel; Fournel, Thierry

    2014-12-01

    We introduce a model allowing convenient calculation of the spectral reflectance and transmittance of duplex prints. It is based on flux transfer matrices and enables retrieving classical Kubelka-Munk formulas, as well as extended formulas for nonsymmetric layers. By making different assumptions on the flux transfers, we obtain two predictive models for the duplex halftone prints: the "duplex Clapper-Yule model," which is an extension of the classical Clapper-Yule model, and the "duplex primary reflectance-transmittance model." The two models can be calibrated from either reflectance or transmittance measurements; only the second model can be calibrated from both measurements, thus giving optimal accuracy for both reflectance and transmittance predictions. The conceptual differences between the two models are deeply analyzed, as well as their advantages and drawbacks in terms of calibration. According to the test carried out in this study with paper printed in inkjet, their predictive performances are good provided appropriate calibration options are selected.

  16. Intercalating nucleic acids: the influence of linker length and intercalator type on their duplex stabilities.

    Science.gov (United States)

    Christensen, Ulf B; Wamberg, Michael; El-Essawy, Farag A G; Ismail, Abd El-Hamid; Nielsen, Christina B; Filichev, Vyacheslav V; Jessen, Carsten H; Petersen, Michael; Pedersen, Erik B

    2004-01-01

    Six new examples of intercalating nucleic acids were synthesized in order to evaluate the dependence of the length of the linker between oligo and intercalator on the thermal stability of their corresponding duplexes and triplexes.

  17. Can Full Duplex reduce the discovery time in D2D Communication?

    DEFF Research Database (Denmark)

    Gatnau, Marta; Berardinelli, Gilberto; Mahmood, Nurul Huda;

    2016-01-01

    Device-to-device (D2D) communication is considered as one of the key technologies to support new types of services, such as public safety and proximity-based applications. D2D communication requires a discovery phase, i.e., the node awareness procedure prior to the communication phase. Conventional...... half duplex transmission may not be sufficient to provide fast discovery and cope with the strict latency targets of future 5G services. On the other hand, in-band full duplex, by allowing simultaneous transmission and reception, may complete the discovery phase faster. In this paper, the potential...... of full duplex in providing fast discovery for the next 5th generation (5G) system supporting D2D communication is investigated. A design for such system is presented and evaluated via simulations, showing that full duplex can accelerate the discovery phase by supporting a higher transmission probability...

  18. Rapid method to detect duplex formation in sequencing by hybridization methods

    Science.gov (United States)

    Mirzabekov, A.D.; Timofeev, E.N.; Florentiev, V.L.; Kirillov, E.V.

    1999-01-19

    A method for determining the existence of duplexes of oligonucleotide complementary molecules is provided. A plurality of immobilized oligonucleotide molecules, each of a specific length and each having a specific base sequence, is contacted with complementary, single stranded oligonucleotide molecules to form a duplex. Each duplex facilitates intercalation of a fluorescent dye between the base planes of the duplex. The invention also provides for a method for constructing oligonucleotide matrices comprising confining light sensitive fluid to a surface and exposing the light-sensitive fluid to a light pattern. This causes the fluid exposed to the light to coalesce into discrete units and adhere to the surface. This places each of the units in contact with a set of different oligonucleotide molecules so as to allow the molecules to disperse into the units. 13 figs.

  19. Electrochemical Investigation of Interaction between a Bifunctional Probe and GG Mismatch Duplex.

    Science.gov (United States)

    Li, Jiao; He, Hanping; Peng, Xiaoqian; Huang, Min; Zhang, Xiuhua; Wang, Shengfu

    2015-01-01

    A bifunctional probe (FecNC), containing a recognition part and an electrochemical active center, was applied to electrochemical detection of GG mismatch duplexes. The preparation of gold electrodes modified by mismatch and complementatry duplexes was characterized by electrochemical impedance spectroscopy (EIS) and optimized for better detection in terms of self-assembly time, hybridization time, and incubation time. The interaction between FecNC and DNA duplexes modified on the surface of a gold electrode was explored by square wave voltammetry (SWV) and EIS. The results showed that the DNA duplexes with GG mismatch on the surface of a gold electrode was easily detected by the largest electrochemical signal of the bifunctional probe because of its selective binding to GG mismatches. The bifunctional probe could offer a simple, effective electrochemical detection of GG mismatches, and theoretical bases for development of electrochemical biosensors. Further, the method would be favorable for diagnosis of genetic diseases.

  20. Artificial neural network aided non-invasive grading evaluation of hepatic fibrosis by duplex ultrasonography

    National Research Council Canada - National Science Library

    Zhang, Li; Li, Qiao-Ying; Duan, Yun-You; Yan, Guo-Zhen; Yang, Yi-Lin; Yang, Rui-Jing

    2012-01-01

    Artificial neural networks (ANNs) are widely studied for evaluating diseases. This paper discusses the intelligence mode of an ANN in grading the diagnosis of liver fibrosis by duplex ultrasonogaphy...

  1. Phase transformations evaluation on a UNS S31803 duplex stainless steel based on nondestructive testing

    Energy Technology Data Exchange (ETDEWEB)

    Macedo Silva, Edgard de, E-mail: edgard@cefetpb.edu.br [Centro federal de Educacao Tecnologica da Paraiba (CEFET PB), Area da Industria, Avenida 1o de Maio, 720 - 58015-430 - Joao Pessoa/PB (Brazil); Costa de Albuquerque, Victor Hugo, E-mail: victor.albuquerque@fe.up.pt [Universidade Federal da Paraiba (UFPB), Departamento de Engenharia Mecanica (DEM), Cidade Universitaria, S/N - 58059-900 - Joao Pessoa/PB (Brazil); Pereira Leite, Josinaldo, E-mail: josinaldo@ct.ufpb.br [Universidade Federal da Paraiba (UFPB), Departamento de Engenharia Mecanica (DEM), Cidade Universitaria, S/N - 58059-900 - Joao Pessoa/PB (Brazil); Gomes Varela, Antonio Carlos, E-mail: varela@cefetpb.edu.br [Universidade Federal da Paraiba (UFPB), Departamento de Engenharia Mecanica (DEM), Cidade Universitaria, S/N - 58059-900 - Joao Pessoa/PB (Brazil); Pinho de Moura, Elineudo, E-mail: elineudo@pq.cnpq.br [Universidade Federal do Ceara (UFC), Departamento de Engenharia Metalurgica e de Materiais, Campus do Pici, Bloco 715, 60455-760 - Fortaleza/CE (Brazil); Tavares, Joao Manuel R.S., E-mail: tavares@fe.up.pt [Faculdade de Engenharia da Universidade do Porto (FEUP), Departamento de Engenharia Mecanica e Gestao Industrial (DEMEGI)/Instituto de Engenharia Mecanica e Gestao Industrial - INEGI, Rua Dr. Roberto Frias, s/n, 4200-465 Porto (Portugal)

    2009-08-15

    Duplex stainless steel presents special mechanical properties such as, for example, mechanical and corrosion strength, becoming competitive in relation to the other types of stainless steel. One of the great problems of duplex stainless steel microstructural changes study is related to embrittlement above 300 deg. C, with the precipitation of the {alpha}' phase occurring over the ferritic microstructure. Aiming to characterise embrittlement of duplex stainless steel, hardening kinetics, from 425 to 475 deg. C, was analysed through the speed of sound, Charpy impact energy, X-ray diffraction, hardness and microscopy parameters. The presence of two hardening stages, detected through the speed of sound, was observed, one being of brittle characteristic and the other ductile. Moreover, the speed of sound showed a direct correlation with the material's hardness. Thus, it is concluded that the speed of sound is a promising nondestructive parameter to follow-up embrittlement in duplex stainless steel.

  2. Management of multiple type Ⅱ endoleaks detected by duplex ultrasound after endovascular abdominal aneurysm repair

    Institute of Scientific and Technical Information of China (English)

    GUO Da-qiao; LI Wei-miao; JIANG Jun-hao; SHI Zhen-yu; WANG Yu-qi; FU Wei-guo

    2012-01-01

    We reported a case of multiple type Ⅱ endoleaks detected by duplex ultrasound after endovascular abdominal aneurysm repair.The patient was undergoing warfarin therapy.Duplex ultrasound was applied as the sole surveillance method during follow-up and provided the concerned information for reintervention.The endoleaks were successfully repaired by coil embolization of the collaterals from the internal iliac artery feeding the fourth lumbar artery.

  3. The influence of sintering time on the properties of PM duplex stainless steel

    OpenAIRE

    Z. Brytan; L.A. Dobrzański; M. Actis Grande; Rosso, M.

    2009-01-01

    Purpose: The purpose of this paper is to analyse the effect of sintering time on the pore morphology, microstructural changes, tensile properties and corrosion resistance of vacuum sintered duplex stainless steel.Design/methodology/approach: In presented study PM duplex stainless steels were obtained through mixing base ferritic stainless steel powder with controlled addition of elemental alloying powders and then sintered in a vacuum furnace with argon backfilling at 1250°C for different tim...

  4. Minor Groove Binding between Norfloxacin and DNA Duplexes in Solution: A Molecular Dynamics Study

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Molecular dynamics were used to investigate the interaction between norfloxacin and DNA duplex. The results showed that norfloxacin was situated in the minor groove of DNA,binding to the TCGA region of d [ATATCGATAT] 2. Specific hydrogen bonds were formed between norfloxacin and guanine base of DNA during the 2 ns MD, which may be the reason for the preferentiality of quinolone antibacterial towards the guanine base of DNA duplex.

  5. RCFD: A Frequency Based Channel Access Scheme for Full Duplex Wireless Networks

    OpenAIRE

    Luvisotto, Michele; Sadeghi, Alireza; Lahouti, Farshad; Vitturi, Stefano; Zorzi, Michele

    2016-01-01

    Recently, several working implementations of in--band full--duplex wireless systems have been presented, where the same node can transmit and receive simultaneously in the same frequency band. The introduction of such a possibility at the physical layer could lead to improved performance but also poses several challenges at the MAC layer. In this paper, an innovative mechanism of channel contention in full--duplex OFDM wireless networks is proposed. This strategy is able to ensure efficient t...

  6. Pitting Corrosion of Super Duplex Stainless Steel - Effect of Isothermal Heat Treament

    OpenAIRE

    Lauritsen, Christian Rene

    2016-01-01

    Super duplex stainless steels (SDSS), with a chromium content of 25 wt$\\%$, contain a duplex structure which consists of ferrite and austenite, and have a pitting resistance equivalent number (PREN) equal or higher than 40. SDSS are affected by the alloying elements, microstructure and fabrication processes. The high degree of alloying elements in SDSS can lead to formation of intermetallic precipitates and secondary phases during heat treatments. Detrimental phases, such as sigma ($\\sigma$) ...

  7. In-Band α-Duplex Scheme for Cellular Networks: A Stochastic Geometry Approach

    KAUST Repository

    Alammouri, Ahmad

    2016-07-13

    In-band full-duplex (FD) communications have been optimistically promoted to improve the spectrum utilization and efficiency. However, the penetration of FD communications to the cellular networks domain is challenging due to the imposed uplink/downlink interference. This paper presents a tractable framework, based on stochastic geometry, to study FD communications in cellular networks. Particularly, we assess the FD communications effect on the network performance and quantify the associated gains. The study proves the vulnerability of the uplink to the downlink interference and shows that FD rate gains harvested in the downlink (up to 97%) come at the expense of a significant degradation in the uplink rate (up to 94%). Therefore, we propose a novel fine-grained duplexing scheme, denoted as -duplex scheme, which allows a partial overlap between the uplink and the downlink frequency bands. We derive the required conditions to harvest rate gains from the -duplex scheme and show its superiority to both the FD and half-duplex (HD) schemes. In particular, we show that the -duplex scheme provides a simultaneous improvement of 28% for the downlink rate and 56% for the uplink rate. Finally, we show that the amount of the overlap can be optimized based on the network design objective.

  8. A duplex kidney with dromedary hump showing altered hilar anatomy

    Directory of Open Access Journals (Sweden)

    Tallapaneni S

    2011-02-01

    Full Text Available A comprehensive knowledge of the wide range of variations of renal vasculature and renal pelvis is mandatory to the anatomists for a better understanding of the embryology. It remains as the key issue in determining the technical feasibility of various endourologic procedures and innumerable intervention techniques besides kidney retrievals for transplantation. In the present case the duplex kidney showed lobulations on the anterior surface just adjacent to the hilar region. The midlateral portion of the convex lateral border of the kidney showed a small focal bulge –dromedary hump. At the hilum reversed anterio-posterior disposition of renal vasculature with anteriorly placed renal artery which bifurcated into two upper and lower anterior segmental branches. The renal vein formed by large tributaries arising from the hilum running towards the inferior vena cava. The renal pelvis was most posteriorly placed which showed a double pelvis. The upper pelvis was seen arising behind the renal vein and the lower pelvis arising inferomedial to the lower anterior segmental artery.Both the pelvises were seen uniting medial to the lower part of hilum and continued as a single ureter which opened into the bladder. The thorough knowledge of these anatomical variations is necessary to avoid iatrogenic injuries and enable the surgeon and radiologists approach unusual situations with confidence rather than surprise.

  9. Hardfacing of duplex stainless steel using melting and diffusion processes

    Science.gov (United States)

    Lailatul, H.; Maleque, M. A.

    2017-03-01

    Duplex stainless steel (DSS) is a material with high potential successes in many new applications such as rail car manufacturing, automotive and chemical industries. Although DSS is widely used in various industries, this material has faced wear and hardness problems which obstruct a wider capability of this material and causes problems in current application. Therefore, development of surface modification has been introduced to produce hard protective layer or coating on DSS. The main aim of this work is to brief review on hard surface layer formation on DSS using melting and diffusion processes. Melting technique using tungsten inert gas (TIG) torch and diffusion technique using gas nitriding are the effective process to meet this requirement. The processing route plays a significant role in developing the hard surface layer for any application with effective cost and environmental factors. The good understanding and careful selection of processing route to form products are very important factors to decide the suitable techniques for surface engineering treatment. In this paper, an attempt is also made to consolidate the important research works done on melting and diffusion techniques of DSS in the past. The advantages and disadvantages between melting and diffusion technique are presented for better understanding on the feasibility of hard surface formation on DSS. Finally, it can be concluded that this work will open an avenue for further research on the application of suitable process for hard surface formation on DSS.

  10. Creep properties of aged duplex stainless steels containing [sigma] phase

    Energy Technology Data Exchange (ETDEWEB)

    Shek, C.H.; Wong, K.W.; Lai, J.K.L. (City Univ. of Hong Kong, Kowloon (Hong Kong). Dept. of Physics and Materials Science); Li, D.J. (Department of Materials Engineering, Dalian University of Technology, Dalian 116 024 (China))

    1999-06-30

    The creep properties of a cast of duplex stainless steel were characterized at temperatures 550-800 C under different loading conditions. For fully aged specimens containing [sigma], the stress exponent for creep was close to 3 and the activation energy was 281[+-]9 kJ mol[sup -1]. The results suggested that the creep mechanism in the samples in this investigation was controlled by dislocation movement. Extensive [sigma]/[gamma][sub 2] interfaces introduced during ageing improved the creep resistance of the material and related to a reduction of the creep rate in Stage II creep and an increase in the creep rupture strength of the material. Microstructural studies revealed the dependence of the creep properties on the morphology of the microstructure. Among the aged specimens containing [sigma], the creep strength and ductility were higher for specimens having larger [gamma] grain thickness measured on the longitudinal plane. This characteristic was related to the crack propagation and interconnection of voids within [gamma] matrix during tertiary creep. With appropriate solution treatment, the creep strength of [sigma]-containing steels can be improved to a value exceeding that of type 316 steels. (orig.) 14 refs.

  11. Beamforming Based Full-Duplex for Millimeter-Wave Communication.

    Science.gov (United States)

    Liu, Xiao; Xiao, Zhenyu; Bai, Lin; Choi, Jinho; Xia, Pengfei; Xia, Xiang-Gen

    2016-07-21

    In this paper, we study beamforming based full-duplex (FD) systems in millimeter-wave (mmWave) communications. A joint transmission and reception (Tx/Rx) beamforming problem is formulated to maximize the achievable rate by mitigating self-interference (SI). Since the optimal solution is difficult to find due to the non-convexity of the objective function, suboptimal schemes are proposed in this paper. A low-complexity algorithm, which iteratively maximizes signal power while suppressing SI, is proposed and its convergence is proven. Moreover, two closed-form solutions, which do not require iterations, are also derived under minimum-mean-square-error (MMSE), zero-forcing (ZF), and maximum-ratio transmission (MRT) criteria. Performance evaluations show that the proposed iterative scheme converges fast (within only two iterations on average) and approaches an upper-bound performance, while the two closed-form solutions also achieve appealing performances, although there are noticeable differences from the upper bound depending on channel conditions. Interestingly, these three schemes show different robustness against the geometry of Tx/Rx antenna arrays and channel estimation errors.

  12. Beamforming Based Full-Duplex for Millimeter-Wave Communication

    Directory of Open Access Journals (Sweden)

    Xiao Liu

    2016-07-01

    Full Text Available In this paper, we study beamforming based full-duplex (FD systems in millimeter-wave (mmWave communications. A joint transmission and reception (Tx/Rx beamforming problem is formulated to maximize the achievable rate by mitigating self-interference (SI. Since the optimal solution is difficult to find due to the non-convexity of the objective function, suboptimal schemes are proposed in this paper. A low-complexity algorithm, which iteratively maximizes signal power while suppressing SI, is proposed and its convergence is proven. Moreover, two closed-form solutions, which do not require iterations, are also derived under minimum-mean-square-error (MMSE, zero-forcing (ZF, and maximum-ratio transmission (MRT criteria. Performance evaluations show that the proposed iterative scheme converges fast (within only two iterations on average and approaches an upper-bound performance, while the two closed-form solutions also achieve appealing performances, although there are noticeable differences from the upper bound depending on channel conditions. Interestingly, these three schemes show different robustness against the geometry of Tx/Rx antenna arrays and channel estimation errors.

  13. Transperitoneal laparoscopic heminephrectomy in duplex kidneys: a one centre experience

    Directory of Open Access Journals (Sweden)

    Mehdi Abedinzadeh

    2012-02-01

    Full Text Available Purpose: The standard treatment for a duplex kidney with poorly functioning upper pole moiety is ipsilateral upper pole heminephrectomy. This procedure is usually performed by open surgery, but with recent developments in techniques of uro-laparoscopy, it can be done with it, safely. In this study we evaluated the results and safety of laparoscopic heminephrectomy in our consecutive cases.Materials and Methods: From February 2001 to May 2007 fourteen unilateral laparoscopic heminephrectomy were performed in our center. Patients' characteristics, presenting symptoms, operative time, and blood loss, early and late complications were all collected retrospectively. Using pre-operative ultrasonography, intravenous pyelography (IVP and CT scanning, unilateral upper pole hydronephrosis was detected in all cases. By DMSA isotope scan hypofunctioning of ipsilateral moieties was detected in all cases.Results: Mean operative time was 203±80 minutes. No major intra-operative or early complications were identified. Mean hospital stay was 4.1 days. On mean follow-up of 32 months no disturbing symptoms or episodes of urinary tract infections (UTIs were detected. Atrophic kidney was detected in one case in post-operative IVP.Conclusion: Laparoscopic heminephrectomy is a valuable minimal invasive procedure that can be performed safely in experienced hands without any important complication. Perfect renal pedicles vascular system manipulation is important for the preservation of renal function post-operatively.

  14. Hot Forging of Nitrogen Alloyed Duplex Stainless Steels

    Institute of Scientific and Technical Information of China (English)

    P.Chandramohan; S.S. Mohamed Nazirudeen; S.S. Ramakrishnan

    2007-01-01

    Duplex stainless steels are gaining global importance because of the need for a high strength corrosion resistant material. Three compositions of this group were selected with three different nitrogen contents viz, 0.15 wt pct (alloy 1), 0.23 wt pct (alloy 2) and 0.32 wt pct (alloy 3). The steels were melted in a high frequency induction furnace and hot forged to various reductions from 16% to 62%. In this work, the effect of hot forging on the ferrite content, hardness, yield strength, impact strength and grain orientation (texture) were studied. Fracture analysis on all the forged specimens using SEM reveals that a size reduction of 48% results in maximum ductility and impact strength as well as minimal ferrite content and grain size. Thus the mechanical properties are found to have a direct correlation to ferrite content and grain size. The highest impact strength was observed in specimens with the smallest grain size, which was observed in specimens forged to 48% reduction in size.

  15. Carburizing of Duplex Stainless Steel (DSS) Under Compression Superplastic Deformation

    Science.gov (United States)

    Ahamad, Nor Wahida; Jauhari, Iswadi

    2012-12-01

    A new surface carburizing technique which combines superplastic deformation with superplastic carburizing (SPC) is introduced. SPC was conducted on duplex stainless steel under compression mode at a fixed 0.5 height reduction strain rates ranging from 6.25 × 10-5 to 1 × 10-3 s-1 and temperature ranging from 1173 K to 1248 K (900 °C to 975 °C). The results are compared with those from conventional and non-superplastic carburizing. The results show that thick hard carburized layers are formed at a much faster rate compared with the other two processes. A more gradual hardness transition from the surface to the substrate is also obtained. The highest carburized layer thickness and surface hardness are attained under SPC process at 1248 K (975 °C) and 6.25 × 10-5 s-1 with a value of (218.3 ± 0.5) μm and (1581.0 ± 5.0) HV respectively. Other than that, SPC also has the highest scratch resistance.

  16. JAEA Fatigue Analysis of EBR-II Duplex Tubing

    Energy Technology Data Exchange (ETDEWEB)

    J. H. Jackson; D. L. Porter; W. R. Lloyd

    2009-07-01

    This work addresses questions brought up concerning the mechanisms associated with fatigue crack growth retardation and/or arrest within the nickel bond layer in duplex 2¼ Cr-1Mo steel superheater tubes. Previous work performed at the Idaho National Laboratory (INL) indicated that the nickel bond layer did not function as a crack arrestor during fatigue crack propagation with the exception of one, isolated case involving an exceptionally low fatigue load and a high temperature (400 0C) environment. Since it is atypical for a fatigue crack to propagate from a relatively soft material (the nickel bond layer) to a harder material (the 2¼ Cr-1Mo steel) there has been speculation that the nickel bond layer was hardened in service. Additionally, there are questions surrounding the nature of the fatigue crack propagation within the nickel bond layer; specifically with regard to the presence of voids seen on micrographs of the bond layer and oxidation within the steel along the edge of the nickel bond layer. There is uncertainty as to the effect of these voids and/or oxide barriers with respect to potential fatigue crack arrest.

  17. Methylation-sensitive restriction enzyme nested real time PCR, a potential approach for sperm DNA identification.

    Science.gov (United States)

    Bai, Lijuan; Yan, Peng; Cao, Ximei; Jia, Linna; Zhang, Ce; Guo, Dawei

    2015-08-01

    Mammal H19 gene is an imprinting gene in which the paternal allele is silenced. On H19 imprinting control region (ICR), one of the mechanisms regulating the paternal allelic specific silence is DNA methylation in somatic cells throughout the individual's whole life. Nevertheless, this pattern of DNA methylation is erased and re-established in germline. As results, in mature sperm H19 ICR shows biallelic methylation instead of paternal specific methylation in somatic cells. Although the data were mainly from experiments on mice the same mechanisms are believed existing in human germline. We designed an experiment to probe the sperm DNA by methylation sensitive restriction enzyme based nested qPCR (MSRE-nested-qPCR). The genomic DNA digested/undigested by HhaI was amplified by outer primers encompassing four HhaI sites on H19 ICR. These PCR products were used as templates for second round real-time PCR to quantify the DNA methylation level. The results showed that DNA methylation level at H19 ICR were 55.27 ± 8.36% in 32 blood samples and 101.94 ± 11.66% in 31 semen samples. Based on our data sperm DNA could be identified if H19 ICR methylation level is over 78.62%.

  18. Effect of Deleterious Phases on Corrosion Resistance of Duplex Stainless Steel (2205

    Directory of Open Access Journals (Sweden)

    AbdulKadar M. Godil

    2013-07-01

    Full Text Available Duplex stainless steel is a Ferritic(BCC-Austenitic(FCC steel, covers the advantages of both Austenitic and Ferritic Stainless steels. They having good mechanical and corrosion resistance properties are widely used in many industries like chemical plants, refineries for critical equipments such as pressure vessels, heatexchangers, water heaters. Major problem occurs with duplex steels when they are worked or heated above about temperature of 280°C. Detrimental phases like Sigma, Chi, Laves and Alpha prime form when the Duplex steels are treated above this temperature and they retard the properties of Duplex stainless steels. They also cause embrittlement above temperature of 475°C called “475°C embrittlement”. During welding of duplex steels, Secondary austenite also forms, which is also one of the harmful phases in duplex steels. Among all of these phases, Sigma (σ is extremely harmful to the corrosion resistance of steel. Due to these limitations duplexgrades are not used above certain temperature ranges. In this experimental work a plate of duplex grade 2205 in hot worked condition was procured from TCR Advanced Engineering Pvt. Ltd., GIDC, Vadodara. Initially chemical composition of the plate was checked with emission spectrometer, tensile test and hardness tests werecarried out for comparing with the standard data. As there was no Sigma phase detected when tested with ASTM 930 in the received sample, Sigma phase was intentionally produced by giving heat treatment in the range of 700-850°C. Sigma phases were quantified with ASTM 930 practice A, by electrolytic etching with 40% NaOH. The effect of Sigma phase on corrosion resistance was measured by ASTM G48. The pitting corrosion resistance was evaluated in terms of average pit depth and overall corrosion rate.

  19. Modeling stopped-flow data for nucleic acid duplex formation reactions: the importance of off-path intermediates.

    Science.gov (United States)

    Sikora, Jacqueline R; Rauzan, Brittany; Stegemann, Rachel; Deckert, Alice

    2013-08-01

    Evidence for unexpected off-path intermediates to DNA duplex formation is presented. These off-path intermediates are shown to involve unimolecular and, in one case, bimolecular structure in one of the single strands of complementary DNA. Three models are developed to account for the observed single-stranded structures that are formed in parallel with duplex formation. These models are applied to the analysis of stopped-flow data for eight different nonself-complementary duplex formation reactions in order to extract the elementary rate constant for formation of the duplex from the complementary random coil single-stranded DNA. The free energy of activation (at 25 °C) for the denaturation of each duplex is calculated from these data and is shown to have a linear correlation to the overall standard free energy for duplex formation (also at 25 °C). Duplexes that contain mismatches obey a parallel linear free-energy (LFE) relationship with a y-intercept that is greater than that of duplexes without mismatches. Slopes near unity for the LFE relationships indicate that all duplexes go through an early, unstructured transition state.

  20. Evaluation of two real-time multiplex PCR screening assays detecting fetal RHD in plasma from RhD negative women to ascertain the requirement for antenatal RhD prophylaxis

    DEFF Research Database (Denmark)

    Clausen, Frederik Banch; Krog, Grethe Risum; Rieneck, Klaus

    2011-01-01

    OBJECTIVE: To evaluate two different multiplex real-time PCR assays detecting fetal RHD for screening of RhD negative women in relation to antenatal RhD prophylaxis. METHODS: We designed a duplex assay for the detection of RHD exon 7 and 10 and a triplex assay for the detection of RHD exon 7, 10 ...

  1. Development and Preliminary Evaluation of a New Real-Time RT-PCR Assay For Detection of Peste des petits Ruminants Virus Genome.

    Science.gov (United States)

    Polci, A; Cosseddu, G M; Ancora, M; Pinoni, C; El Harrak, M; Sebhatu, T T; Ghebremeskel, E; Sghaier, S; Lelli, R; Monaco, F

    2015-06-01

    A duplex real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for a simple and rapid diagnosis of Peste des petits ruminants (PPR). qRT-PCR primers and TaqMan probe were designed on a conserved region of nucleocapsid protein (Np) of PPR virus (PPRV) genome. An in vitro transcript of the target region was constructed and tested to determine analytical sensitivity. Commercial heterologous Armored RNA(®) was used as an internal positive control (IPC) for either RNA isolation or RT-PCR steps. The detection limit of the newly designed duplex real-time RT-PCR (qRT-PCR PPR_Np) was approximately 20 copies/μl with a 95% probability. No amplification signals were recorded when the qRT-PCR PPR_Np was applied to viruses closely related or clinically similar to PPRV- or to PPR-negative blood samples. A preliminary evaluation of the diagnostic performance was carried out by testing a group of 43 clinical specimens collected from distinct geographic areas of Africa and Middle East. qRT-PCR PPR_Np showed higher sensitivity than the conventional gel-based RT-PCR assays, which have been used as reference standards. Internal positive control made it possible to identify the occurrence of 5 false-negative results caused by the amplification failure, thus improving the accuracy of PPRV detection. © 2013 Blackwell Verlag GmbH.

  2. Comparison of Real-Time Multiplex Human Papillomavirus (HPV) PCR Assays with INNO-LiPA HPV Genotyping Extra Assay▿

    OpenAIRE

    Else, Elizabeth A.; Swoyer, Ryan; Zhang, Yuhua; Taddeo, Frank J.; Bryan, Janine T.; Lawson, John; Van Hyfte, Inez; Roberts, Christine C.

    2011-01-01

    Real-time type-specific multiplex human papillomavirus (HPV) PCR assays were developed to detect HPV DNA in samples collected for the efficacy determination of the quadrivalent HPV (type 6, 11, 16, and 18) L1 virus-like particle (VLP) vaccine (Gardasil). Additional multiplex (L1, E6, and E7 open reading frame [ORF]) or duplex (E6 and E7 ORF) HPV PCR assays were developed to detect high-risk HPV types, including HPV type 31 (HPV31), HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, and H...

  3. Full-Duplex Digital Communication on a Single Laser Beam

    Science.gov (United States)

    Hazzard, D. A.; MacCannell, J. A.; Lee, G.; Selves, E. R.; Moore, D.; Payne, J. A.; Garrett, C. D.; Dahlstrom, N.; Shay, T. M.

    2006-01-01

    A proposed free-space optical communication system would operate in a full-duplex mode, using a single constant-power laser beam for transmission and reception of binary signals at both ends of the free-space optical path. The system was conceived for two-way data communication between a ground station and a spacecraft in a low orbit around the Earth. It has been estimated that in this application, a data rate of 10 kb/s could be achieved at a ground-station-to-spacecraft distance of 320 km, using a laser power of only 100 mW. The basic system concept is also applicable to terrestrial free-space optical communications. The system (see figure) would include a diode laser at one end of the link (originally, the ground station) and a liquid-crystal- based retroreflecting modulator at the other end of the link (originally, the spacecraft). At the laser end, the beam to be transmitted would be made to pass through a quarter-wave plate, which would convert its linear polarization to right circular polarization. For transmission of data from the laser end to the retroreflector end, the laser beam would be modulated with subcarrier phase-shift keying (SC-PSK). The transmitted beam would then pass through an aperture- sharing element (ASE) - basically, a mirror with a hole in it, used to separate the paths of the transmitted and received light beams. The transmitted beam would continue outward through a telescope (which, in the original application, would be equipped with a spacecraft-tracking system) that would launch the transmitted beam along the free-space optical path to the retroreflector end.

  4. Thermodynamics of HMGB1 interaction with duplex DNA.

    Science.gov (United States)

    Müller, S; Bianchi, M E; Knapp, S

    2001-08-28

    The high mobility group protein HMGB1 is a small, highly abundant protein that binds to DNA in a non-sequence-specific manner. HMGB1 consists of 2 DNA binding domains, the HMG boxes A and B, followed by a short basic region and a continuous stretch of 30 glutamate or aspartate residues. Isothermal titration calorimetry was used to characterize the binding of HMGB1 to the double-stranded model DNAs poly(dAdT).(dTdA) and poly(dGdC).(dCdG). To elucidate the contribution of the different structural motifs to DNA binding, calorimetric measurements were performed comparing the single boxes A and B, the two boxes plus or minus the basic sequence stretch (AB(bt) and AB), and the full-length HMGB1 protein. Thermodynamically, binding of HMGB1 and all truncated constructs to duplex DNA was characterized by a positive enthalpy change at 15 degrees C. From the slopes of the temperature dependence of the binding enthalpies, heat capacity changes of -0.129 +/- 0.02 and -0.105 +/- 0.05 kcal mol(-1) K(-1) were determined for box A and full-length HMGB1, respectively. Significant differences in the binding characteristics were observed using full-length HMGB1, suggesting an important role for the acid tail in modulating DNA binding. Moreover, full-length HMGB1 binds differently these two DNA templates: binding to poly(dAdT).(dTdA) was cooperative, had a larger apparent binding site size, and proceeded with a much larger unfavorable binding enthalpy than binding to poly(dGdC).(dCdG).

  5. Microstructure and Antiwear Property of Laser Cladding Ni–Co Duplex Coating on Copper

    Directory of Open Access Journals (Sweden)

    Yiyong Wang

    2016-07-01

    Full Text Available Ni–Co duplex coatings were cladded onto Cu to improve the antiwear properties of Cu products. Prior to laser cladding, n-Al2O3/Ni layers were introduced as interlayers between laser cladding coatings and Cu substrates to improve the laser absorptivity of these substrates and ensure defect-free laser cladding coatings. The structure and morphology of the coatings were characterized by scanning electron microscopy and optical microscopy, and the phases of the coatings were analyzed by X-ray diffraction. Their hardness was measured using a microhardness tester. Experimental results showed that defect-free composite coatings were obtained and that the coatings were metallurgically bonded to the substrates. The surface of the Ni–Co duplex coatings comprised a Co-based solid solution, Cr7C3, (Fe,Ni23C6, and other strengthening phases. The microhardness and wear resistance of the duplex coatings were significantly improved compared with the Cu substrates. The average microhardness of the cladded coatings was 845.6 HV, which was approximately 8.2 times greater than that of the Cu substrates (102.6 HV. The volume loss of the Cu substrates was approximately 7.5 times greater than that of the Ni–Co duplex coatings after 60 min of sliding wear testing. The high hardness of and lack of defects in the Ni–Co duplex coatings reduced the plastic deformation and adhesive wear of the Cu substrates, resulting in improved wear properties.

  6. A kinematic model for the formation of duplex systems with a perfectly planar roof thrust

    Science.gov (United States)

    Contreras, Juan; Suter, Max

    1997-03-01

    We present a cross-sectional kinematic forward model for the formation of duplexes with a perfectly planar roof thrust. The major assumptions are a constant dip and constant spacing of the ramps in the undeformed state and sequential deformation in the direction of tectonic transport, with equal displacement along each ramp. The model is based on a coordinate transformation that simulates flexural slip parallel to the active fault surface. This causes angular parallel folds and keeps the layer thickness constant, except in the forelimbs of the horses. Attempts by previous workers to simulate the formation of duplexes with a perfectly planar roof thrust, on the other hand, were based on the assumptions of constant bed thickness and bed length, or a different topology of the axial planes delimiting the forelimbs of the horses, and resulted in corrugated roof thrusts. We show that it is not possible to form a flat roof duplex type and preserve the forelimb thickness of the horses under flexural slip parallel to the active fault. We describe duplexes by three parameters which are the separation s between ramps, the ramp length l, and the displacement u along the ramps. In a {u}/{s} vs {l}/{s} diagram, duplexes with a perfectly planar roof thrust, resulting from numerical experiments with our kinematic algorithm, occupy specific families of straight lines. Our results are independent of the dip or internal geometry of the thrust ramps.

  7. DNA Duplexes with Hydrophobic Modifications Inhibit Fusion between HIV-1 and Cell Membranes

    Science.gov (United States)

    Xu, Liang; Cai, Lifeng; Chen, Xueliang; Jiang, Xifeng; Chong, Huihui; Zheng, Baohua; Wang, Kun; He, Junlin; Chen, Wei; Zhang, Tao; Cheng, Maosheng; He, Yuxian

    2013-01-01

    Discovery of new drugs for the treatment of AIDS typically possessing unique structures associated with novel mechanisms of action has been of great importance due to the quick drug-resistant mutations of HIV-1 strains. The work presented in this report describes a novel class of DNA duplex-based HIV-1 fusion inhibitors. Hydrophobic groups were introduced into a DNA duplex skeleton either at one end, at both ends, or in the middle. These modified DNA duplexes inhibited fusion between HIV-1 and human cell membranes at micro- or submicromolar concentrations. Respective inhibitors adopted an aptamer pattern instead of a base-pairing interaction pattern. Structure-activity relationship studies of the respective DNA duplexes showed that the rigid and negatively charged DNA skeletons, in addition to the presence of hydrophobic groups, were crucial to the anti-HIV-1 activity of these compounds. A fluorescent resonance energy transfer (FRET)-based inhibitory assay showed that these duplex inhibitors interacted with the primary pocket in the gp41 N-terminal heptad repeat (NHR) instead of interacting with the lipid bilayers. PMID:23896466

  8. Characterization of duplex stainless steel weld metals obtained by hybrid plasma-gas metal arc welding

    Directory of Open Access Journals (Sweden)

    Koray Yurtisik

    2013-09-01

    Full Text Available Despite its high efficiency, autogenous keyhole welding is not well-accepted for duplex stainless steels because it causes excessive ferrite in as-welded duplex microstructure, which leads to a degradation in toughness and corrosion properties of the material. Combining the deep penetration characteristics of plasma arc welding in keyhole mode and metal deposition capability of gas metal arc welding, hybrid plasma - gas metal arc welding process has considered for providing a proper duplex microstructure without compromising the welding efficiency. 11.1 mm-thick standard duplex stainless steel plates were joined in a single-pass using this novel technique. Same plates were also subjected to conventional gas metal arc and plasma arc welding processes, providing benchmarks for the investigation of the weldability of the material. In the first place, the hybrid welding process enabled us to achieve less heat input compared to gas metal arc welding. Consequently, the precipitation of secondary phases, which are known to be detrimental to the toughness and corrosion resistance of duplex stainless steels, was significantly suppressed in both fusion and heat affected zones. Secondly, contrary to other keyhole techniques, proper cooling time and weld metal chemistry were achieved during the process, facilitating sufficient reconstructive transformation of austenite in the ferrite phase.

  9. Microstructure and Antiwear Property of Laser Cladding Ni–Co Duplex Coating on Copper

    Science.gov (United States)

    Wang, Yiyong; Liang, Zhipeng; Zhang, Junwei; Ning, Zhe; Jin, Hui

    2016-01-01

    Ni–Co duplex coatings were cladded onto Cu to improve the antiwear properties of Cu products. Prior to laser cladding, n-Al2O3/Ni layers were introduced as interlayers between laser cladding coatings and Cu substrates to improve the laser absorptivity of these substrates and ensure defect-free laser cladding coatings. The structure and morphology of the coatings were characterized by scanning electron microscopy and optical microscopy, and the phases of the coatings were analyzed by X-ray diffraction. Their hardness was measured using a microhardness tester. Experimental results showed that defect-free composite coatings were obtained and that the coatings were metallurgically bonded to the substrates. The surface of the Ni–Co duplex coatings comprised a Co-based solid solution, Cr7C3, (Fe,Ni)23C6, and other strengthening phases. The microhardness and wear resistance of the duplex coatings were significantly improved compared with the Cu substrates. The average microhardness of the cladded coatings was 845.6 HV, which was approximately 8.2 times greater than that of the Cu substrates (102.6 HV). The volume loss of the Cu substrates was approximately 7.5 times greater than that of the Ni–Co duplex coatings after 60 min of sliding wear testing. The high hardness of and lack of defects in the Ni–Co duplex coatings reduced the plastic deformation and adhesive wear of the Cu substrates, resulting in improved wear properties. PMID:28773755

  10. 等位基因特异性引物延伸法在婴儿型和幼儿型神经元蜡样质脂褐质沉积病产前诊断中的应用%Prenatal diagnostic testing for infantile and late-infantile neuronal ceroid lipofusinoses (NCL) using allele specific primer extension (ASPE)

    Institute of Scientific and Technical Information of China (English)

    Nanbert ZHONG; Weina JU; Dorota MOROZIEWICZ; Anetta WRONSKA; Marilyn LI; Krystyna WISNIEWSKI; Susan Sklower BROOKS; Edmund JENKINS; W. Ted BROWN

    2005-01-01

    SUMMARY Infantile (INCL, NCL1) and late-infantile (LINCL, NCL2) neuronal ceroid lipofuscinoses have been found to result from genetic deficiency of genes CLN1 and CLN2, respectively. The application of molecular analyses can facilitate prenatal diagnosis for families affected by NCL1 or NCL2, in which the familial mutation(s) have been identified. Molecular testing with allele-specific primer extension and DNA sequencing was performed in nine pregnancies, four from two NCL1 families and five from five NCL2 families. Lysosomal enzyme activity assays were carried out as well.Four fetuses from three pregnancies in NCL1 families were found to be carriers for a mutation 451C-T in the CLN1 gene and one was normal. Prenatal testing of three NCL2 families who carried mutation R208X in the CLN2 gene showed that all fetuses were carriers. In NCL2 families who carried either mutation IVS5-1C or/and IVS5-1A two normal pregnancies were detected. Our studies indicate that DNA testing, which may provide definitive prenatal diagnosis for NCL, may be used in combination with lysosomal enzyme activity analyses.

  11. Duplex polymerase chain reaction methods for rapid determination of 3 foodborne bacterial pathogens%二重PCR法快速检测3种食源性致病菌

    Institute of Scientific and Technical Information of China (English)

    蔡军; 欧静堃; 李慧; 胡梦龙; 傅洋; 刘冬雪

    2014-01-01

    Objective To establish a duplex PCR system for rapid detecting foodborne bacterial pathogens such as Staphylococcus aureus, Salmonella spp. and Shigella spp. in food, respectively. Method Six pairs of specific primers were designed according to the specific genes femA and nuc of Staphylococcus aureus, genes invA and hilA of Salmonella spp., and genes ipaB and ipaH of Shigella spp. respectively. The corresponding detection specificity and sensitivity of each pair of primers were measured, and each duplex PCR reaction systems were combinatorial optimized. Results The duplex PCR detection systems for rapid detecting the above-mentioned foodborne bacterial pathogens were preliminary established, the whole detection was performed in less than 18 h with all of the detection sensitivities reached 10 pg DNA/reaction. Conclusion The duplex PCR detection systems showed many advantages, including with high sensitivity and strong specificity, convenient and efficient to operate. So it has a prospective application in market.%目的:建立快速检测食品中金黄色葡萄球菌(Staphylococcus aureus)、沙门氏菌属(Salmonella spp.)及志贺氏菌属(Shigella spp.)的二重PCR方法。方法分别针对金黄色葡萄球菌femA和nuc、沙门氏菌属invA和hilA 及志贺氏菌属 ipaB 和 ipaH 设计6对特异性引物,检测每对引物的特异性及灵敏度,组合优化各自二重PCR反应体系。结果初步建立了针对这3类致病菌的二重PCR检测方法,整个检测过程不超过18 h,检测灵敏度均可达10 pg DNA/reaction。结论所建立的针对3类致病菌的二重PCR检测方法具有灵敏度高、特异性强和方便高效等优点,具有良好的市场应用前景。

  12. Apta-PCR.

    Science.gov (United States)

    Pinto, Alessandro; Polo, Pedro Nadal; Rubio, Miriam Jauest; Svobodova, Marketa; Lerga, Teresa Mairal; O'Sullivan, Ciara K

    2016-01-01

    Real-time Apta-PCR is a methodology that can be used for a wide variety of applications ranging from food quality control to clinical diagnostics. This method takes advantage of the combination of the sensitivity of nucleic acid amplification with the selectivity of aptamers. Ultra-low detection of target analyte can potentially be achieved, or, improved detection limits can be achieved with aptamers of low-medium affinity. Herein, we describe a generic methodology coined real-time Apta-PCR, using a model target (β-conglutin) and a competitive format, which can be adapted for the detection of any target which an aptamer has been selected for.

  13. Simultaneous detection of hemagglutinin and neuraminidase genes of novel influenza A (H7N9) by duplex real-time reverse transcription polymerase chain reaction.

    Science.gov (United States)

    Li, Yan; Wu, Tao; Qi, Xian; Ge, Yiyue; Guo, Xiling; Wu, Bin; Yu, Huiyan; Zhu, Yefei; Shi, Zhiyang; Wang, Hua; Cui, Lunbiao; Zhou, Minghao

    2013-12-01

    A novel reassortant influenza A (H7N9) virus emerged recently in China. In this study, a duplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay was developed for the simultaneous detection of hemagglutinin (HA) and neuraminidase (NA) genes of H7N9 influenza viruses. The sensitivity of the assay was determined to be 10 RNA copies per reaction for both HA and NA genes. No cross-reactivity was observed with other influenza virus subtypes or respiratory tract viruses. One hundred and forty-six clinical and environmental specimens were tested and compared with reference methods and were found to be consistent. The assay is suitable for large-scale screening due to short turnaround times and high specificity, sensitivity, and reproducibility.

  14. Fluorescence studies on the interaction of ethidium bromide with duplex, triplex and quadruplex DNA structures

    Institute of Scientific and Technical Information of China (English)

    孙雪光; 曹恩华; 何裕建; 秦静芬

    1999-01-01

    Under different conditions, oligonucleotides can form several alternative DNA structures such as duplex, triplex and quadruplex. All these structures can interact with ethidium bromide (EB) and make its fluorescence intensity change. The fluorescence spectra and other related parameters provided by static fluorescence techniques showed that the interaction mechanisms between EB and these structures were not always the same. Among them, B type duplex and triplex DNA adopt an intercalative mode when binding to the EB, which has a relatively high efficiency of energy transfer and the fluorescence of EB cannot be quenched easily. While for the parallel duplex DNA, the interaction mode is an outside binding in which energy transfer can hardly happen and its fluorescence intensity as well as Stern-Volmer constant is almost the same to the free EB. For the quadruplex, the binding mechanism to EB is more complex. Results from the energy transfer and quenching studies indicate that the two interaction modes note

  15. In-Band Full-Duplex Communications for Cellular Networks with Partial Uplink/Downlink Overlap

    KAUST Repository

    AlAmmouri, Ahmad

    2015-12-06

    In-band full-duplex (FD) communications have been optimistically promoted to improve the spectrum utilization in cellular networks. However, the explicit impact of spatial interference, imposed by FD communications, on uplink and downlink transmissions has been overlooked in the literature. This paper presents an extensive study of the explicit effect of FD communications on the uplink and downlink performances. For the sake of rigorous analysis, we develop a tractable framework based on stochastic geometry toolset. The developed model accounts for uplink truncated channel inversion power control in FD cellular networks. The study shows that FD communications improve the downlink throughput at the expense of significant degradation in the uplink throughput. Therefore, we propose a novel fine-grained duplexing scheme, denoted as α-duplex scheme, which allows a partial overlap between uplink and downlink frequency bands. To this end, we show that the amount of the overlap can be optimized via adjusting α to achieve a certain design objective.

  16. Vertebral artery dissection with compelling evidence on duplex ultrasound presenting only with neck pain

    Directory of Open Access Journals (Sweden)

    Siepmann T

    2016-11-01

    Full Text Available Timo Siepmann, Monique Borchert, Kristian Barlinn Department of Neurology, Carl Gustav Carus University Hospital, Technische Universität Dresden, Dresden, Germany Abstract: Vertebral artery dissection (VAD is among the most common identifiable etiologies of stroke in young adults and poses a diagnostic challenge due to nonspecific symptoms and substantial variability of imaging results. Here, we present a case of unspecific neck pain as isolated symptom of VAD with unusually compelling evidence on duplex ultrasound. This observation has clinical relevance as the absence of any neurological symptoms in our patient highlights the necessity of considering cervical artery dissection in patients presenting with unspecific symptoms such as neck pain, even if isolated. Furthermore, our image of intramural hematoma on duplex ultrasound has been captured in an unusual, clear and distinct fashion and might therefore be a useful reference image in the clinical assessment of patients with a suspicion of cervical artery dissection. Keywords: vertebrobasilar, cervical, duplex sonography, magnetic resonance imaging

  17. Interference alignment for degrees of freedom improvement in 3-relay half-duplex systems

    KAUST Repository

    Park, Seongho

    2011-12-01

    In a half-duplex relaying, the capacity pre-log factor is a major drawback in spectral efficiency. This paper proposes a linear precoding scheme and an alternate relaying protocol in a dual-hop half-duplex system where three relays help the communication between the source and the destination. In our proposed scheme, we consider a phase incoherent method in relays in which the source alternately transmits message signals to the different relays. In addition, we propose a linear interference alignment scheme which can eliminate the inter-relay interference resulted from the phase incoherence of relaying. Based on our analysis of degrees of freedom and our simulation results, we show that our proposed scheme achieves additional degrees of freedom compared to the conventional half-duplex relaying. © 2011 IEEE.

  18. Fracture Toughness and Thermal Shock Properties of Calcium Aluminate Composite with Controlled Duplex Structure

    Institute of Scientific and Technical Information of China (English)

    W.J.Wei; H.J.Liaw

    2004-01-01

    This study used monocalcium aluminate (CaAl2O4,CA) for producing ceramic composites with duplex microstructures by hydration and gelation reactions of the aluminate, and compared the properties with those made by a die-pressing process of mixed powders. Densification of the composite was optimized. The microstructure of sintered bodies, the fracture strengths and toughness of the composites with and without thermal shocking was characterized by different techniques. Experimental results show that the green body with the addition of CA resulted glomerated platelets or uniform distribution of platy CA6 grains. The former, which appeared a duplex microstructure consisted of dense matrix and distributed clusters of CA6 platelets, performed an improvement on toughness and thermal shock resistance. Toughness mechanisms of samples with duplex microstructure are also investigated.

  19. Alternate transmission relaying based on interference alignment in 3-relay half-duplex MIMO systems

    KAUST Repository

    Park, Seongho

    2012-09-01

    In a half-duplex relaying, the capacity pre-log factor 1/2 is a major drawback in spectral efficiency. This paper proposes a linear precoding/decoding scheme and an alternate relaying protocol in a dual-hop half-duplex system where three relays help the communication between the source and the destination. In our proposed scheme, we consider a phase incoherent method in relays in which the source alternately transmits message signals to the different relays. In addition, we propose a linear interference alignment scheme which can suppress the inter-relay interference resulting from the phase incoherence of relaying. Based on our analysis of degrees of freedom and our simulation results, we show that our proposed scheme achieves additional degrees of freedom compared to the conventional half-duplex relaying. © 2012 IEEE.

  20. Effect of Temperature and Cl-Concentration on Pitting of 2205 Duplex Stainless Steel

    Institute of Scientific and Technical Information of China (English)

    DONG Chaofang; LUO Hong; XIAO Kui; SUN Ting; LIU Qian; LI Xiaogang

    2011-01-01

    The electrochemical behaviors of 2205 duplex stainless steel in NaCl solution with different temperatures and concentrations were studied by gravimetric tests,potentiodynamic polarization,electrochemical impedance spectroscopy and scanning electron microscopy.The experimental results show that temperature and chloride concentration have a great influence on the pitting resistance of 2205 duplex stainless steels.They not only effect the corrosion rate of pitting,but also change the shape of the pits.When NaCl solution was in low concentration and temperature below the critical pitting temperature,pits were very small and scattered with hemisphere-like shape.On the contrary,the pits of 2205 duplex stainless steel were large and sometimes had a lacy cover when the NaCl concentration was higher and the temperature was 70℃.

  1. Mixer-Duplexer-Antenna Leaky-Wave System Based on Periodic Space-Time Modulation

    CERN Document Server

    Taravati, Sajjad

    2016-01-01

    We present a mixer-duplexer-antenna leaky-wave system based on periodic space-time modulation. This system operates as a full transceiver, where the upconversion and downconversion mixing operations are accomplished via space-time transitions, the duplexing operation is induced by the nonreciprocal nature of the structure, and the radiation operation is provided by the leaky-wave nature of the wave. A rigorous electromagnetic solution is derived for the dispersion relation and field distributions. The system is implemented in the form of a spatio-temporally modulated microstrip leaky-wave structure incorporating an array of sub-wavelengthly spaced varactors modulated by a harmonic wave. In addition to the overall mixer-duplexer-antenna operation, frequency beam scanning at fixed input frequency is demonstrated as one of the interesting features of the system. A prototype is realized and demonstrated by full-wave and experimental results.

  2. Throughput maximization for buffer-aided hybrid half-/full-duplex relaying with self-interference

    KAUST Repository

    Khafagy, Mohammad Galal

    2015-06-01

    In this work, we consider a two-hop cooperative setting where a source communicates with a destination through an intermediate relay node with a buffer. Unlike the existing body of work on buffer-aided half-duplex relaying, we consider a hybrid half-/full-duplex relaying scenario with loopback interference in the full-duplex mode. Depending on the channel outage and buffer states that are assumed available at the transmitters, the source and relay may either transmit simultaneously or revert to orthogonal transmission. Specifically, a joint source/relay scheduling and relaying mode selection mechanism is proposed to maximize the end-to-end throughput. The throughput maximization problem is converted to a linear program where the exact global optimal solution is efficiently obtained via standard convex/linear numerical optimization tools. Finally, the theoretical findings are corroborated with event-based simulations to provide the necessary performance validation.

  3. Does Dopplersignal enhancement with Levovist improve the diagnostic confidence of duplex scanning of the iliac arteries?--(A pilot study with correlation to intravascular ultrasound)

    DEFF Research Database (Denmark)

    Vogt, K C; Jensen, F; Schroeder, T V

    1998-01-01

    To evaluate whether echo-enhancement with Levovist improves the diagnostic confidence of duplex scanning of the iliac arteries.......To evaluate whether echo-enhancement with Levovist improves the diagnostic confidence of duplex scanning of the iliac arteries....

  4. A simple real-time polymerase chain reaction (PCR)-based assay for authentication of the Chinese Panax ginseng cultivar Damaya from a local ginseng population.

    Science.gov (United States)

    Wang, H; Wang, J; Li, G

    2016-06-27

    Panax ginseng is one of the most important medicinal plants in the Orient. Owing to its increasing demand in the world market, cultivated ginseng has become the main source of medicinal material. Among the Chinese ginseng cultivars, Damaya commands higher prices and is grown in significant proportions among the local ginseng population. Due to the lack of rapid and accurate authentication methods, Damaya is distributed among different cultivars in the local ginseng population in China. Here, we identified a unique, Damaya-specific single nucleotide polymorphism (SNP) site present in the second intron of mitochondrial cytochrome c oxidase subunit 2 (cox2). Based on this SNP, a Damaya cultivar-specific primer was designed and an allele-specific polymerase chain reaction (PCR) was optimized for the effective molecular authentication of Damaya. We designed a method by combining a simple DNA isolation method with real-time allele-specific PCR using SYBR Green I fluorescent dye, and proved its efficacy in clearly discriminated Damaya cultivar from other Chinese ginseng cultivars according to the allelic discrimination analysis. Hence, this study provides a simple and rapid assay for the differentiation and conservation of Damaya from the local Chinese ginseng population.

  5. Explanatory chapter: PCR primer design.

    Science.gov (United States)

    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. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Structural, Dynamical, and Electronic Transport Properties of Modified DNA Duplexes Containing Size-Expanded Nucleobases

    Energy Technology Data Exchange (ETDEWEB)

    Fuentes-Cabrera, Miguel A [ORNL; Orozco, Modesto [Institut de Recerca Biomedica, Parc Cientific de Barcelona, Barcelona, Spain; Luque, Javier [Universitat de Barcelona; Sumpter, Bobby G [ORNL; Blas, Jose [Universidad de Castilla-La Mancha; Ordejon, Pablo J [ORNL; Huertas, Oscar [Universitat de Barcelona; Tabares, Carolina [Universitat de Barcelona

    2011-01-01

    Among the distinct strategies proposed to expand the genetic alphabet, sizeexpanded nucleobases are promising for the development of modified DNA duplexes with improved biotechnological properties. In particular, duplexes built up by replacing canonical bases with the corresponding benzo-fused counterparts could be valuable as molecular nanowires. In this context, this study reports the results of classical molecular dynamics simulations carried out to examine the structural and dynamical features of size-expanded DNAs, including both hybrid duplexes containing mixed pairs of natural and benzo-fused bases (xDNA) and pure size-expanded (xxDNA) duplexes. Furthermore, the electronic structure of both natural and size-expanded duplexes is examined by means of density functional computations. The results confirm that the structural and flexibility properties of the canonical DNA are globally little affected by the presence of benzo-fused bases. Themost relevant differences are found in the enhanced size of the grooves, and the reduction in the twist. However, the analysis also reveals subtle structural effects related to the nature and sequence of benzo-fused bases in the duplex. On the other hand, electronic structure calculations performed for xxDNAs confirm the reduction in the HOMOLUMO gap predicted from the analysis of the natural bases and their size-expanded counterparts, which suggests that pure size-expanded DNAs can be good conductors. A more complex situation is found for xDNAs, where fluctuations in the electrostatic interaction between base pairs exerts a decisive influence on the modulation of the energy gap.

  7. Plastomes of the green algae Hydrodictyon reticulatum and Pediastrum duplex (Sphaeropleales, Chlorophyceae

    Directory of Open Access Journals (Sweden)

    Hilary A. McManus

    2017-05-01

    Full Text Available Background Comparative studies of chloroplast genomes (plastomes across the Chlorophyceae are revealing dynamic patterns of size variation, gene content, and genome rearrangements. Phylogenomic analyses are improving resolution of relationships, and uncovering novel lineages as new plastomes continue to be characterized. To gain further insight into the evolution of the chlorophyte plastome and increase the number of representative plastomes for the Sphaeropleales, this study presents two fully sequenced plastomes from the green algal family Hydrodictyaceae (Sphaeropleales, Chlorophyceae, one from Hydrodictyon reticulatum and the other from Pediastrum duplex. Methods Genomic DNA from Hydrodictyon reticulatum and Pediastrum duplex was subjected to Illumina paired-end sequencing and the complete plastomes were assembled for each. Plastome size and gene content were characterized and compared with other plastomes from the Sphaeropleales. Homology searches using BLASTX were used to characterize introns and open reading frames (orfs ≥ 300 bp. A phylogenetic analysis of gene order across the Sphaeropleales was performed. Results The plastome of Hydrodictyon reticulatum is 225,641 bp and Pediastrum duplex is 232,554 bp. The plastome structure and gene order of H. reticulatum and P. duplex are more similar to each other than to other members of the Sphaeropleales. Numerous unique open reading frames are found in both plastomes and the plastome of P. duplex contains putative viral protein genes, not found in other Sphaeropleales plastomes. Gene order analyses support the monophyly of the Hydrodictyaceae and their sister relationship to the Neochloridaceae. Discussion The complete plastomes of Hydrodictyon reticulatum and Pediastrum duplex, representing the largest of the Sphaeropleales sequenced thus far, once again highlight the variability in size, architecture, gene order and content across the Chlorophyceae. Novel intron insertion sites and unique

  8. Dwell-Time Distribution, Long Pausing and Arrest of Single-Ribosome Translation through the mRNA Duplex

    Directory of Open Access Journals (Sweden)

    Ping Xie

    2015-10-01

    Full Text Available Proteins in the cell are synthesized by a ribosome translating the genetic information encoded on the single-stranded messenger RNA (mRNA. It has been shown that the ribosome can also translate through the duplex region of the mRNA by unwinding the duplex. Here, based on our proposed model of the ribosome translation through the mRNA duplex we study theoretically the distribution of dwell times of the ribosome translation through the mRNA duplex under the effect of a pulling force externally applied to the ends of the mRNA to unzip the duplex. We provide quantitative explanations of the available single molecule experimental data on the distribution of dwell times with both short and long durations, on rescuing of the long paused ribosomes by raising the pulling force to unzip the duplex, on translational arrests induced by the mRNA duplex and Shine-Dalgarno(SD-like sequence in the mRNA. The functional consequences of the pauses or arrests caused by the mRNA duplex and the SD sequence are discussed and compared with those obtained from other types of pausing, such as those induced by “hungry” codons or interactions of specific sequences in the nascent chain with the ribosomal exit tunnel.

  9. Impact of Transport Control Protocol on Full Duplex Performance in 5G Networks

    DEFF Research Database (Denmark)

    Gatnau, Marta; Berardinelli, Gilberto; Mahmood, Nurul Huda

    2016-01-01

    Full duplex (FD) communication has attracted the attention of the industry and the academia as an important feature in the design of the future 5th generation (5G) wireless communication system. Such technology allows a device to simultaneously transmit and receive in the same frequency band......, with the potential of providing higher throughput and lower latency compared to traditional half duplex (HD) systems. In this paper, the interaction between Transport Control Protocol (TCP) and FD in 5G ultra-dense small cell networks is studied. TCP is a well-known transport layer protocol for providing reliability...

  10. Problems in repair-welding of duplex-treated tool steels

    Directory of Open Access Journals (Sweden)

    T. Muhič

    2009-01-01

    Full Text Available The present paper addresses problems in laser welding of die-cast tools used for aluminum pressure die-castings and plastic moulds. To extend life cycle of tools various surface improvements are used. These surface improvements significantly reduce weldability of the material. This paper presents development of defects in repair welding of duplex-treated tool steel. The procedure is aimed at reduction of defects by the newly developed repair laser welding techniques. Effects of different repair welding process parameters and techniques are considered. A microstructural analysis is conducted to detect defect formation and reveal the best laser welding method for duplex-treated tools.

  11. Texture and microstructure development in a cold-rolled duplex stainless steel annealed at 800 C

    Energy Technology Data Exchange (ETDEWEB)

    Shek, C.H. (Dept. of Mechanical Engineering, Univ. of Hong Kong (Hong Kong)); Shen, G.J. (Analysis and Test Centre, Southeast Univ., Nanjing (China)); Lai, J.K.L. (Dept. of Applied Science, City Polytechnic of Hong Kong (Hong Kong)); Duggan, B.J. (Dept. of Mechanical Engineering, Univ. of Hong Kong (Hong Kong))

    1994-01-01

    A cast of austenitic-ferritic duplex stainless steel was rolled 85% and then annealed at 800 C to study the microstructural and texture changes in the material caused by these mechanical and thermal treatments. The austenite in the duplex alloy behaves as typical low SFE materials when rolled as well as annealed but larger spreads in orientation were observed, compared with rolled and annealed brass. The cold-rolled ferrite mainly consists of long microbands and has texture similar to cold-rolled carbon steel. Upon annealing, the ferrite undergoes recovery only and the rolling texture is thus retained. (orig.)

  12. Microstructural and Tribological Characterization of Duplex Coatings with Additional Ion Bombardment

    Institute of Scientific and Technical Information of China (English)

    B.(S)kori(c); D.Kaka(s); M.Rakita

    2004-01-01

    A duplex surface treatment involves the sequential application of two surface technologies to produce a surface composition with combined properties. A typical duplex process involves plasma nitriding and the PVD coating treatment of steels. In the paper are presented characteristics of hard coatings, type TiN, produced by classic technology PVD (physical vapour deposition) and IBAD (ion beam assisted deposition). Subsequent ion implantation was provided with N5+ions. The dependence of friction coefficient was investigated by means of tribometer (pin-on-ring). The sliding pair was TiN thin coating on steel pin combined with steel ring without coating. The ring was produced from hardenable steel.

  13. Design of duplex low carbon steels for improved strength: weight applications

    Energy Technology Data Exchange (ETDEWEB)

    Koo, J.

    1977-08-01

    Duplex ferrite-martensite (DFM) steels with excellent combinations of strength and formability have been selected from first principles, and their microstructure-property relationships established through a systematic investigation of a series of ternary Fe/X/0.1 C steels (X being varying amount of Cr and Si). Duplex processing consists of initial austenitization and quenching to form 100% martensite, followed by annealing in the (..cap alpha.. + ..gamma..) range and subsequent quenching. The resultant DFM morphology is controlled by the type and amount of alloying element X.

  14. Influence Of Heat Treatment On Duplex Stainless Steel To Study The Material Properties

    Directory of Open Access Journals (Sweden)

    Jithin M

    2015-02-01

    Full Text Available Abstract The various heat treatment processes are annealing normalizing hardening tempering spheroidising surface hardening flame and induction hardening nitriding cyaniding carbonitriding carburizing etc Heat treatment on duplex stainless steel is to improve ductility toughness strength hardness and to relieve internal stress developed in the material. Here basically the experiment of hardness test impact test wear test and compression is done to get idea about heat treated duplex stainless steel which has extensive uses in all industries and scientific research and development fields.

  15. Towards economic design of a pressure vessel made of duplex stainless steel

    OpenAIRE

    Veljkovic, Milan, ed. lit.; Gozzi, Jonas

    2005-01-01

    The feasibility of manufacturing pressure vessels from duplex stainless steel (grade EN 1.4462, duplex 2205: 0.02%C, 22%Cr, 5.7%Ni, 3.1%Mo, 0.17%N) was evaluated by measurements of the mechanical properties of the parent metal and welds, and by finite element modelling. The tensile stress-strain properties of 4 mm sheet, and of the weld metal and the HAZ were measured in the transverse and rolling directions. Behaviour under biaxial stress was studied by pressurising circular sheet specimens ...

  16. Microstructural and Tribological Characterization of Duplex Coatings with Additional Ion Bombardment

    Institute of Scientific and Technical Information of China (English)

    B.Skorie; D.Kakas; M.akita

    2004-01-01

    A duplex surface treatment involves the sequential application of two surface technologies to produce a surface composition with combined properties. A typical duplex process involves plasma nitriding and the PVD coating treatment of steels. In the paper are presented characteristics of hard coatings, type TiN, produced by classic technology PVD (physical vapour deposition) and IBAD (ion beam assisted deposition). Subsequent ion implantation was provided with N5+ ions. The dependence of friction coefficient was investigated by means of tribometer (pin-on-ring). The sliding pair was TiN thin coating on steel pin combined with steel ring without coating. The ring was produced from hardenable steel.

  17. Design and Synthesis of a Highly Stable Six-hydrogen-bonded Self-assembly Yellowish Green Electroluminescent Molecular Duplex

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    This paper describes the design, synthesis and characterization of a hydrogen-bonded molecular duplex with 1,8-naphthalimide fluorescent pendants. The two oligoamide molecular strands, with complementary hydrogen bond sequences of DDADAA and AADADD, caa form an ultra stable self-assembly duplex. Its molecular structure was confirmed by 1H NMR and ESI-MS, and its photoluminescence properties were determined. The resulting duplex exhibited a dramatically enhanced photoluminescence (PL) quantum efficiency of 63.7% compared to the corresponding 1,8-naphthalimide segment (32.4%), suggesting that the formation of the duplex with larger molecular weight could successfully inhibit the quenching of the fluorescent p