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Sample records for multiplex pcr-based reverse

  1. Simultaneous direct identification of genital microorganisms in voided urine using multiplex PCR-based reverse line blot assays.

    PubMed

    McKechnie, Michelle L; Kong, Fanrong; Gilbert, Gwendolyn L

    2013-01-01

    Our aim was to develop and evaluate sensitive methods that would allow simultaneous direct identification of multiple potential pathogens in clinical specimens for diagnosis and epidemiological studies, using a multiplex PCR-based reverse line blot assay. We have previously developed assays suitable for detection of bacterial respiratory and systemic pathogens. In this chapter we describe, in detail, a method developed to identify 14 genital microorganisms, for use in epidemiological studies of genital infection or colonization, using first voided urine specimens. The 14 urogenital pathogens or putative pathogens studied were Trichomonas vaginalis, Streptococcus pneumoniae, Neisseria gonorrhoeae, N. meningitidis, Chlamydia trachomatis, Ureaplasma parvum, U. urealyticum, Mycoplasma hominis, M. genitalium, Gardnerella vaginalis, Haemophilus influenzae, herpes simplex virus 1 and 2, and adenovirus. Two species-specific primer pairs and probes were designed for each target. The method was validated using a reference strain or a well-characterized clinical isolate of each target organism. In a clinical study among men attending sexual health clinics in Sydney, we used the assay to compare rates of detection of the 14 organisms in men with urethritis with those in asymptomatic controls and found the method to be sensitive, specific, convenient, and relatively inexpensive. PMID:23104293

  2. Capsular typing of Streptococcus pneumoniae isolated in an Algerian hospital using a new multiplex PCR-based scheme.

    PubMed

    Ziane, Hanifa; Manageiro, Vera; Ferreira, Eugénia; Bektache, Soumia; Tazir, Mohamed; Caniça, Manuela

    2015-12-01

    We developed a new sequential multiplex-PCR-based typing scheme (MPBTS) for pneumococcal capsular classification. The serogroup/type of 37 control isolates obtained by the Quellung reaction, MPBTS, and nucleotide sequencing, were fully concordant. The serogroups/types of 75 invasive isolates determined by MPBTS, presented 100% specificity and 96% sensitivity, when compared with the Quellung reaction. PMID:26546733

  3. Phylogeographic analysis of hemorrhagic fever with renal syndrome patients using multiplex PCR-based next generation sequencing.

    PubMed

    Kim, Won-Keun; Kim, Jeong-Ah; Song, Dong Hyun; Lee, Daesang; Kim, Yong Chul; Lee, Sook-Young; Lee, Seung-Ho; No, Jin Sun; Kim, Ji Hye; Kho, Jeong Hoon; Gu, Se Hun; Jeong, Seong Tae; Wiley, Michael; Kim, Heung-Chul; Klein, Terry A; Palacios, Gustavo; Song, Jin-Won

    2016-01-01

    Emerging and re-emerging infectious diseases caused by RNA viruses pose a critical public health threat. Next generation sequencing (NGS) is a powerful technology to define genomic sequences of the viruses. Of particular interest is the use of whole genome sequencing (WGS) to perform phylogeographic analysis, that allows the detection and tracking of the emergence of viral infections. Hantaviruses, Bunyaviridae, cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) in humans. We propose to use WGS for the phylogeographic analysis of human hantavirus infections. A novel multiplex PCR-based NGS was developed to gather whole genome sequences of Hantaan virus (HTNV) from HFRS patients and rodent hosts in endemic areas. The obtained genomes were described for the spatial and temporal links between cases and their sources. Phylogenetic analyses demonstrated geographic clustering of HTNV strains from clinical specimens with the HTNV strains circulating in rodents, suggesting the most likely site and time of infection. Recombination analysis demonstrated a genome organization compatible with recombination of the HTNV S segment. The multiplex PCR-based NGS is useful and robust to acquire viral genomic sequences and may provide important ways to define the phylogeographical association and molecular evolution of hantaviruses. PMID:27221218

  4. Phylogeographic analysis of hemorrhagic fever with renal syndrome patients using multiplex PCR-based next generation sequencing

    PubMed Central

    Kim, Won-Keun; Kim, Jeong-Ah; Song, Dong Hyun; Lee, Daesang; Kim, Yong Chul; Lee, Sook-Young; Lee, Seung-Ho; No, Jin Sun; Kim, Ji Hye; Kho, Jeong Hoon; Gu, Se Hun; Jeong, Seong Tae; Wiley, Michael; Kim, Heung-Chul; Klein, Terry A.; Palacios, Gustavo; Song, Jin-Won

    2016-01-01

    Emerging and re-emerging infectious diseases caused by RNA viruses pose a critical public health threat. Next generation sequencing (NGS) is a powerful technology to define genomic sequences of the viruses. Of particular interest is the use of whole genome sequencing (WGS) to perform phylogeographic analysis, that allows the detection and tracking of the emergence of viral infections. Hantaviruses, Bunyaviridae, cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) in humans. We propose to use WGS for the phylogeographic analysis of human hantavirus infections. A novel multiplex PCR-based NGS was developed to gather whole genome sequences of Hantaan virus (HTNV) from HFRS patients and rodent hosts in endemic areas. The obtained genomes were described for the spatial and temporal links between cases and their sources. Phylogenetic analyses demonstrated geographic clustering of HTNV strains from clinical specimens with the HTNV strains circulating in rodents, suggesting the most likely site and time of infection. Recombination analysis demonstrated a genome organization compatible with recombination of the HTNV S segment. The multiplex PCR-based NGS is useful and robust to acquire viral genomic sequences and may provide important ways to define the phylogeographical association and molecular evolution of hantaviruses. PMID:27221218

  5. Multiplex PCR-based detection of Leptospira in environmental water samples obtained from a slum settlement.

    PubMed

    Vital-Brazil, Juliana Magalhães; Balassiano, Ilana Teruszkin; Oliveira, Fabiano Sutter de; Costa, Alberto Dias de Souza; Hillen, Leandro; Pereira, Martha Maria

    2010-05-01

    The aim of this study was to apply a molecular protocol to detect leptospiral DNA in environmental water samples. The study was carried out in a peri-urban settlement in Petrópolis, state of Rio de Janeiro. A multiplex PCR method employing the primers LipL32 and 16SrRNA was used. Three out of 100 analysed samples were positive in the multiplex PCR, two were considered to have saprophytic leptospires and one had pathogenic leptospires. The results obtained supported the idea that multiplex PCR can be used to detect Leptospira spp in water samples. This method was also able to differentiate between saprophytic and pathogenic leptospires and was able to do so much more easily than conventional methodologies. PMID:20512254

  6. A colony multiplex quantitative PCR-Based 3S3DBC method and variations of it for screening DNA libraries.

    PubMed

    An, Yang; Toyoda, Atsushi; Zhao, Chen; Fujiyama, Asao; Agata, Kiyokazu

    2015-01-01

    A DNA library is a collection of DNA fragments cloned into vectors and stored individually in host cells, and is a valuable resource for molecular cloning, gene physical mapping, and genome sequencing projects. To take the best advantage of a DNA library, a good screening method is needed. After describing pooling strategies and issues that should be considered in DNA library screening, here we report an efficient colony multiplex quantitative PCR-based 3-step, 3-dimension, and binary-code (3S3DBC) method we used to screen genes from a planarian genomic DNA fosmid library. This method requires only 3 rounds of PCR reactions and only around 6 hours to distinguish one or more desired clones from a large DNA library. According to the particular situations in different research labs, this method can be further modified and simplified to suit their requirements. PMID:25646755

  7. A Colony Multiplex Quantitative PCR-Based 3S3DBC Method and Variations of It for Screening DNA Libraries

    PubMed Central

    An, Yang; Toyoda, Atsushi; Zhao, Chen; Fujiyama, Asao; Agata, Kiyokazu

    2015-01-01

    A DNA library is a collection of DNA fragments cloned into vectors and stored individually in host cells, and is a valuable resource for molecular cloning, gene physical mapping, and genome sequencing projects. To take the best advantage of a DNA library, a good screening method is needed. After describing pooling strategies and issues that should be considered in DNA library screening, here we report an efficient colony multiplex quantitative PCR-based 3-step, 3-dimension, and binary-code (3S3DBC) method we used to screen genes from a planarian genomic DNA fosmid library. This method requires only 3 rounds of PCR reactions and only around 6 hours to distinguish one or more desired clones from a large DNA library. According to the particular situations in different research labs, this method can be further modified and simplified to suit their requirements. PMID:25646755

  8. Development of a PCR-Based Reverse Genetics System for an Attenuated Duck Tembusu Virus Strain

    PubMed Central

    Wu, Xiaogang; Shi, Ying; Yan, Dawei; Li, Xuesong; Yan, Pixi; Gao, Xuyuan; Zhang, Yuee; Yu, Lei; Ren, Chaochao; Li, Guoxin; Yan, Liping; Teng, Qiaoyang; Li, Zejun

    2016-01-01

    The infectious disease caused by the duck Tembusu virus (DTMUV) has resulted in massive economic losses to the Chinese duck industry in China since 2010. Research on the molecular basis of DTMUV pathogenicity has been hampered by the lack of a reliable reverse genetics system for this virus. Here we developed a PCR-based reverse genetics system with high fidelity for the attenuated DTMUV strain FX2010-180P. The rescued virus was characterized by using both indirect immunofluorescence assays (IFA) and whole genome sequencing. The rescued virus (rFX2010-180P) grew to similar titers as compared with the wild-type virus in DF-1 cells, and had similar replication and immunogenicity properties in ducks. To determine whether exogenous proteins could be expressed from DTMUV, both an internal ribosomal entry site (IRES) and the enhanced green fluorescent protein (eGFP) gene were introduced between the NS5 gene and the 3' non-coding sequence of FX2010-180P. A recombinant DTMUV expressing eGFP was rescued, but eGFP expression was unstable after 4 passages in DF-1 cells due to a deletion of 1,294 nucleotides. The establishment of a reliable reverse genetics system for FX2010-180P provides a foundation for future studies of DTMUV. PMID:27248497

  9. Organic Substances Interfere with Reverse Transcription-Quantitative PCR-Based Virus Detection in Water Samples

    PubMed Central

    Katayama, Hiroyuki; Furumai, Hiroaki

    2014-01-01

    Reverse transcription (RT)-PCR-based virus detection from water samples is occasionally hampered by organic substances that are coconcentrated during virus concentration procedures. To characterize these organic substances, samples containing commercially available humic acid, which is known to inhibit RT-PCR, and river water samples were subjected to adsorption-elution-based virus concentration using an electronegative membrane. In this study, the samples before, during, and after the concentration were analyzed in terms of organic properties and virus detection efficiencies. Two out of the three humic acid solutions resulted in RT-quantitative PCR (qPCR) inhibition that caused >3-log10-unit underestimation of spiked poliovirus. Over 60% of the organics contained in the two solutions were recovered in the concentrate, while over 60% of the organics in the uninhibited solution were lost during the concentration process. River water concentrates also caused inhibition of RT-qPCR. Organic concentrations in the river water samples increased by 2.3 to 3.9 times after the virus concentration procedure. The inhibitory samples contained organic fractions in the 10- to 100-kDa size range, which are suspected to be RT-PCR inhibitors. According to excitation-emission matrices, humic acid-like and protein-like fractions were also recovered from river water concentrates, but these fractions did not seem to affect virus detection. Our findings reveal that detailed organic analyses are effective in characterizing inhibitory substances. PMID:25527552

  10. New multiplex PCR-based protocol allowing indirect diagnosis of FSHD on single cells: can PGD be offered despite high risk of recombination?

    PubMed Central

    Barat-Houari, Mouna; Nguyen, Karine; Bernard, Rafaëlle; Fernandez, Céline; Vovan, Catherine; Bareil, Corinne; Van Kien, Philippe Khau; Thorel, Delphine; Tuffery-Giraud, Sylvie; Vasseur, Francis; Attarian, Shahram; Pouget, Jean; Girardet, Anne; Lévy, Nicolas; Claustres, Mireille

    2010-01-01

    Molecular pathophysiology of facioscapulohumeral muscular dystrophy (FSHD) involves the heterozygous contraction of the number of tandemly repeated D4Z4 units at chromosome 4q35.2. FSHD is associated with a range of 1–10 D4Z4 units instead of 11–150 in normal controls. Several factors complicate FSHD molecular diagnosis, especially the cis-segregation of D4Z4 contraction with a 4qA allele, whereas D4Z4 shortening is silent both on alleles 4qB and 10q. Discrimination of pathogenic 4q-D4Z4 alleles from highly homologous 10q-D4Z4 arrays requires the use of the conventional Southern blot, which is not suitable at the single-cell level. Preimplantation genetic diagnosis (PGD) is a frequent request from FSHD families with several affected relatives. We aimed to develop a rapid and sensitive PCR-based multiplex approach on single cells to perform an indirect familial segregation study of pathogenic alleles. Among several available polymorphic markers at 4q35.2, the four most proximal (D4S2390, D4S1652, D4S2930 and D4S1523, <1.23 Mb) showing the highest heterozygote frequencies (67–91%) were selected. Five recombination events in the D4S2390-D4S1523 interval were observed among 144 meioses. In the D4S2390-D4Z4 interval, no recombination event occurred among 28 FSHD meioses. Instead, a particular haplotype segregated with both clinical and molecular status, allowing the characterization of an at-risk allele in each tested FSHD family (maximal LOD score 2.98 for θ=0.0). This indirect protocol can easily complement conventional techniques in prenatal diagnosis. Although our multiplex PCR-based approach technically fulfils guidelines for single-cell analysis, the relatively high recombination risk hampers its application to PGD. PMID:19935833

  11. Allele distribution of 15 PCR-based loci in the Rwanda Tutsi population by multiplex amplification and capillary electrophoresis.

    PubMed

    Regueiro, M; Carril, J C; Pontes, M L; Pinheiro, M F; Luis, J R; Caeiro, B

    2004-06-30

    A genetic study of 15 autosomal STRs is carried out (D2S1338, D3S1358, D5S818, D7S820, D8S1 79, D13S317, D16S359, D18S51, D19S433, D21S11, CSF1PO, FGA, TPOX, THO1, VWA) in a sample of unrelated Tutsis. The molecular phenotypes were determined by means of multiplex strategies (AmpFlSTR Identifiler PCR Amplification Kit, Applied Biosystems) followed by capillary electrophoresis. PMID:15177631

  12. Detection of Bacillus cereus with enteropathogenic potential by multiplex real-time PCR based on SYBR Green I.

    PubMed

    Wehrle, Esther; Didier, Andrea; Moravek, Maximilian; Dietrich, Richard; Märtlbauer, Erwin

    2010-06-01

    In order to meet the growing demand for fast and reliable detection of potentially toxinogenic Bacillus cereus, we developed a multiplex real-time PCR assay based on SYBR Green I with subsequent melting curve analysis. We designed and selected primers specific for genes of toxins responsible for diarrhoea (nheA, hblD and cytK1) and emesis (ces). A panel of 337 Bacillus strains was applied to the novel method on Light Cycler 2.0 with average melting temperature (T(m)) values of 73.85 degrees C (nheA), 87.01 degrees C (hblD), 78.66 degrees C (ces) and 82.19 degrees C (cytK1). An adapted version of the assay was also successfully run on Light Cycler 480 using one third (113 strains) of the total test panel. Verification of PCR results by conventional PCR as well as immunoassays and cytotoxicity tests gave an overall excellent correlation. Distinct melting peaks were only observed in B. cereus and B. cereus group strains but not in other Bacilli and Gram-positive or Gram-negative bacteria. Artificial contamination of three different food matrices with distinct bacterial counts revealed a detection limit of 10(1) CFU/g B. cereus cells after overnight enrichment. Thus, the novel multiplex real-time PCR turned out to be a reliable method for identification of B. cereus with enteropathogenic potential. PMID:19944752

  13. Detection of Salmonella spp, Salmonella Enteritidis and Typhimurium in naturally infected broiler chickens by a multiplex PCR-based assay

    PubMed Central

    Paião, F.G.; Arisitides, L.G.A.; Murate, L.S.; Vilas-Bôas, G.T.; Vilas-Boas, L.A.; Shimokomaki, M.

    2013-01-01

    The presence of Salmonella in the intestinal tract, on the chickens skin and among their feathers, may cause carcasses contamination during slaughtering and processing and possibly it is responsible by the introduction of this microorganism in the slaughterhouses. A rapid method to identify and monitor Salmonella and their sorovars in farm is becoming necessary. A pre-enriched multiplex polymerase chain reaction (m-PCR) assay employing specific primers was developed and used to detect Salmonella at the genus level and to identify the Salmonella enterica serovar Enteritidis (S. Enteritidis) and Salmonella enterica serovar Typhimurium (S. Typhimurium) in broiler chicken swab samples. The method was validated by testing DNA extract from 90 fresh culture cloacal swab samples from poultry chicken cultured in phosphate buffer peptone water at 37 °C for 18 h. The final results showed the presence of Salmonella spp. in 25% of samples, S. Enteritidis was present in 12% of the Salmonella-positive samples and S. Typhimurium in 3% of the samples. The m-PCR assay developed in this study is a specific and rapid alternative method for the identification of Salmonella spp. and allowed the observation of specific serovar contamination in the field conditions within the locations where these chickens are typically raised. PMID:24159281

  14. Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing.

    PubMed

    Ståhlberg, Anders; Krzyzanowski, Paul M; Jackson, Jennifer B; Egyud, Matthew; Stein, Lincoln; Godfrey, Tony E

    2016-06-20

    Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants. PMID:27060140

  15. Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing

    PubMed Central

    Ståhlberg, Anders; Krzyzanowski, Paul M.; Jackson, Jennifer B.; Egyud, Matthew; Stein, Lincoln; Godfrey, Tony E.

    2016-01-01

    Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants. PMID:27060140

  16. A multiplex PCR-based method to identify strongylid parasite larvae recovered from ovine faecal cultures and/or pasture samples.

    PubMed

    Bisset, S A; Knight, J S; Bouchet, C L G

    2014-02-24

    A multiplex PCR-based method was developed to overcome the limitations of microscopic examination as a means of identifying individual infective larvae from the wide range of strongylid parasite species commonly encountered in sheep in mixed sheep-cattle grazing situations in New Zealand. The strategy employed targets unique species-specific sequence markers in the second internal transcribed spacer (ITS-2) region of ribosomal DNA of the nematodes and utilises individual larval lysates as reaction templates. The basic assay involves two sets of reactions designed to target the ten strongylid species most often encountered in ovine faecal cultures under New Zealand conditions (viz. Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus axei, Trichostrongylus colubriformis, Trichostrongylus vitrinus, Cooperia curticei, Cooperia oncophora, Nematodirus spathiger, Chabertia ovina, and Oesophagostomum venulosum). Five species-specific primers, together with a pair of "generic" (conserved) primers, are used in each of the reactions. Two products are generally amplified, one by the generic primer pair regardless of species (providing a positive PCR control) and the other (whose size is indicative of the species present) by the appropriate species-specific primer in combination with one or other of the generic primers. If necessary, any larvae not identified by these reactions can subsequently be tested using primers designed specifically to detect those species less frequently encountered in ovine faecal cultures (viz. Ostertagia ostertagi, Ostertagia leptospicularis, Cooperia punctata, Nematodirus filicollis, and Bunostomum trigonocephalum). Results of assays undertaken on >5500 nematode larvae cultured from lambs on 16 different farms distributed throughout New Zealand indicated that positive identifications were initially obtained for 92.8% of them, while a further 4.4% of reactions gave a generic but no visible specific product and 2.8% gave no discernible

  17. Detection of Aeromonas salmonicida by reverse transcription-multiplex polymerase chain reaction.

    PubMed

    Rattanachaikunsopon, Pongsak; Phumkhachorn, Parichat

    2012-01-01

    Aeromonas salmonicida is one of the major fish pathogens causing economically devastating losses in aquaculture. A. salmonicida subsp. salmonicida is a typical A. salmonicida causing furunculosis, while the other subspecies are atypical strains causing ulcer diseases. PCR-based methods of detecting A. salmonicida suffer from the drawback that they do not distinguish living (pathogenic) from dead cells. In this study, a method of detecting A. salmonicida was developed based on reverse transcription-multiplex PCR (RT-MPCR) using two sets of primers, SV1/SV2 and SF1/SF2, specific to the vapA gene and the fstB gene of A. salmonicida respectively. This method was found to detect A. salmonicida specifically with detection limits of 10 CFU in pure culture and 30 CFU in the presence of tissue debris. It was also found distinguish not only between viable and nonviable cells but also between typical and atypical strains of A. salmonicida. Using RT-MPCR, two DNA fragments, of 542 and 1,258 bp, were amplified from RNA of typical A. salmonicida, whereas only one DNA fragment, of 542 bp, was amplified from the RNA of the atypical ones. The proposed assay was also used successfully to detect A. salmonicida in artificially infected rainbow trout (Oncorhyncus mykiss). PMID:22484927

  18. How Well Does Physician Selection of Microbiologic Tests Identify Clostridium difficile and other Pathogens in Paediatric Diarrhoea? Insights Using Multiplex PCR-Based Detection

    PubMed Central

    Stockmann, Chris; Rogatcheva, Margarita; Harrel, Brian; Vaughn, Mike; Crisp, Rob; Poritz, Mark; Thatcher, Stephanie; Korgenski, Ernest K; Barney, Trenda; Daly, Judy; Pavia, Andrew T

    2014-01-01

    The objective of this study was to compare the aetiologic yield of standard of care microbiologic testing ordered by physicians with that of a multiplex PCR platform. Stool specimens obtained from children and young adults with gastrointestinal illness were evaluated by standard laboratory methods and a developmental version of the FilmArray Gastrointestinal Diagnostic System (FilmArray GI Panel), a rapid multiplex PCR platform that detects 23 bacterial, viral, and protozoal agents. Results were classified according to the microbiologic tests requested by the treating physician. A median of 3 (range 1-10) microbiologic tests were performed by the clinical laboratory during 378 unique diarrhoeal episodes. A potential aetiologic agent was identified in 46% of stool specimens by standard laboratory methods and in 65% of specimens tested using the FilmArray GI Panel (P<0.001). For those patients who only had Clostridium difficile testing requested, an alternative pathogen was identified in 29% of cases with the FilmArray GI Panel. Notably, 11 (12%) cases of norovirus were identified among children who only had testing for C. difficile ordered. Among those who had C. difficile testing ordered in combination with other tests, an additional pathogen was identified in 57% of stool specimens with the FilmArray GI Panel. For patients who had no C. difficile testing performed, the FilmArray GI Panel identified a pathogen in 63% of cases, including C. difficile in 8%. Physician-specified laboratory testing may miss important diarrhoeal pathogens. Additionally, standard laboratory testing is likely to underestimate co-infections with multiple infectious diarrhoeagenic agents. PMID:25599941

  19. Multiplex, construct-specific, and real-time PCR-based analytical methods for Bt rice with cry1Ac gene.

    PubMed

    Randhawa, Gurinder Jit; Singh, Monika

    2012-01-01

    Qualitative and quantitative analytical methods based on PCR for Bacillus thuringiensis (Bt) rice hybrid, namely, MRP 5401 Bt expressing a modified version of the Bt cry1Ac gene, are reported here. Multiplex PCR assays were developed to target the cry1Ac transgene, Cauliflower mosaic virus (CaMV) 35S promoter, Agrobacterium tumefaciens nopaline synthase (nos) terminator, the neomycin phosphotransferase II (nptLL) marker gene, and an endogenous a-tubulin (TubA) gene in Bt rice. The 3.178 kb region of inserted gene construct comprising the region of the CaMV 35S promoter and cry1Ac gene was amplified, and the construct integrity was confirmed by the nested PCR. The LOD for cry1Ac gene-specific simplex PCR was 0.01%, as established using Bt rice DNA dilutions with 100, 10, 1.0, 0.1, 0.05, 0.01, and 0.001% genetically modified trait. A real-time PCR assay was also developed to quantify the cry1Ac gene. The method performance of the reported real-time PCR assay was in line with the acceptance criteria of Codex Alimentarius Commission ALINORM 10/33/23, with LOD and LOQ values of 0.05%. The reliable PCR assays prior to commercial release of Bt rice would facilitate efficient regulatory compliance for identification of genetic trait, labeling requirements, and effective risk assessment and management. They could also address consumers' concerns and legal disputes that may arise. PMID:22468358

  20. Multiplex PCR based screening for micro/partial deletions in the AZF region of Y-chromosome in severe oligozoospermic and azoospermic infertile men in Iran

    PubMed Central

    Motovali-Bashi, Majid; Rezaei, Zahra; Dehghanian, Fariba; Rezaei, Halimeh

    2015-01-01

    Background: Infertility is a health problem which affects about 10-20% of married couples. Male factor infertility is involved approximately 50% of infertile couples. Most of male infertility is regarding to deletions in the male-specific region of the Y chromosome. Objective: In this study, the occurrence of deletions in the AZF region and association between infertility and paternal age were investigated in Iranian men population. Materials and Methods: To assess the occurrence of Y chromosomal microdeletions and partial deletions of the AZF region, 100 infertile men and 100 controls with normal spermatogenesis were analyzed. AZFa, AZFb, AZFc and partial deletions within the AZFc region were analyzed using multiplex PCR method. Finally, the association between paternal age and male infertility was evaluated. Results: No AZFa, AZFb or AZFc deletions were found in the control group. Seven infertile men had deletions as the following: one AZFb, five AZFc, and one AZFab. Partial deletions of AZFc (gr/gr) in 9 of the 100 infertile men (9/100, 9%) and 1 partial AZFc deletions (gr/gr) in the control group (1/100, 1%) were observed. In addition, five b2/b3 deletions in five azoospermic subjects (5/100, 5%) and 2 partial AZFc deletions (b2/b3) in the control group (2/100, 2%) were identified. Moreover, the risk of male infertility was influenced by the paternal age. Conclusion: The results of this study suggested that the frequency of Y chromosome AZF microdeletions increased in subjects with severe spermatogenic failure and gr/gr deletion associated with spermatogenic failure. PMID:26568761

  1. Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses.

    PubMed

    Waggoner, Jesse J; Gresh, Lionel; Mohamed-Hadley, Alisha; Ballesteros, Gabriela; Davila, Maria Jose Vargas; Tellez, Yolanda; Sahoo, Malaya K; Balmaseda, Angel; Harris, Eva; Pinsky, Benjamin A

    2016-07-01

    Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses. PMID:27184629

  2. Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses

    PubMed Central

    Waggoner, Jesse J.; Gresh, Lionel; Mohamed-Hadley, Alisha; Ballesteros, Gabriela; Davila, Maria Jose Vargas; Tellez, Yolanda; Sahoo, Malaya K.; Balmaseda, Angel; Harris, Eva

    2016-01-01

    Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses. PMID:27184629

  3. Performance analysis of spatial multiplexing MIMO system with time reversal technology

    NASA Astrophysics Data System (ADS)

    Shrestha, Sanjeeb; Dou, Zheng; Khan, Zayed

    2013-03-01

    This paper deals with the performance analysis of Spatial Multiplexing(SM) multiple input multiple output (MIMO) system with time reversal (TR) technology. Focus is given on the spatial multiplexing gain of MIMO than the diversity gain aspect with the notion that the idea of diversity is inseparably associated with the uncertainty of the channel. If transmitter knows Channel State Information (CSI) before transmission, potential benefits can be harvested. TR is used here, to provide Channel State Information (CSI) at the transmitter before transmission. With the features of temporal and spatial focusing, TR not only can provide immunity against fading for spatially multiplexed data stream but also help boost its Multi Stream Interference (MSI) limited performance by mitigating it. The performance analysis of SM-MIMOTR is carried out with the aim of average minimum error probability for quantity of interest data rate. The interest date rate is 19.07 Mbps, where as the average minimum error probably is set to be that of Single Input Multi Output (SIMO) maximum ratio combining system (MRC). BER of Single Input Single Output (SISO) system is also simulated for making comparison tangible. Simulation study shows that Bit Error Rate (BER) performance of the system with the data rate of interest nearly coincides with that of SIMO system at the range of 10-15db and is better than SISO in all simulated Eb/No points. Additionally, from the standpoint of tread off curve, between diversity gain and spatial multiplexing gain, the non linearity nature still holds.

  4. Rapid discrimination of rabies viruses isolated from various host species in Brazil by multiplex reverse transcription-polymerase chain reaction.

    PubMed

    Sato, Go; Tanabe, Hitomi; Shoji, Youko; Itou, Takuya; Ito, Fumio H; Sato, Tetsuo; Sakai, Takeo

    2005-08-01

    Rabies is carried mainly by mammalian carnivores and vampire bats in Latin America. However, rabies virus (RV) has been isolated in recent years from not only vampire bats in rural areas but also from several non-vampire bat species in urban areas, respectively. Therefore, rapid molecular screening is necessary for efficient epidemiology of these RVs. In this study, we investigated the usefulness of multiplex reverse transcription-polymerase chain reaction (RT-PCR) for determining the origins of 54 RV isolates from various host species in Brazil. And to evaluate the multiplex RT-PCR as a potential diagnostic tool, we investigated the sensitivity of this method. In addition, we compared the results with a phylogenetic tree developed from sequences of the RV glycoprotein (G protein) gene. Multiplex RT-PCR products showed five different sizes of products, whereas the phylogenic tree showed six groups. Of these six groups, four corresponded with the four sizes of the multiplex RT-PCR products. The other two groups showed correspondance with another one size of the multiplex RT-PCR products, indicating that multiplex RT-PCR results reflected the lineage of the 54 isolates. This study also showed that this method can detect trace amounts of RNA. In conclusion, this multiplex RT-PCR method allows the rapid, specific, and simultaneous detection of RVs isolated from various host species in Brazil. PMID:16036175

  5. Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

    PubMed Central

    O'Sullivan, Matthew V. N.; Zhou, Fei; Sintchenko, Vitali; Kong, Fanrong; Gilbert, Gwendolyn L.

    2011-01-01

    Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction followed by probe hybridization on a nylon membrane, which is re-usable. Probes are 5' amine modified to allow fixation to the membrane. Primers are 5' biotin modified which allows detection of hybridized PCR products using streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film. With low setup and consumable costs, this technique is inexpensive (approximately US$2 per sample), high throughput (multiple membranes can be processed simultaneously) and has a short turnaround time (approximately 10 hours). The technique can be utilized in a number of ways. Multiple probes can be designed to detect sequence variation within a single amplified product, or multiple products can be amplified simultaneously, with one (or more) probes used for subsequent detection. A combination of both approaches can also be used within a single assay. The ability to include multiple probes for a single target sequence makes the assay highly specific. Published applications of mPCR/RLB include detection of antibiotic resistance genes1,2, typing of methicillin-resistant Staphylococcus aureus3-5 and Salmonella sp6, molecular serotyping of Streptococcus pneumoniae7,8, Streptococcus agalactiae9 and enteroviruses10,11, identification of Mycobacterium sp12, detection of genital13-15 and respiratory tract16 and other17 pathogens and detection and identification of mollicutes18. However, the versatility of the technique means the applications are virtually limitless and not restricted to molecular analysis of micro-organisms. The five steps in mPCR/RLB are a) Primer and Probe design, b) DNA extraction and PCR amplification c) Preparation of the membrane, d) Hybridization and detection, and e) Regeneration of the Membrane. PMID:21847083

  6. Simultaneous detection of four garlic viruses by multiplex reverse transcription PCR and their distribution in Indian garlic accessions.

    PubMed

    Majumder, S; Baranwal, V K

    2014-06-01

    Indian garlic is infected with Onion yellow dwarf virus (OYDV), Shallot latent virus (SLV), Garlic common latent virus (GarCLV) and allexiviruses. Identity and distribution of garlic viruses in various garlic accessions from different geographical regions of India were investigated. OYDV and allexiviruses were observed in all the garlic accessions, while SLV and GarCLV were observed only in a few accessions. A multiplex reverse transcription (RT)-PCR method was developed for the simultaneous detection and identification of OYDV, SLV, GarCLV and Allexivirus infecting garlic accessions in India. This multiplex protocol standardized in this study will be useful in indexing of garlic viruses and production of virus free seed material. PMID:24598229

  7. Simultaneous detection of papaya ringspot virus, papaya leaf distortion mosaic virus, and papaya mosaic virus by multiplex real-time reverse transcription PCR.

    PubMed

    Huo, P; Shen, W T; Yan, P; Tuo, D C; Li, X Y; Zhou, P

    2015-12-01

    Both the single infection of papaya ringspot virus (PRSV), papaya leaf distortion mosaic virus (PLDMV) or papaya mosaic virus (PapMV) and double infection of PRSV and PLDMV or PapMV which cause indistinguishable symptoms, threaten the papaya industry in Hainan Island, China. In this study, a multiplex real-time reverse transcription PCR (RT-PCR) was developed to detect simultaneously the three viruses based on their distinctive melting temperatures (Tms): 81.0±0.8°C for PRSV, 84.7±0.6°C for PLDMV, and 88.7±0.4°C for PapMV. The multiplex real-time RT-PCR method was specific and sensitive in detecting the three viruses, with a detection limit of 1.0×10(1), 1.0×10(2), and 1.0×10(2) copies for PRSV, PLDMV, and PapMV, respectively. Indeed, the reaction was 100 times more sensitive than the multiplex RT-PCR for PRSV, and 10 times more sensitive than multiplex RT-PCR for PLDMV. Field application of the multiplex real-time RT-PCR demonstrated that some non-symptomatic samples were positive for PLDMV by multiplex real-time RT-PCR but negative by multiplex RT-PCR, whereas some samples were positive for both PRSV and PLDMV by multiplex real-time RT-PCR assay but only positive for PLDMV by multiplex RT-PCR. Therefore, this multiplex real-time RT-PCR assay provides a more rapid, sensitive and reliable method for simultaneous detection of PRSV, PLDMV, PapMV and their mixed infections in papaya. PMID:26666186

  8. Combining reverse-transcription multiplex PCR and microfluidic electrophoresis to simultaneously detect seven mosquito-transmitted zoonotic encephalomyelitis viruses.

    PubMed

    Wang, Yu; Ostlund, Eileen N; Jun, Yang; Nie, Fu-Ping; Li, Ying-Guo; Johnson, Donna J; Lin, Rui; Li, Zheng-Guo

    2016-06-01

    Several mosquito-transmitted viruses are causative agents for zoonotic encephalomyelitis. Rapid identification of these viruses in mosquito populations is an effective method for surveying these diseases. To detect multiple mosquito-transmitted viral agents, including West Nile virus, Saint Louis encephalitis virus, Venezuelan equine encephalomyelitis virus, Western equine encephalomyelitis virus, Eastern equine encephalomyelitis virus, Highlands J virus and Japanese encephalitis virus, an assay using multiplex reverse-transcription PCR combined with microfluidic electrophoresis was developed and evaluated. Tailed nested primers were used in the assay to amplify specific viral genomic segments, and products with specific length were further analyzed by using a microfluidic electrophoresis chip. The assay exhibited good specificity and analytical sensitivity (10(2) copies/µL). This technology can be helpful in the quarantine and surveillance of exotic encephalomyelitis viruses which are transmitted by mosquitoes. PMID:27256022

  9. Primer Extension Reactions for the PCR- based α- complementation Assay

    PubMed Central

    Achuthan, Vasudevan; DeStefano, Jeffrey J.

    2016-01-01

    The PCR- based- α- complementation assay is an effective technique to measure the fidelity of polymerases, especially RNA-dependent RNA polymerases (RDRP) and Reverse Transcriptases (RT). It has been successfully employed to determine the fidelity of the poliovirus polymerase 3D-pol (DeStefano, 2010) as well as the human immunodeficiency virus Reverse Transcriptase (HIV RT) (Achuthan et al., 2014). A major advantage of the assay is that since the PCR step is involved, even the low yield of products obtained after two rounds of low yield of RNA synthesis (for RDRP) or reverse transcription (for RT) can be measured using the assay. The assay also mimics the reverse transcription process, since both RNA- and DNA- directed RT synthesis steps are performed. We recently used this assay to show that the HIV RT, at physiologically relevant magnesium concentration, has accuracy in the same range as other reverse transcriptases (Achuthan et al., 2014). Here, we describe in detail how to prepare the inserts using the primer extension reactions. The prepared inserts are then processed further in the PCR- based- α- complementation assay.

  10. Focused human gene expression profiling using dual-color reverse transcriptase multiplex ligation-dependent probe amplification.

    PubMed

    Haks, Mariëlle C; Goeman, Jelle J; Magis-Escurra, Cecile; Ottenhoff, Tom H M

    2015-09-29

    To investigate the human immune response to newly developed or existing vaccines, or during infection/disease on a population scale, we have recently developed a dual-color Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification (dcRT-MLPA) assay, which can rapidly profile mRNA expression of multiple host genes. dcRT-MLPA has a dynamic range and sensitivity comparable to real-time QPCR and RNA-Sequencing. Since this assay is high-throughput, it is an exceptionally suitable technique for monitoring host biomarkers in semi-large scale human cohorts, such as cross sectional studies with multiple groups, or longitudinal studies with multiple time points. Multicomponent host biomarker signatures with excellent predictive values can easily be identified using lasso regression analysis, while exploring additional data adjustment methods like RUV-2 may further optimize the identification of informative host biomarker signatures. dcRT-MLPA also allows comparisons of gene expression patterns across different human populations to explore the impact of geographical diversity on for example vaccine induced responses. The use of dcRT-MLPA is not limited to peripheral blood but can be adapted to analyze host biomarkers derived from any tissue or body fluids, further demonstrating the versatility of the dcRT-MLPA platform. Several examples will be given and discussed. PMID:25917681

  11. Development of multiplex real-time reverse transcriptase PCR assays for detecting eight medically important flaviviruses in mosquitoes.

    PubMed

    Chao, Day-Yu; Davis, Brent S; Chang, Gwong-Jen J

    2007-02-01

    A multiplex real-time reverse transcriptase PCR has been developed for the rapid detection and identification of eight medically important flaviviruses from laboratory-reared, virus-infected mosquito pools. The method used involves the gene-specific amplification of yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and dengue virus (DENV) serotypes 1 to 4 (DENV-1 to DENV-4, respectively) by use of the flavivirus consensus amplimers located at the RNA-dependent RNA polymerase domain of nonstructural protein 5. Virus-specific amplicons were detected by four newly characterized TaqMan fluorogenic probes (probes specific for YFV, JEV, WNV, and SLEV) and four previously published probes specific for DENV-1 to -4 (L. J. Chien, T. L. Liao, P. Y. Shu, J. H. Huang, D. J. Gubler, and G. J. Chang, J. Clin. Microbiol. 44:1295-1304, 2006). This assay had a specificity of 100% and various sensitivities of at least 3.5 PFU/ml for YFV, 2.0 PFU/ml for JEV, 10.0 PFU/ml for WNV, and 10.0 PFU/ml for SLEV. Additionally, we have developed an in vitro transcription system to generate RNase-resistant RNA templates for each of these eight viruses. These templates can be incorporated into the assay as RNA copy number controls and/or as external controls for RNA-spiked mosquito pools for quality assurance purposes. Although further study with mosquitoes collected in the field is needed, the incorporation of this assay into mosquito surveillance could be used as an early-warning system for the detection of medically important flaviviruses, particularly when the cocirculation of multiple viruses in the same region is suspected. PMID:17108075

  12. Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses.

    PubMed

    Simmons, Monika; Myers, Todd; Guevara, Carolina; Jungkind, Donald; Williams, Maya; Houng, Huo-Shu

    2016-07-01

    Dengue virus (DENV) and chikungunya virus (CHIKV) are important human pathogens with common transmission vectors and similar clinical presentations. Patient care may be impacted by the misdiagnosis of DENV and CHIKV in areas where both viruses cocirculate. In this study, we have developed and validated a one-step multiplex reverse transcriptase PCR (RT-PCR) to simultaneously detect, quantify, and differentiate between four DENV serotypes (pan-DENV) and chikungunya virus. The assay uses TaqMan technology, employing two forward primers, three reverse primers, and four fluorophore-labeled probes in a single-reaction format. Coextracted and coamplified RNA was used as an internal control (IC), and in vitro-transcribed DENV and CHIKV RNAs were used to generate standard curves for absolute quantification. The diagnostic 95% limits of detection (LOD) within the linear range were 50 and 60 RNA copies/reaction for DENV (serotypes 1 to 4) and CHIKV, respectively. Our assay was able to detect 53 different strains of DENV, representing four serotypes, and six strains of CHIKV. No cross-reactivity was observed with related flaviviruses and alphaviruses, To evaluate diagnostic sensitivity and specificity, 89 clinical samples positive or negative for DENV (serotypes 1 to 4) and CHIKV by the standard virus isolation method were tested in our assay. The multiplex RT-PCR assay showed 95% sensitivity and 100% specificity for DENV and 100% sensitivity and specificity for CHIKV. With an assay turnaround time of less than 2 h, including extraction of RNA, the multiplex quantitative RT-PCR assay provides rapid diagnosis for the differential detection of two clinically indistinguishable diseases, whose geographical occurrence is increasingly overlapping. PMID:27098955

  13. Is real-time PCR-based diagnosis similar in performance to routine parasitological examination for the identification of Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica from stool samples? Evaluation of a new commercial multiplex PCR assay and literature review.

    PubMed

    Laude, A; Valot, S; Desoubeaux, G; Argy, N; Nourrisson, C; Pomares, C; Machouart, M; Le Govic, Y; Dalle, F; Botterel, F; Bourgeois, N; Cateau, E; Leterrier, M; Le Pape, P; Morio, F

    2016-02-01

    Microscopy is the reference standard for routine laboratory diagnosis in faecal parasitology but there is growing interest in alternative methods to overcome the limitations of microscopic examination, which is time-consuming and highly dependent on an operator's skills and expertise. Compared with microscopy, DNA detection by PCR is simple and can offer a better turnaround time. However, PCR performances remain difficult to assess as most studies have been conducted on a limited number of positive clinical samples and used in-house PCR methods. Our aim was to evaluate a new multiplex PCR assay (G-DiaParaTrio; Diagenode Diagnostics), targeting Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica. To minimize the turnaround time, PCR was coupled with automated DNA extraction (QiaSymphony; Qiagen). The PCR assay was evaluated using a reference panel of 185 samples established by routine microscopic examination using a standardized protocol including Ziehl-Neelsen staining and adhesin detection by ELISA (E. histolytica II; TechLab). This panel, collected from 12 French parasitology laboratories, included 135 positive samples for G. intestinalis (n = 38), C. parvum/C. hominis (n = 26), E. histolytica (n = 5), 21 other gastrointestinal parasites, together with 50 negative samples. In all, the G-DiaParaTrio multiplex PCR assay identified 38 G. intestinalis, 25 C. parvum/C. hominis and five E. histolytica leading to sensitivity/specificity of 92%/100%, 96%/100% and 100%/100% for G. intestinalis, C. parvum/C. hominis and E. histolytica, respectively. This new multiplex PCR assay offers fast and reliable results, similar to microscopy-driven diagnosis for the detection of these gastrointestinal protozoa, allowing its implementation in routine clinical practice. PMID:26548509

  14. A multiplexed reverse transcriptase PCR assay for identification of viral respiratory pathogens at point-of-care

    SciTech Connect

    Letant, S E; .Ortiz, J I; Tammero, L; Birch, J M; Derlet, R W; Cohen, S; Manning, D; McBride, M T

    2007-04-11

    We have developed a nucleic acid-based assay that is rapid, sensitive, specific, and can be used for the simultaneous detection of 5 common human respiratory pathogens including influenza A, influenza B, parainfluenza type 1 and 3, respiratory syncytial virus, and adenovirus group B, C, and E. Typically, diagnosis on an un-extracted clinical sample can be provided in less than 3 hours, including sample collection, preparation, and processing, as well as data analysis. Such a multiplexed panel would enable rapid broad-spectrum pathogen testing on nasal swabs, and therefore allow implementation of infection control measures, and timely administration of antiviral therapies. This article presents a summary of the assay performance in terms of sensitivity and specificity. Limits of detection are provided for each targeted respiratory pathogen, and result comparisons are performed on clinical samples, our goal being to compare the sensitivity and specificity of the multiplexed assay to the combination of immunofluorescence and shell vial culture currently implemented at the UCDMC hospital. Overall, the use of the multiplexed RT-PCR assay reduced the rate of false negatives by 4% and reduced the rate of false positives by up to 10%. The assay correctly identified 99.3% of the clinical negatives, 97% of adenovirus, 95% of RSV, 92% of influenza B, and 77% of influenza A without any extraction performed on the clinical samples. The data also showed that extraction will be needed for parainfluenza virus, which was only identified correctly 24% of the time on un-extracted samples.

  15. Multiplex detection of agricultural pathogens

    DOEpatents

    Siezak, Thomas R.; Gardner, Shea; Torres, Clinton; Vitalis, Elizabeth; Lenhoff, Raymond J.

    2013-01-15

    Described are kits and methods useful for detection of agricultural pathogens in a sample. Genomic sequence information from agricultural pathogens was analyzed to identify signature sequences, e.g., polynucleotide sequences useful for confirming the presence or absence of a pathogen in a sample. Primer and probe sets were designed and optimized for use in a PCR based, multiplexed Luminex assay and/or an array assay to successfully identify the presence or absence of pathogens in a sample.

  16. Multiplex detection of agricultural pathogens

    DOEpatents

    McBride, Mary Teresa; Slezak, Thomas Richard; Messenger, Sharon Lee

    2010-09-14

    Described are kits and methods useful for detection of seven agricultural pathogens (BPSV; BHV; BVD; FMDV; BTV; SVD; and VESV) in a sample. Genomic sequence information from 7 agricultural pathogens was analyzed to identify signature sequences, e.g., polynucleotide sequences useful for confirming the presence or absence of a pathogen in a sample. Primer and probe sets were designed and optimized for use in a PCR based, multiplexed Luminex assay to successfully identify the presence or absence of pathogens in a sample.

  17. Design of Multiplexed Detection Assays for Identification of Avian Influenza A Virus Subtypes Pathogenic to Humans by SmartCycler Real-Time Reverse Transcription-PCR ▿

    PubMed Central

    Wang, Wei; Ren, Peijun; Mardi, Sek; Hou, Lili; Tsai, Cheguo; Chan, Kwok Hung; Cheng, Peter; Sheng, Jun; Buchy, Philippe; Sun, Bing; Toyoda, Tetsuya; Lim, Wilina; Peiris, J. S. Malik; Zhou, Paul; Deubel, Vincent

    2009-01-01

    Influenza A virus (IAV) epidemics are the result of human-to-human or poultry-to-human transmission. Tracking seasonal outbreaks of IAV and other avian influenza virus (AIV) subtypes that can infect humans, aquatic and migratory birds, poultry, and pigs is essential for epidemiological surveillance and outbreak alerts. In this study, we performed four real-time reverse transcription-PCR (rRT-PCR) assays for identification of the IAV M and hemagglutinin (HA) genes from six known AIVs infecting pigs, birds, and humans. IAV M1 gene-positive samples tested by single-step rRT-PCR and a fluorogenic Sybr green I detection system were further processed for H5 subtype identification by using two-primer-set multiplex and Sybr green I rRT-PCR assays. H5 subtype-negative samples were then tested with either a TaqMan assay for subtypes H1 and H3 or a TaqMan assay for subtypes H2, H7, and H9 and a beacon multiplex rRT-PCR identification assay. The four-tube strategy was able to detect 10 RNA copies of the HA genes of subtypes H1, H2, H3, H5, and H7 and 100 RNA copies of the HA gene of subtype H9. At least six H5 clades of H5N1 viruses isolated in Southeast Asia and China were detected by that test. Using rRT-PCR assays for the M1 and HA genes in 202 nasopharyngeal swab specimens from children with acute respiratory infections, we identified a total of 39 samples positive for the IAV M1 gene and subtypes H1 and H3. When performed with a portable SmartCycler instrument, the assays offer an efficient, flexible, and reliable platform for investigations of IAV and AIV in remote hospitals and in the field. PMID:18971359

  18. A two-tube multiplex reverse transcription PCR assay for simultaneous detection of viral and bacterial pathogens of infectious diarrhea.

    PubMed

    Wang, Ji; Xu, Ziqian; Niu, Peihua; Zhang, Chen; Zhang, Jingyun; Guan, Li; Kan, Biao; Duan, Zhaojun; Ma, Xuejun

    2014-01-01

    Diarrhea caused by viral and bacterial infections is a major health problem in developing countries. The purpose of this study is to develop a two-tube multiplex PCR assay using automatic electrophoresis for simultaneous detection of 13 diarrhea-causative viruses or bacteria, with an intended application in provincial Centers for Diseases Control and Prevention, China. The assay was designed to detect rotavirus A, norovirus genogroups GI and GII, human astrovirus, enteric adenoviruses, and human bocavirus (tube 1), and Salmonella, Vibrio parahaemolyticus, diarrheagenic Escherichia coli, Campylobacter jejuni, Shigella, Yersinia, and Vibrio cholera (tube 2). The analytical specificity was examined with positive controls for each pathogen. The analytical sensitivity was evaluated by performing the assay on serial tenfold dilutions of in vitro transcribed RNA, recombinant plasmids, or bacterial culture. A total of 122 stool samples were tested by this two-tube assay and the results were compared with those obtained from reference methods. The two-tube assay achieved a sensitivity of 20-200 copies for a single virus and 10(2)-10(3) CFU/mL for bacteria. The clinical performance demonstrated that the two-tube assay had comparable sensitivity and specificity to those of reference methods. In conclusion, the two-tube assay is a rapid, cost-effective, sensitive, specific, and high throughput method for the simultaneous detection of enteric bacteria and virus. PMID:24711998

  19. Mononeuritis multiplex

    MedlinePlus

    Mononeuropathy multiplex; Multifocal neuropathy; Peripheral neuropathy - mononeuritis multiplex ... Shy ME. Peripheral neuropathies. In: Goldman L, Ausiello D, eds. Goldman's Cecil Medicine . 23rd ed. Philadelphia, PA: Elsevier Saunders; 2007:chap 446.

  20. Use of an internal positive control in a multiplex reverse transcription-PCR to detect West Nile virus RNA in mosquito pools.

    PubMed

    Eisler, Diane L; McNabb, Alan; Jorgensen, Danielle R; Isaac-Renton, Judith L

    2004-02-01

    We report on the use of West Nile virus Armored RNA as an internal positive control (IPC) for the extraction and reverse transcription-PCR (RT-PCR) of RNA extracted from field-collected mosquitoes and on a multiplex real-time Taqman RT-PCR to simultaneously detect the 3' noncoding region of West Nile virus and the West Nile virus NS5-2 region comprising the IPC. Mosquito pools from the province of British Columbia, Canada (n = 635), were tested in duplicate and found to be negative for West Nile virus and positive for the IPC. Known West Nile virus-positive supernatants from mosquito pools from the provinces of Alberta and Manitoba were tested in duplicate and found to be positive for both regions of the West Nile virus genome. The mean cycle threshold (Ct) value for the IPC in batch extraction controls +/- 2 standard deviations was found to be 36.43 +/- 1.78 cycles. IPCs of 98.4% (624) of West Nile virus-negative pools fell within this range, indicating the reproducibility of RNA extraction and RT-PCR for pools varying in mosquito genus and number. A comparison of mosquito pool genera revealed no significant genus effect on the Ct value of the IPC. The incorporation of West Nile virus Armored RNA as an IPC allows monitoring of RNA extraction and RT-PCR and detection of false-negative results due to failures in these processes or to PCR inhibition, respectively. PMID:14766868

  1. Simultaneous Detection of Rift Valley Fever, Bluetongue, Rinderpest, and Peste des Petits Ruminants Viruses by a Single-Tube Multiplex Reverse Transcriptase-PCR Assay Using a Dual-Priming Oligonucleotide System▿

    PubMed Central

    Yeh, Jung-Yong; Lee, Ji-Hye; Seo, Hyun-Ji; Park, Jee-Yong; Moon, Jin-San; Cho, In-Soo; Choi, In-Soo; Park, Seung-Yong; Song, Chang-Seon; Lee, Joong-Bok

    2011-01-01

    The aim of this study was to develop a highly sensitive and specific one-step multiplex reverse transcriptase PCR assay for the simultaneous and differential detection of Rift Valley Fever virus (RVFV), bluetongue virus (BTV), rinderpest virus (RPV), and Peste des petits ruminants virus (PPRV). These viruses cause mucosal lesions in cattle, sheep, and goats, and they are difficult to differentiate from one another based solely on their clinical presentation in suspected disease cases. In this study, we developed a multiplex reverse transcriptase PCR to detect these viruses using a novel dual-priming oligonucleotide (DPO). The DPO contains two separate priming regions joined by a polydeoxyinosine linker, which blocks extension of nonspecifically primed templates and consistently allows high PCR specificity even under less-than-optimal PCR conditions. A total of 19 DPO primers were designed to detect and discriminate between RVFV, BTV, RPV, and PPRV by the generation of 205-, 440-, 115-, and 243-bp cDNA products, respectively. The multiplex reverse transcriptase PCR described here enables the early diagnosis of these four viruses and may also be useful as part of a testing regime for cattle, sheep, or goats exhibiting similar clinical signs, including mucosal lesions. PMID:21307219

  2. Development and validation of a multiplex reverse transcription quantitative PCR (RT-qPCR) assay for the rapid detection of Citrus tristeza virus, Citrus psorosis virus, and Citrus leaf blotch virus.

    PubMed

    Osman, Fatima; Hodzic, Emir; Kwon, Sun-Jung; Wang, Jinbo; Vidalakis, Georgios

    2015-08-01

    A single real-time multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay for the simultaneous detection of Citrus tristeza virus (CTV), Citrus psorosis virus (CPsV), and Citrus leaf blotch virus (CLBV) was developed and validated using three different fluorescently labeled minor groove binding qPCR probes. To increase the detection reliability, coat protein (CP) genes from large number of different isolates of CTV, CPsV and CLBV were sequenced and a multiple sequence alignment was generated with corresponding CP sequences from the GenBank and a robust multiplex RT-qPCR assay was designed. The capacity of the multiplex RT-qPCR assay in detecting the viruses was compared to singleplex RT-qPCR designed specifically for each virus and was assessed using multiple virus isolates from diverse geographical regions and citrus species as well as graft-inoculated citrus plants infected with various combination of the three viruses. No significant difference in detection limits was found and specificity was not affected by the inclusion of the three assays in a multiplex RT-qPCR reaction. Comparison of the viral load for each virus using singleplex and multiplex RT-qPCR assays, revealed no significant differences between the two assays in virus detection. No significant difference in Cq values was detected when using one-step and two-step multiplex RT-qPCR detection formats. Optimizing the RNA extraction technique for citrus tissues and testing the quality of the extracted RNA using RT-qPCR targeting the cytochrome oxidase citrus gene as an RNA specific internal control proved to generate better diagnostic assays. Results showed that the developed multiplex RT-qPCR can streamline viruses testing of citrus nursery stock by replacing three separate singleplex assays, thus reducing time and labor while retaining the same sensitivity and specificity. The three targeted RNA viruses are regulated pathogens for California's mandatory "Section 3701

  3. Development of a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction assay for the differential diagnosis of Feline leukemia virus vaccine and wild strains.

    PubMed

    Ho, Chia-Fang; Chan, Kun-Wei; Yang, Wei-Cheng; Chiang, Yu-Chung; Chung, Yang-Tsung; Kuo, James; Wang, Chi-Young

    2014-05-19

    A multiplex amplification refractory mutation system reverse transcription polymerase chain reaction (ARMS RT-PCR) was developed for the differential diagnosis of Feline leukemia virus (FeLV) vaccine and wild-type strains based on a point mutation between the vaccine strain (S) and the wild-type strain (T) located in the p27 gene. This system was further upgraded to obtain a real-time ARMS RT-PCR (ARMS qRT-PCR) with a high-resolution melt analysis (HRMA) platform. The genotyping of various strains of FeLV was determined by comparing the HRMA curves with the defined wild-type FeLV (strain TW1), and the results were expressed as a percentage confidence. The detection limits of ARMS RT-PCR and ARMS qRT-PCR combined with HRMA were 100 and 1 copies of transcribed FeLV RNA per 0.5 ml of sample, respectively. No false-positive results were obtained with 6 unrelated pathogens and 1 feline cell line. Twelve FeLV Taiwan strains were correctly identified using ARMS qRT-PCR combined with HRMA. The genotypes of the strains matched the defined FeLV wild-type strain genotype with at least 91.17% confidence. A higher degree of sequence polymorphism was found throughout the p27 gene compared with the long terminal repeat region. In conclusion, the current study describes the phylogenetic relationship of the FeLV Taiwan strains and demonstrates that the developed ARMS RT-PCR assay is able to be used to detect the replication of a vaccine strain that has not been properly inactivated, thus acting as a safety check for the quality of FeLV vaccines. PMID:24842287

  4. Multiplex detection of respiratory pathogens

    DOEpatents

    McBride, Mary; Slezak, Thomas; Birch, James M.

    2012-07-31

    Described are kits and methods useful for detection of respiratory pathogens (influenza A (including subtyping capability for H1, H3, H5 and H7 subtypes) influenza B, parainfluenza (type 2), respiratory syncytial virus, and adenovirus) in a sample. Genomic sequence information from the respiratory pathogens was analyzed to identify signature sequences, e.g., polynucleotide sequences useful for confirming the presence or absence of a pathogen in a sample. Primer and probe sets were designed and optimized for use in a PCR based, multiplexed Luminex assay to successfully identify the presence or absence of pathogens in a sample.

  5. Steatocystoma multiplex.

    PubMed

    Chu, David H

    2003-10-01

    A 25-year-old man with a 20-year history of asymptomatic nodules on his arms and trunk, which histopathological analysis showed to be consistent with steatocystoma multiplex, is presented. Steatocystoma multiplex is a disorder characterized by multiple, asymptomatic, dermal cysts that usually occur on the trunk and proximal aspects of the extremities. Steatocystoma multiplex with acral predominance has only recently been described. Development of steatocystomas has been hypothesized to be due to alterations in the structure of keratin 17. Treatment for lesions has included surgical excision or drainage, oral retinoids, and liquid nitrogen cryotherapy. PMID:14594591

  6. Straightforward and sensitive RT-qPCR based gene expression analysis of FFPE samples

    PubMed Central

    Zeka, Fjoralba; Vanderheyden, Katrien; De Smet, Els; Cuvelier, Claude A.; Mestdagh, Pieter; Vandesompele, Jo

    2016-01-01

    Fragmented RNA from formalin-fixed paraffin-embedded (FFPE) tissue is a known obstacle to gene expression analysis. In this study, the impact of RNA integrity, gene-specific reverse transcription and targeted cDNA preamplification was quantified in terms of reverse transcription polymerase chain reaction (RT-qPCR) sensitivity by measuring 48 protein coding genes on eight duplicate cultured cancer cell pellet FFPE samples and twenty cancer tissue FFPE samples. More intact RNA modestly increased gene detection sensitivity by 1.6 fold (earlier detection by 0.7 PCR cycles, 95% CI = 0.593–0.850). Application of gene-specific priming instead of whole transcriptome priming during reverse transcription further improved RT-qPCR sensitivity by a considerable 4.0 fold increase (earlier detection by 2.0 PCR cycles, 95% CI = 1.73–2.32). Targeted cDNA preamplification resulted in the strongest increase of RT-qPCR sensitivity and enabled earlier detection by an average of 172.4 fold (7.43 PCR cycles, 95% CI = 6.83–7.05). We conclude that gene-specific reverse transcription and targeted cDNA preamplification are adequate methods for accurate and sensitive RT-qPCR based gene expression analysis of FFPE material. The presented methods do not involve expensive or complex procedures and can be easily implemented in any routine RT-qPCR practice. PMID:26898768

  7. Multiplex Pyrosequencing.

    PubMed

    Pourmand, Nader; Elahi, Elahe; Davis, Ronald W; Ronaghi, Mostafa

    2002-04-01

    We describe here the development of a new and simple single-tube multiplex Pyrosequencing assay. Genomic DNA or cDNA was employed to PCR amplify region(s) using biotinylated and normal primer(s). Subsequent to capture of PCR products on streptavidin-coated beads, single-stranded DNA separation and hybridization of multiple sequencing primers, Pyrosequencing was performed. The obtained pyrogram resulted in a unique pattern in which the intensity of the signal determined the number of incorporated nucleotide(s). Here, we demonstrate the use of this multiplex Pyrosequencing for single nucleotide polymorphisms genotyping and microbial typing. PMID:11917037

  8. Hungarian population data on seven PCR-based loci.

    PubMed

    Budowle, B; Woller, J; Koons, B W; Furedi, S; Errera, J D; Padar, Z

    1996-07-01

    Hungarian population data for the loci LDLR, GYPA, HBGG, D7S8, Gc, HLA-DQA1, and D1S80 were generated. The genotype frequency distributions for the loci do not deviate from Hardy Weinberg expectations. Furthermore, there was little evidence for departures from expectations of independence between the loci. Using a test for homogeneity all the loci were similar between two Hungarian population samples and only the HLA-DQA1 locus was statistically different between Hungarians and US Caucasians. There generally would be little forensic differences, whether a Hungarian or a US Caucasian database was used, for estimating multiple locus profile frequencies for the seven PCR-based loci. PMID:8754580

  9. [PCR-based detection of pathogens in clinical rheumatology].

    PubMed

    Ehrenstein, B; Reischl, U

    2016-05-01

    In the differential diagnostics of autoimmune-mediated rheumatic diseases, rheumatologists often have to consider infections (e. g. Lyme arthritis) or reactive diseases (e. g. reactive arthritis after urogenital bacterial infections). Furthermore, infections with an atypical presentation or caused by atypical pathogens (opportunistic infections) can complicate the immunosuppressive therapy of autoimmune diseases. For this purpose not only conventional microbiological culture methods but also PCR-based methods are increasingly being applied for the direct detection of pathogens in clinical specimens. The aim of this overview is to present commonly used PCR methods in the clinical practice of rheumatology and to describe their benefits and limitations compared to culture-based detection methods. PMID:26892924

  10. PCR-based polymorphisms in neurofibromatosis type 1 (NFI)

    SciTech Connect

    Lai, P.S.; Chee, S.; Low, P.S.

    1994-09-01

    Neurofibromatosis type 1 (NF1) is one of the most common genetic disorders in humans with an incidence of 1 in 3,000. The NF1 gene is located on chromosome 17q 11.2 and encodes an ubiquitously expressed transcript of about 13kb. Direct mutation detection is difficult in this disorder due to the large gene size, high mutation rate and variety of mutations. We have studied the allele frequencies of seven PCR-based polymorphisms. Six of the probes used flank the NF1 gene, namely p11.3C4.2/Msp I (proximal), pEW206/Msp I (distal), p2.f9.8/Rsa I (distal), pEW207/Bgl II (distal), pEW207/Hind III (distal) and pHHH202/Rsa I (proximal). An intragenic RFLP, pEvi 2B-B/Eco R1 polymorphism in intron 27, was also analyzed by PCR. Allele frequencies for 48 normal unrelated individuals were obtained as follows: A1 = 0.40, A2 = 0.6 (p11.3C4.2/Msp I), A1 = 0.44, A2 = 0.56 (pEW206/Msp I), A1 = 0.17, A2 = 0.83 (p2.F9.8/Rsa I), A1 = 0.64, A2 = 0.36 (pEW207/Bgl I), A1 = 0.45, A2 = 0.55 (pEvi 2B-B/Eco RI). Heterozygosity rates of the alleles ranged from 20.8% to 51.7%. Using a combination of these markers, seven local families with NF1 were studied. Normal Mendelian segregation of alleles was observed in these families and no recombination was detected so far. These PCR-based markers were found to be useful for linkage analysis in our families.

  11. Multiplex PCR Tests for Detection of Pathogens Associated with Gastroenteritis

    PubMed Central

    Zhang, Hongwei; Morrison, Scott; Tang, Yi-Wei

    2016-01-01

    Synopsis A wide range of enteric pathogens can cause infectious gastroenteritis. Conventional diagnostic algorithms including culture, biochemical identification, immunoassay and microscopic examination are time consuming and often lack sensitivity and specificity. Advances in molecular technology have as allowed its use as clinical diagnostic tools. Multiplex PCR based testing has made its way to gastroenterology diagnostic arena in recent years. In this article we present a review of recent laboratory developed multiplex PCR tests and current commercial multiplex gastrointestinal pathogen tests. We will focus on two FDA cleared commercial syndromic multiplex tests: Luminex xTAG GPP and Biofire FimArray GI test. These multiplex tests can detect and identify multiple enteric pathogens in one test and provide results within hours. Multiplex PCR tests have shown superior sensitivity to conventional methods for detection of most pathogens. The high negative predictive value of these multiplex tests has led to the suggestion that they be used as screening tools especially in outbreaks. Although the clinical utility and benefit of multiplex PCR test are to be further investigated, implementing these multiplex PCR tests in gastroenterology diagnostic algorithm has the potential to improve diagnosis of infectious gastroenteritis. PMID:26004652

  12. PCR-based identification of drowning: four case reports.

    PubMed

    Rácz, Evelin; Könczöl, Franciska; Tóth, Dénes; Patonai, Zoltán; Porpáczy, Zoltán; Kozma, Zsolt; Poór, Viktor S; Sipos, Katalin

    2016-09-01

    Proper diagnosis in drowning victims is often difficult due to the lack of signs specific to drowning. The diatom test is a widely used procedure for the diagnosis. Some types of water contain only minimal amounts of diatom cells which may provide false-negative results, while a negative diatom test result does not exclude drowning. In proving drowning, we used a polymerase chain reaction (PCR)-based biological method in addition to the conventional methods. DNA was extracted from postmortem spleen tissues and water of the drowning site. Samples were tested with algae (diatoms and small green algae)- and cyanobacteria (blue-green algae)-specific primers. We present here multiple drowning cases in which diatom tests of the postmortem tissue samples and the water were negative. In each case, the presence of phytoplanktonic DNA strengthened the autopsy diagnosis of drowning even in the absence of visible diatoms. In the future, the PCR method may be of consideration as a possible supplement of the diatom test in the examination of presumed drowning cases. PMID:27080711

  13. A Ribeiroia spp. (Class: Trematoda) - Specific PCR-based diagnostic

    USGS Publications Warehouse

    Reinitz, D.M.; Yoshino, T.P.; Cole, R.A.

    2007-01-01

    Increased reporting of amphibian malformations in North America has been noted with concern in light of reports that amphibian numbers and species are declining worldwide. Ribeiroia ondatrae has been shown to cause a variety of types of malformations in amphibians. However, little is known about the prevalence of R. ondatrae in North America. To aid in conducting field studies of Ribeiroia spp., we have developed a polymerase chain reaction (PCR)-based diagnostic. Herein, we describe the development of an accurate, rapid, simple, and cost-effective diagnostic for detection of Ribeiroia spp. infection in snails (Planorbella trivolvis). Candidate oligonucleotide primers for PCR were designed via DNA sequence analyses of multiple ribosomal internal transcribed spacer-2 regions from Ribeiroia spp. and Echinostoma spp. Comparison of consensus sequences determined from both genera identified areas of sequence potentially unique to Ribeiroia spp. The PCR reliably produced a diagnostic 290-base pair (bp) product in the presence of a wide concentration range of snail or frog DNA. Sensitivity was examined with DNA extracted from single R. ondatrae cercaria. The single-tube PCR could routinely detect less than 1 cercariae equivalent, because DNA isolated from a single cercaria could be diluted at least 1:50 and still yield a positive result via gel electrophoresis. An even more sensitive nested PCR also was developed that routinely detected 100 fg of the 290-bp fragment. The assay did not detect furcocercous cercariae of certain Schistosomatidae, Echinostoma sp., or Sphaeridiotrema globulus nor adults of Clinostomum sp. or Cyathocotyle bushiensis. Field testing of 137 P. trivolvis identified 3 positives with no overt environmental cross-reactivity, and results concurred with microscopic examinations in all cases. ?? American Society of Parasitologists 2007.

  14. Quenching of Unincorporated Amplification Signal Reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification Enabling Bright, Single-Step, Closed-Tube, and Multiplexed Detection of RNA Viruses.

    PubMed

    Ball, Cameron S; Light, Yooli K; Koh, Chung-Yan; Wheeler, Sarah S; Coffey, Lark L; Meagher, Robert J

    2016-04-01

    Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read "quasar"), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA. PMID:26980448

  15. Evaluation of Two PCR-Based Swine-Specific Fecal Source Tracking Assays (Poster)

    EPA Science Inventory

    Several PCR-based methods have been proposed to identify swine fecal pollution in environmental waters. However, the specificity and distribution of these targets have not been adequately assessed. Consequently, the utility of these assays in identifying swine fecal contamination...

  16. One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees.

    PubMed

    Malandraki, Ioanna; Varveri, Christina; Olmos, Antonio; Vassilakos, Nikon

    2015-03-01

    A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total nucleic acids (TNAs) extraction was performed according to a modified CTAB protocol. Primers and TaqMan MGB probes for specific detection of the two viroids were designed in this study, whereas for phytoplasma detection published universal primers and probe were used, with the difference that the later was modified to carry a MGB quencher. The pathogens were detected simultaneously in 10-fold serial dilutions of TNAs from infected plant material into TNAs of healthy plant up to dilutions 10(-5) for viroids and 10(-4) for phytoplasmas. The multiplex real-time assay was at least 10 times more sensitive than conventional protocols for viroid and phytoplasma detection. Simultaneous detection of the three targets was achieved in composite samples at least up to a ratio of 1:100 triple-infected to healthy tissue, demonstrating that the developed assay has the potential to be used for rapid and massive screening of viroids and phytoplasmas of pome fruit trees in the frame of certification schemes and surveys. PMID:25479356

  17. CRISPR-STAT: an easy and reliable PCR-based method to evaluate target-specific sgRNA activity.

    PubMed

    Carrington, Blake; Varshney, Gaurav K; Burgess, Shawn M; Sood, Raman

    2015-12-15

    CRISPR/Cas9 has emerged as a versatile genome-engineering tool that relies on a single guide RNA (sgRNA) and the Cas9 enzyme for genome editing. Simple, fast and economical methods to generate sgRNAs have made targeted mutagenesis routine in cultured cells, mice, zebrafish and other model systems. Pre-screening of sgRNAs for target efficacy is desirable both for successful mutagenesis and minimizing wasted animal husbandry on targets with poor activity. Here, we describe an easy, quick and cost-effective fluorescent polymerase chain reaction (PCR)-based method, CRISPR Somatic Tissue Activity Test (CRISPR-STAT), to determine target-specific efficiency of sgRNA. As a proof of principle, we validated our method using 28 sgRNAs with known and varied levels of germline transmission efficiency in zebrafish by analysis of their somatic activity in injected embryos. Our data revealed a strong positive correlation between the fluorescent PCR profiles of the injected embryos and the germline transmission efficiency. Furthermore, the assay was sensitive enough to evaluate multiplex gene targeting. This method is easy to implement by laboratories with access to a capillary sequencer. Although we validated the method using CRISPR/Cas9 and zebrafish, it can be applied to other model systems and other genome targeting nucleases. PMID:26253739

  18. CRISPR-STAT: an easy and reliable PCR-based method to evaluate target-specific sgRNA activity

    PubMed Central

    Carrington, Blake; Varshney, Gaurav K.; Burgess, Shawn M.; Sood, Raman

    2015-01-01

    CRISPR/Cas9 has emerged as a versatile genome-engineering tool that relies on a single guide RNA (sgRNA) and the Cas9 enzyme for genome editing. Simple, fast and economical methods to generate sgRNAs have made targeted mutagenesis routine in cultured cells, mice, zebrafish and other model systems. Pre-screening of sgRNAs for target efficacy is desirable both for successful mutagenesis and minimizing wasted animal husbandry on targets with poor activity. Here, we describe an easy, quick and cost-effective fluorescent polymerase chain reaction (PCR)-based method, CRISPR Somatic Tissue Activity Test (CRISPR-STAT), to determine target-specific efficiency of sgRNA. As a proof of principle, we validated our method using 28 sgRNAs with known and varied levels of germline transmission efficiency in zebrafish by analysis of their somatic activity in injected embryos. Our data revealed a strong positive correlation between the fluorescent PCR profiles of the injected embryos and the germline transmission efficiency. Furthermore, the assay was sensitive enough to evaluate multiplex gene targeting. This method is easy to implement by laboratories with access to a capillary sequencer. Although we validated the method using CRISPR/Cas9 and zebrafish, it can be applied to other model systems and other genome targeting nucleases. PMID:26253739

  19. Detection of African swine fever, classical swine fever, and foot-and-mouth disease viruses in swine oral fluids by multiplex reverse transcription real-time polymerase chain reaction.

    PubMed

    Grau, Frederic R; Schroeder, Megan E; Mulhern, Erin L; McIntosh, Michael T; Bounpheng, Mangkey A

    2015-03-01

    African swine fever (ASF), classical swine fever (CSF), and foot-and-mouth disease (FMD) are highly contagious animal diseases of significant economic importance. Pigs infected with ASF and CSF viruses (ASFV and CSFV) develop clinical signs that may be indistinguishable from other diseases. Likewise, various causes of vesicular disease can mimic clinical signs caused by the FMD virus (FMDV). Early detection is critical to limiting the impact and spread of these disease outbreaks, and the ability to perform herd-level surveillance for all 3 diseases rapidly and cost effectively using a single diagnostic sample and test is highly desirable. This study assessed the feasibility of simultaneous ASFV, CSFV, and FMDV detection by multiplex reverse transcription real-time polymerase chain reaction (mRT-qPCR) in swine oral fluids collected through the use of chewing ropes. Animal groups were experimentally infected independently with each virus, observed for clinical signs, and oral fluids collected and tested throughout the course of infection. All animal groups chewed on the ropes readily before and after onset of clinical signs and before onset of lameness or serious clinical signs. ASFV was detected as early as 3 days postinoculation (dpi), 2-3 days before onset of clinical disease; CSFV was detected at 5 dpi, coincident with onset of clinical disease; and FMDV was detected as early as 1 dpi, 1 day before the onset of clinical disease. Equivalent results were observed in 4 independent studies and demonstrate the feasibility of oral fluids and mRT-qPCR for surveillance of ASF, CSF, and FMD in swine populations. PMID:25776540

  20. A Targeted Q-PCR-Based Method for Point Mutation Testing by Analyzing Circulating DNA for Cancer Management Care.

    PubMed

    Thierry, Alain R

    2016-01-01

    Circulating cell-free DNA (cfDNA) is a valuable source of tumor material available with a simple blood sampling enabling a noninvasive quantitative and qualitative analysis of the tumor genome. cfDNA is released by tumor cells and exhibits the genetic and epigenetic alterations of the tumor of origin. Circulating cell-free DNA (cfDNA) analysis constitutes a hopeful approach to provide a noninvasive tumor molecular test for cancer patients. Based upon basic research on the origin and structure of cfDNA, new information on circulating cell-free DNA (cfDNA) structure, and specific determination of cfDNA fragmentation and size, we revisited Q-PCR-based method and recently developed a the allele-specific-Q-PCR-based method with blocker (termed as Intplex) which is the first multiplexed test for cfDNA. This technique, named Intplex(®) and based on a refined Q-PCR method, derived from critical observations made on the specific structure and size of cfDNA. It enables the simultaneous determination of five parameters: the cfDNA total concentration, the presence of a previously known point mutation, the mutant (tumor) cfDNA concentration (ctDNA), the proportion of mutant cfDNA, and the cfDNA fragmentation index. Intplex(®) has enabled the first clinical validation of ctDNA analysis in oncology by detecting KRAS and BRAF point mutations in mCRC patients and has demonstrated that a blood test could replace tumor section analysis for the detection of KRAS and BRAF mutations. The Intplex(®) test can be adapted to all mutations, genes, or cancers and enables rapid, highly sensitive, cost-effective, and repetitive analysis. As regards to the determination of mutations on cfDNA Intplex(®) is limited to the mutational status of known hotspot mutation; it is a "targeted approach." However, it offers the opportunity in detecting quantitatively and dynamically mutation and could constitute a noninvasive attractive tool potentially allowing diagnosis, prognosis, theranostics

  1. Comparison of Two Widely Used Human Papillomavirus Detection and Genotyping Methods, GP5+/6+-Based PCR Followed by Reverse Line Blot Hybridization and Multiplex Type-Specific E7-Based PCR.

    PubMed

    Clifford, Gary M; Vaccarella, Salvatore; Franceschi, Silvia; Tenet, Vanessa; Umulisa, M Chantal; Tshomo, Ugyen; Dondog, Bolormaa; Vorsters, Alex; Tommasino, Massimo; Heideman, Daniëlle A M; Snijders, Peter J F; Gheit, Tarik

    2016-08-01

    GP5+/6+-based PCR followed by reverse line blot hybridization (GP5+/6+RLB) and multiplex type-specific PCR (E7-MPG) are two human papillomavirus (HPV) genotyping methodologies widely applied in epidemiological research. We investigated their relative analytical performance in 4,662 samples derived from five studies in Bhutan, Rwanda, and Mongolia coordinated by the International Agency for Research on Cancer (IARC). A total of 630 samples were positive by E7-MPG only (13.5%), 24 were positive by GP5+/6+RLB only (0.5%), and 1,014 were positive (21.8%) by both methods. Ratios of HPV type-specific positivity of the two tests (E7-MPG:GP5+/6+RLB ratio) were calculated among 1,668 samples that were HPV positive by one or both tests. E7-MPG:GP5+/6+RLB ratios were >1 for all types and highly reproducible across populations and sample types. E7-MPG:GP5+/6+RLB ratios were highest for HPV53 (7.5) and HPV68 (7.1). HPV16 (1.6) and HPV18 (1.7) had lower than average E7-MPG:GP5+/6+RLB ratios. Among E7-MPG positive infections, median mean fluorescence intensity (MFI; a semiquantitative measure of viral load) tended to be higher among samples positive for the same virus type by GP5+/6+RLB than for those negative for the same type by GP5+/6+RLB. Exceptions, however, included HPV53, -59, and -82, for which the chances of being undetected by GP5+/6+RLB appeared to be MFI independent. Furthermore, the probability of detecting an additional type by E7-MPG was higher when another type was already detected by GP5+/6+RLB, suggesting the existence of masking effects due to competition for GP5+/6+ PCR primers. In conclusion, this analysis is not an evaluation of clinical performance but may inform choices for HPV genotyping methods in epidemiological studies, when the relative merits and dangers of sensitivity versus specificity for individual types should be considered, as well as the potential to unmask nonvaccine types following HPV vaccination. PMID:27225411

  2. MIG-seq: an effective PCR-based method for genome-wide single-nucleotide polymorphism genotyping using the next-generation sequencing platform

    PubMed Central

    Suyama, Yoshihisa; Matsuki, Yu

    2015-01-01

    Restriction-enzyme (RE)-based next-generation sequencing methods have revolutionized marker-assisted genetic studies; however, the use of REs has limited their widespread adoption, especially in field samples with low-quality DNA and/or small quantities of DNA. Here, we developed a PCR-based procedure to construct reduced representation libraries without RE digestion steps, representing de novo single-nucleotide polymorphism discovery, and its genotyping using next-generation sequencing. Using multiplexed inter-simple sequence repeat (ISSR) primers, thousands of genome-wide regions were amplified effectively from a wide variety of genomes, without prior genetic information. We demonstrated: 1) Mendelian gametic segregation of the discovered variants; 2) reproducibility of genotyping by checking its applicability for individual identification; and 3) applicability in a wide variety of species by checking standard population genetic analysis. This approach, called multiplexed ISSR genotyping by sequencing, should be applicable to many marker-assisted genetic studies with a wide range of DNA qualities and quantities. PMID:26593239

  3. Proposal of a quantitative PCR-based protocol for an optimal Pseudomonas aeruginosa detection in patients with cystic fibrosis

    PubMed Central

    2013-01-01

    Background The lung of patients with cystic fibrosis (CF) is particularly sensitive to Pseudomonas aeruginosa. This bacterium plays an important role in the poor outcome of CF patients. During the disease progress, first acquisition of P. aeruginosa is the key-step in the management of CF patients. Quantitative PCR (qPCR) offers an opportunity to detect earlier the first acquisition of P. aeruginosa by CF patients. Given the lack of a validated protocol, our goal was to find an optimal molecular protocol for detection of P. aeruginosa in CF patients. Methods We compared two formerly described qPCR formats in early detection of P. aeruginosa in CF sputum samples: a qPCR targeting oprL gene, and a multiplex PCR targeting gyrB and ecfX genes. Results Tested in vitro on a large panel of P. aeruginosa isolates and others gram-negative bacilli, oprL qPCR exhibited a better sensitivity (threshold of 10 CFU/mL versus 730 CFU/mL), whereas the gyrB/ecfX qPCR exhibited a better specificity (90% versus 73%). These results were validated ex vivo on 46 CF sputum samples positive for P. aeruginosa in culture. Ex vivo assays revealed that qPCR detected 100 times more bacterial cells than culture-based method did. Conclusion Based on these results, we proposed a reference molecular protocol combining the two qPCRs, which offers a sensitivity of 100% with a threshold of 10 CFU/mL and a specificity of 100%. This combined qPCR-based protocol can be adapted and used for other future prospective studies. PMID:24088260

  4. USE OF BACTEROIDES PCR-BASED METHODS TO EXAMINE FECAL CONTAMINATION SOURCES IN TROPICAL COASTAL WATERS

    EPA Science Inventory

    Several library independent Microbial Source Tracking methods have been developed to rapidly determine the source of fecal contamination. Thus far, none of these methods have been tested in tropical marine waters. In this study, we used a Bacteroides 16S rDNA PCR-based...

  5. Evaluation of Two PCR-based Swine-specific Fecal Source Tracking Assays (Abstract)

    EPA Science Inventory

    Several PCR-based methods have been proposed to identify swine fecal pollution in environmental waters. However, the utility of these assays in identifying swine fecal contamination on a broad geographic scale is largely unknown. In this study, we evaluated the specificity, distr...

  6. DEVELOPMENT OF AN IMPROVED PCR-BASED TECHNIQUE FOR DETECTION OF PHYTOPHTHORA CACTORUM IN STRAWBERRY PLANTS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Specific and rapid plant pathogen detection methods can aid in strawberry disease management decisions. PCR-based diagnostics for Phytophthora cactorum and other strawberry pathogens are hindered by PCR inhibitors and lack of species-specific PCR primers. We developed a DNA extraction and purificati...

  7. Extraction of genomic DNA from yeasts for PCR-based applications.

    PubMed

    Lõoke, Marko; Kristjuhan, Kersti; Kristjuhan, Arnold

    2011-05-01

    We have developed a quick and low-cost genomic DNA extraction protocol from yeast cells for PCR-based applications. This method does not require any enzymes, hazardous chemicals, or extreme temperatures, and is especially powerful for simultaneous analysis of a large number of samples. DNA can be efficiently extracted from different yeast species (Kluyveromyces lactis, Hansenula polymorpha, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris, and Saccharomyces cerevisiae). The protocol involves lysis of yeast colonies or cells from liquid culture in a lithium acetate (LiOAc)-SDS solution and subsequent precipitation of DNA with ethanol. Approximately 100 nanograms of total genomic DNA can be extracted from 1 × 10(7) cells. DNA extracted by this method is suitable for a variety of PCR-based applications (including colony PCR, real-time qPCR, and DNA sequencing) for amplification of DNA fragments of ≤ 3500 bp. PMID:21548894

  8. A PCR-based method to quantify fungal growth during pretreatment of lignocellulosic biomass.

    PubMed

    Simeng, Zhou; Sacha, Grisel; Isabelle, Herpoël-Gimbert; Marie-Noëlle, Rosso

    2015-08-01

    Filamentous fungi have shown great potential in the pretreatment of lignocellulosic biomass and their use in bio-processes based on Solid State Fermentation (SSF) opens promising perspectives for plant biomass valorization. Obviously, quantification of the fungal biomass throughout the fermentation is a crucial parameter for SSF evaluation and control, both at the laboratory and industrial scale. Here we provide a qPCR-based method as a reliable alternative to conventional methods to estimate mycelial growth during plant biomass treatment. For the three strains analyzed, the lowest detection limit ranged from 58 to 272 μg mycelium dry weight per gram biomass (dry weight). We show that the qPCR-based method allows fungal growth monitoring during fermentation and provides relevant information for selection of the most appropriate fungal strains in specific SSF/reactor conditions. PMID:26031470

  9. Performance of PCR-based and Bioluminescent assays for mycoplasma detection.

    PubMed

    Falagan-Lotsch, Priscila; Lopes, Talíria Silva; Ferreira, Nívea; Balthazar, Nathália; Monteiro, Antônio M; Borojevic, Radovan; Granjeiro, José Mauro

    2015-11-01

    Contaminated eukaryotic cell cultures are frequently responsible for unreliable results. Regulatory entities request that cell cultures must be mycoplasma-free. Mycoplasma contamination remains a significant problem for cell cultures and may have an impact on biological analysis since they affect many cell parameters. The gold standard microbiological assay for mycoplasma detection involves laborious and time-consuming protocols. PCR-based and Bioluminescent assays have been considered for routine cell culture screening in research laboratories since they are fast, easy and sensitive. Thus, the aim of this work is to compare the performance of two popular commercial assays, PCR-based and Bioluminescent assays, by assessing the level of mycoplasma contamination in cell cultures from Rio de Janeiro Cell Bank (RJCB) and also from customers' laboratories. The results obtained by both performed assays were confirmed by scanning electron microscopy. In addition, we evaluated the limit of detection of the PCR kit under our laboratory conditions and the storage effects on mycoplasma detection in frozen cell culture supernatants. The performance of both assays for mycoplasma detection was not significantly different and they showed very good agreement. The Bioluminescent assay for mycoplasma detection was slightly more dependable than PCR-based due to the lack of inconclusive results produced by the first technique, especially considering the ability to detect mycoplasma contamination in frozen cell culture supernatants. However, cell lines should be precultured for four days or more without antibiotics to obtain safe results. On the other hand, a false negative result was obtained by using this biochemical approach. The implementation of fast and reliable mycoplasma testing methods is an important technical and regulatory issue and PCR-based and Bioluminescent assays may be good candidates. However, validation studies are needed. PMID:26296900

  10. Development and evaluation of new primers for PCR-based identification of Prevotella intermedia.

    PubMed

    Zhou, Yanbin; Liu, Dali; Wang, Yiwei; Zhu, Cailian; Liang, Jingping; Shu, Rong

    2014-08-01

    The aim of this study was to develop new Prevotella intermedia-specific PCR primers based on the 16S rRNA. The new primer set, Pi-192 and Pi-468, increased the accuracy of PCR-based P. intermedia identification and could be useful in the detection of P. intermedia as well as epidemiological studies on periodontal disease. PMID:24875331

  11. Multiplex PageRank.

    PubMed

    Halu, Arda; Mondragón, Raúl J; Panzarasa, Pietro; Bianconi, Ginestra

    2013-01-01

    Many complex systems can be described as multiplex networks in which the same nodes can interact with one another in different layers, thus forming a set of interacting and co-evolving networks. Examples of such multiplex systems are social networks where people are involved in different types of relationships and interact through various forms of communication media. The ranking of nodes in multiplex networks is one of the most pressing and challenging tasks that research on complex networks is currently facing. When pairs of nodes can be connected through multiple links and in multiple layers, the ranking of nodes should necessarily reflect the importance of nodes in one layer as well as their importance in other interdependent layers. In this paper, we draw on the idea of biased random walks to define the Multiplex PageRank centrality measure in which the effects of the interplay between networks on the centrality of nodes are directly taken into account. In particular, depending on the intensity of the interaction between layers, we define the Additive, Multiplicative, Combined, and Neutral versions of Multiplex PageRank, and show how each version reflects the extent to which the importance of a node in one layer affects the importance the node can gain in another layer. We discuss these measures and apply them to an online multiplex social network. Findings indicate that taking the multiplex nature of the network into account helps uncover the emergence of rankings of nodes that differ from the rankings obtained from one single layer. Results provide support in favor of the salience of multiplex centrality measures, like Multiplex PageRank, for assessing the prominence of nodes embedded in multiple interacting networks, and for shedding a new light on structural properties that would otherwise remain undetected if each of the interacting networks were analyzed in isolation. PMID:24205186

  12. High-throughput real-time PCR-based genotyping without DNA purification

    PubMed Central

    2012-01-01

    Background While improvements in genotyping technology have allowed for increased throughput and reduced time and expense, protocols remain hindered by the slow upstream steps of isolating, purifying, and normalizing DNA. Various methods exist for genotyping samples directly through blood, without having to purify the DNA first. These procedures were designed to be used on smaller throughput systems, however, and have not yet been tested for use on current high-throughput real-time (q)PCR based genotyping platforms. In this paper, a method of quantitative qPCR-based genotyping on blood without DNA purification was developed using a high-throughput qPCR platform. Findings The performances of either DNA purified from blood or the same blood samples without DNA purification were evaluated through qPCR-based genotyping. First, 60 different mutations prevalent in the Ashkenazi Jewish population were genotyped in 12 Ashkenazi Jewish individuals using the QuantStudio™12K Flex Real-Time PCR System. Genotyping directly from blood gave a call rate of 99.21%, and an accuracy of 100%, while the purified DNA gave a call rate of 92.49%, and an accuracy of 99.74%. Although no statistical difference was found for these parameters, an F test comparing the standard deviations of the wild type clusters for the two different methods indicated significantly less variation when genotyping directly from blood instead of after DNA purification. To further establish the ability to perform high-throughput qPCR based genotyping directly from blood, 96 individuals of Ashkenazi Jewish decent were genotyped for the same 60 mutations (5,760 genotypes in 5 hours) and resulted in a call rate of 98.38% and a diagnostic accuracy of 99.77%. Conclusion This study shows that accurate qPCR-based high-throughput genotyping can be performed without DNA purification. The direct use of blood may further expedite the entire genotyping process, reduce costs, and avoid tracking errors which can occur during

  13. Multiplex gas chromatography

    NASA Technical Reports Server (NTRS)

    Valentin, Jose R.

    1990-01-01

    The principles of the multiplex gas chromatography (GC) technique, which is a possible candidate for chemical analysis of planetary atmospheres, are discussed. Particular attention is given to the chemical modulators developed by present investigators for multiplex GC, namely, the thermal-desorption, thermal-decomposition, and catalytic modulators, as well as to mechanical modulators. The basic technique of multiplex GC using chemical modulators and a mechanical modulator is demonstrated. It is shown that, with the chemical modulators, only one gas stream consisting of the carrier in combination with the components is being analyzed, resulting in a simplified instrument that requires relatively few consumables. The mechanical modulator demonstrated a direct application of multiplex GC for the analysis of gases in atmosphere of Titan at very low pressures.

  14. Apollo Multiplexer operations manual

    SciTech Connect

    Miller, M.M.

    1985-04-01

    This report describes the operation of the the Apollo Multiplexer, a microprocessor based communications device designed to process data between an Apollo computer and up to four Gandalf PACXIV data switches. Details are given on overall operation, hardware, and troubleshooting. The reader should gain sufficient knowledge from this report to understand the operation of the multiplexer and effectively analyze and correct any problems that might occur.

  15. Downlink data multiplexer

    NASA Technical Reports Server (NTRS)

    Holland, S. Douglas (Inventor); Steele, Glen F. (Inventor); Romero, Denise M. (Inventor); Koudelka, Robert David (Inventor)

    2008-01-01

    A data multiplexer that accommodates both industry standard CCSDS data packets and bits streams and standard IEEE 1394 data is described. The multiplexer provides a statistical allotment of bandwidth to the channels in turn, preferably four, but expandable in increments of four up to sixteen. A microcontroller determines bandwidth requested by the plurality of channels, as well as the bandwidth available, and meters out the available bandwidth on a statistical basis employing flow control to the input channels.

  16. Multiplexed chirp waveform synthesizer

    DOEpatents

    Dudley, Peter A.; Tise, Bert L.

    2003-09-02

    A synthesizer for generating a desired chirp signal has M parallel channels, where M is an integer greater than 1, each channel including a chirp waveform synthesizer generating at an output a portion of a digital representation of the desired chirp signal; and a multiplexer for multiplexing the M outputs to create a digital representation of the desired chirp signal. Preferably, each channel receives input information that is a function of information representing the desired chirp signal.

  17. Coevolution and Correlated Multiplexity in Multiplex Networks

    NASA Astrophysics Data System (ADS)

    Kim, Jung Yeol; Goh, K.-I.

    2013-08-01

    Distinct channels of interaction in a complex networked system define network layers, which coexist and cooperate for the system’s function. Towards understanding such multiplex systems, we propose a modeling framework based on coevolution of network layers, with a class of minimalistic growing network models as working examples. We examine how the entangled growth of coevolving layers can shape the network structure and show analytically and numerically that the coevolution can induce strong degree correlations across layers, as well as modulate degree distributions. We further show that such a coevolution-induced correlated multiplexity can alter the system’s response to the dynamical process, exemplified by the suppressed susceptibility to a social cascade process.

  18. Compact spatial multiplexers for mode division multiplexing.

    PubMed

    Chen, Haoshuo; van Uden, Roy; Okonkwo, Chigo; Koonen, Ton

    2014-12-29

    Spatial multiplexer (SMUX) for mode division multiplexing (MDM) has evolved from mode-selective excitation, multiple-spot and photonic-lantern based solutions in order to minimize both mode-dependent loss (MDL) and coupler insertion loss (CIL). This paper discusses the implementation of all the three solutions by compact components in a small footprint. Moreover, the compact SMUX can be manufactured in mass production and packaged to assure high reliability. First, push-pull scheme and center launch based SMUXes are demonstrated on two mostly-popular photonic integration platforms: Silicon-on-insulator (SOI) and Indium Phosphide (InP) for selectively exciting LP01 and LP11 modes. 2-dimensional (2D) top-coupling by using vertical emitters is explored to provide a coupling interface between a few-mode fiber (FMF) and the photonic integrated SMUX. SOI-based grating couplers and InP-based 45° vertical mirrors are proposed and researched as vertical emitters in each platform. Second, a 3-spot SMUX is realized on an InP-based circuit through employing 45° vertical mirrors. Third, as a newly-emerging photonic integration platform, laser-inscribed 3D waveguide (3DW) technology is applied for a fully-packaged dual-channel 6-mode SMUX including two 6-core photonic lantern structures as mode multiplexer and demultiplexer, respectively. PMID:25607130

  19. An improved, PCR-based strategy for the detection of Trypanosoma cruzi in human blood samples.

    PubMed

    Ribeiro-dos-Santos, G; Nishiya, A S; Sabino, E C; Chamone, D F; Saez-Alquézar, A

    1999-10-01

    Attempts were made to improve the PCR-based detection of Trypanosoma cruzi in blood samples, primarily for screening blood donors. Samples were obtained from candidate donors who were reactive in one or two of three serological tests for Chagas disease (and therefore considered 'indeterminate') or in all three tests (3+). Each sample was then examined using three different, PCR-based techniques: 'PCR-I' (in which the target DNA is a nuclear repetitive sequence); 'PCR-II' [amplifying a conserved region of the T. cruzi kinetoplast DNA (kDNA)]; and 'PCR-III' (a new strategy in which the target kDNA is amplified by 'nested' PCR). Among the samples from 3+ individuals, PCR-I, PCR-II and PCR-III amplified two (3.8%) out of 52, four (4.5%) out of 88, and 27 (25.7%) out of 105 samples tested, respectively. Seven, 69 and 70 samples from 'indeterminate' subjects were tested by PCR-I, PCR-II and PCR-III, respectively; there was not a single positive result by PCR-I or PCR-II, but three (4.3%) of the samples tested by PCR-III were positive. In a reconstruction experiment, in conditions in which PCR-I and PCR-II could not detect 10,000 parasites/ml, PCR-III was able to detect one parasite/ml. Although all three PCR-based strategies examined had rather poor sensitivities, PCR-III was far more sensitive than PCR-I or PCR-II. PMID:10715696

  20. A PCR-based method to identify Entomophaga spp. infections in North American grasshoppers.

    PubMed

    Casique-Valdes, Rebeca; Sanchez-Peña, Sergio; Ivonne Torres-Acosta, R; Bidochka, Michael J

    2012-01-01

    A PCR-based method was developed for the detection and identification of two species of grasshopper-specific pathogens belonging to the genus Entomophaga in North America, Entomophaga calopteni and Entomophaga macleodii. Two separate sets of primers specific for amplification of a DNA product from each species of Entomophaga as well as a positive control were utilized. Grasshoppers were collected from two sites in Mexico during an epizootic with grasshoppers found in "summit disease", typical of Entomophaga infections. There was a preponderance of Melanopline grasshoppers infected by E. calopteni. The described method is an accurate tool for identification of North American grasshopper infections by Entomophaga species. PMID:22146240

  1. Application of a PCR-based approach to identify sex in Hawaiian honeycreepers (Drepanidinae)

    USGS Publications Warehouse

    Jarvi, S.I.; Banko, P.C.

    2000-01-01

    The application of molecular techniques to conservation genetics issues can provide important guidance criteria for management of endangered species. The results from this study establish that PCR-based approaches for sex determination developed in other bird species (Griffiths and Tiwari 1995; Griffiths et al. 1996, 1998; Ellegren 1996) can be applied with a high degree of confidence to at least four species of Hawaiian honeycreepers. This provides a rapid, reliable method with which population managers can optimize sex ratios within populations of endangered species that are subject to artificial manipulation through captive breeding programmes or geographic translocation.

  2. PCR-based immortalization and screening of hierarchical pools of cDNAs.

    PubMed Central

    D'Esposito, M; Mazzarella, R; Pengue, G; Jones, C; D'Urso, M; Schlessinger, D

    1994-01-01

    Starting from sequences of at least 60 bp, PCR-based screening has been developed to recover cDNAs from libraries without the necessity for hybridization or extensive DNA extraction steps. The method maintains the indefinite availability of even scarce cDNA libraries and provides an estimate of the relative abundance of the mRNA species. Isolation of a cDNA clone can be done in less than a week. cDNAs were isolated that were cognate for fragments of expressed sequences and for an exon predicted from genomic sequence. Images PMID:7984433

  3. Rational design and PCR-based synthesis of an artificial Schizophyllum commune xylanase gene.

    PubMed Central

    Graham, R W; Atkinson, T; Kilburn, D G; Miller, R C; Warren, R A

    1993-01-01

    A synthetic gene encoding the Schizophyllum commune xylanase XynA was constructed by a novel PCR-based procedure. Three long oligonucleotides were synthesized and used in combination with flanking PCR primers to generate a 607 base pair gene which contained 31 unique locations for restriction enzyme cleavage. The amino acid sequence was tailored for expression in Escherichia coli by using only those codons found in highly expressed E. coli genes. The availability of the gene will facilitate analysis of the structure and function of this and other beta-(1,4) xylanases. Images PMID:8177740

  4. New PCR-Based Open Reading Frame Typing Method for Easy, Rapid, and Reliable Identification of Acinetobacter baumannii International Epidemic Clones without Performing Multilocus Sequence Typing

    PubMed Central

    Suzuki, Masahiro; Hosoba, Eriko; Matsui, Mari

    2014-01-01

    Antimicrobial resistance issues have become a global health concern. The rapid identification of multidrug-resistant microbes, which depends on microbial genomic information, is essential for overcoming growing antimicrobial resistance challenges. However, genotyping methods, such as multilocus sequence typing (MLST), for identifying international epidemic clones of Acinetobacter baumannii are not easily performed as routine tests in ordinary clinical laboratories. In this study, we aimed to develop a novel genotyping method that can be performed in ordinary microbiology laboratories. Several open reading frames (ORFs) specific to certain bacterial genetic lineages or species, together with their unique distribution patterns on the chromosomes showing a good correlation with the results of MLST, were selected in A. baumannii and other Acinetobacter spp. by comparing their genomic data. The distribution patterns of the ORFs were visualized by agarose gel electrophoresis after multiplex PCR amplification and digitized. A. baumannii sequence types (STs) corresponding to international clones I and II were successfully discriminated from other STs and Acinetobacter species by detecting the distribution patterns of their ORFs using the multiplex PCR developed here. Since bacterial STs can be easily expressed as digitized numeric data with plus (+) expressed as 1 and minus (−) expressed as 0, the results of the method can be easily compared with those obtained by different tests or laboratories. This PCR-based ORF typing (POT) method can easily and rapidly identify international epidemic clones of A. baumannii and differentiate this microbe from other Acinetobacter spp. Since this POT method is easy enough to be performed even in ordinary clinical laboratories, it would also contribute to daily infection control measures and surveillance. PMID:24899031

  5. Performance of PCR-based assays targeting Bacteroidales genetic markers of human fecal pollution in sewage and fecal samples

    EPA Science Inventory

    There are numerous PCR-based methods available to characterize human fecal pollution in ambient waters. Each assay employs distinct oligonucleotides and many target different genes and microorganisms leading to potential variations in method performance. Laboratory comparisons ...

  6. Multiplexed Biosensors for Mycotoxins.

    PubMed

    Maragos, Chris M

    2016-07-01

    Significant progress has been made in the development of biosensors that can be used to detect low-MW toxins produced by fungi (mycotoxins). The number of formats that have been investigated is impressive and is an indication of the importance attached to finding easy-to-use, accurate, and rapid methods for detecting these toxins in commodities and foods. This review explores the details of multiplexed biosensors based on many formats, including multiplexed immunoassays, suspension arrays, membrane-based devices (flow-through and immunochromatographic), and planar microarrays. Each assay format has its own strengths and areas that need improvement. Certain formats, such as multiplexed immunochromatographic devices, are well developed and relatively easy to use, and in some cases, commercial products are being sold. Others, such as the suspension arrays and microarrays, are laboratory-based assays that, although more complicated, are also more amenable to a larger scale of multiplexing. The diversity of such efforts and the multitude of formats under investigation suggest that multiple solutions will be found to satisfy the need for multiplexed toxin detection. PMID:27455928

  7. A PCR-based genetic linkage map of human chromosome 16

    SciTech Connect

    Shen, Y.; Kozman, H.M.; Thompson, A.

    1994-07-01

    A high-resolution cytogenetic-based physical map and a genetic linkage map of human chromosome 16 have been developed based on 79 PCR-typable genetic markers and 2 Southern-based RFLP markers. The PCR-based markers were previously-characterized polymorphic (AC){sub n} repeats. Two approaches have led to the characterization of 47 highly informative genetic markers spread along chromosome 16, some of which are closely linked to disease loci. In addition, 22 markers (D16S401-423) previously genetically mapped were also physically mapped. Ten markers characterized by other laboratories were physically mapped and genotyped on the CEPH families. These 32 markers were incorporated into the PCR-based map. Seventy-two markers have heterozygosities >0.50 and 51 of these markers >0.70. By multipoint linkage analysis a framework genetic map and a comprehensive genetic map were constructed. The length of the sex-averaged framework genetic map if 152.1 cM. The average distance and the median distance between markers on this map are 3.2 and 2.7 cM, respectively, and the largest gap is 15.9 cM. These maps were anchored to the high-resolution cytogenetic map (on average 1.5 Mb per interval). Together these integrated genetic and physical maps of human chromosome 16 provide the basis for the localization and ultimately the isolation of disease genes that map to this chromosome. 1 fig., 3 tabs.

  8. A Novel PCR-Based Approach for Accurate Identification of Vibrio parahaemolyticus.

    PubMed

    Li, Ruichao; Chiou, Jiachi; Chan, Edward Wai-Chi; Chen, Sheng

    2016-01-01

    A PCR-based assay was developed for more accurate identification of Vibrio parahaemolyticus through targeting the bla CARB-17 like element, an intrinsic β-lactamase gene that may also be regarded as a novel species-specific genetic marker of this organism. Homologous analysis showed that bla CARB-17 like genes were more conservative than the tlh, toxR and atpA genes, the genetic markers commonly used as detection targets in identification of V. parahaemolyticus. Our data showed that this bla CARB-17-specific PCR-based detection approach consistently achieved 100% specificity, whereas PCR targeting the tlh and atpA genes occasionally produced false positive results. Furthermore, a positive result of this test is consistently associated with an intrinsic ampicillin resistance phenotype of the test organism, presumably conferred by the products of bla CARB-17 like genes. We envision that combined analysis of the unique genetic and phenotypic characteristics conferred by bla CARB-17 shall further enhance the detection specificity of this novel yet easy-to-use detection approach to a level superior to the conventional methods used in V. parahaemolyticus detection and identification. PMID:26858713

  9. A Novel PCR-Based Approach for Accurate Identification of Vibrio parahaemolyticus

    PubMed Central

    Li, Ruichao; Chiou, Jiachi; Chan, Edward Wai-Chi; Chen, Sheng

    2016-01-01

    A PCR-based assay was developed for more accurate identification of Vibrio parahaemolyticus through targeting the blaCARB-17 like element, an intrinsic β-lactamase gene that may also be regarded as a novel species-specific genetic marker of this organism. Homologous analysis showed that blaCARB-17 like genes were more conservative than the tlh, toxR and atpA genes, the genetic markers commonly used as detection targets in identification of V. parahaemolyticus. Our data showed that this blaCARB-17-specific PCR-based detection approach consistently achieved 100% specificity, whereas PCR targeting the tlh and atpA genes occasionally produced false positive results. Furthermore, a positive result of this test is consistently associated with an intrinsic ampicillin resistance phenotype of the test organism, presumably conferred by the products of blaCARB-17 like genes. We envision that combined analysis of the unique genetic and phenotypic characteristics conferred by blaCARB-17 shall further enhance the detection specificity of this novel yet easy-to-use detection approach to a level superior to the conventional methods used in V. parahaemolyticus detection and identification. PMID:26858713

  10. A multiplexed quantum memory.

    PubMed

    Lan, S-Y; Radnaev, A G; Collins, O A; Matsukevich, D N; Kennedy, T A; Kuzmich, A

    2009-08-01

    A quantum repeater is a system for long-distance quantum communication that employs quantum memory elements to mitigate optical fiber transmission losses. The multiplexed quantum memory (O. A. Collins, S. D. Jenkins, A. Kuzmich, and T. A. B. Kennedy, Phys. Rev. Lett. 98, 060502 (2007)) has been shown theoretically to reduce quantum memory time requirements. We present an initial implementation of a multiplexed quantum memory element in a cold rubidium gas. We show that it is possible to create atomic excitations in arbitrary memory element pairs and demonstrate the violation of Bell's inequality for light fields generated during the write and read processes. PMID:19654771

  11. Dual phase multiplex polymerase chain reaction

    DOEpatents

    Pemov, Alexander; Bavykin, Sergei

    2008-10-07

    Highly specific and sensitive methods were developed for multiplex amplification of nucleic acids on supports such as microarrays. Based on a specific primer design, methods include five types of amplification that proceed in a reaction chamber simultaneously. These relate to four types of multiplex amplification of a target DNA on a solid support, directed by forward and reverse complex primers immobilized to the support and a fifth type--pseudo-monoplex polymerase chain reaction (PCR) of multiple targets in solution, directed by a single pair of unbound universal primers. The addition of the universal primers in the reaction mixture increases the yield over the traditional "bridge" amplification on a solid support by approximately ten times. Methods that provide multitarget amplification and detection of as little as 0.45-4.5.times.10.sup.-12 g (equivalent to 10.sup.2-10.sup.3 genomes) of a bacterial genomic DNA are disclosed.

  12. Validation of a Short Tandem Repeat Multiplex Typing System for Genetic Individualization of Domestic Cat Samples

    PubMed Central

    Coomber, Nikia; David, Victor A.; O’Brien, Stephen J.; Menotti-Raymond, Marilyn

    2007-01-01

    Aim To conduct developmental validation studies on a polymerase chain reaction (PCR) based short tandem repeat (STR) multiplex typing system, developed for the purpose of genetic individualization and parentage testing in domestic cat samples. Methods To evaluate reproducibility of the typing system, the multiplex was amplified using DNA extracted from hair, blood, and buccal samples obtained from the same individual (n = 13). Additional studies were performed to evaluate the system’s species’ specificity, using 26 North American mammalian species and two prokaryotes Sacchromyces and Escherichia coli, sensitivity, and ability to identify DNA mixtures. Patterns of Mendelian inheritance and mutation rates for the 11 loci were directly examined in a large multi-generation domestic cat pedigree (n = 263). Results Our studies confirm that the multiplex system was species-specific for feline DNA and amplified robustly with as little as 125 picograms of genomic template DNA, demonstrating good product balance. The multiplex generated all components of a two DNA mixture when the minor component was one tenth of the major component at a threshold of 50 relative fluorescence units. The multiplex was reproducible in multiple tissue types of the same individual. Mutation rates for the 11 STR were within the range of sex averaged rates observed for Combined DNA Index System (CODIS) loci. Conclusion The cat STR multiplex typing system is a robust and reliable tool for the use of forensic DNA analysis of domestic cat samples. PMID:17696310

  13. Multiplex detection of food allergens and gluten.

    PubMed

    Cho, Chung Y; Nowatzke, William; Oliver, Kerry; Garber, Eric A E

    2015-05-01

    To help safeguard the food supply and detect the presence of undeclared food allergens and gluten, most producers and regulatory agencies rely on commercial test kits. Most of these are ELISAs with a few being PCR-based. These methods are very sensitive and analyte specific, requiring different assays to detect each of the different food allergens. Mass spectrometry offers an alternative approach whereby multiple allergens may be detected simultaneously. However, mass spectrometry requires expensive equipment, highly trained analysts, and several years before a quantitative approach can be achieved. Using multianalyte profiling (xMAP®) technology, a commercial multiplex test kit based on the use of established antibodies was developed for the simultaneous detection of up to 14 different food allergens plus gluten. The assay simultaneously detects crustacean seafood, egg, gluten, milk, peanut, soy, and nine tree nuts (almond, Brazil nut, cashew, coconut, hazelnut, macadamia, pine nut, pistachio, and walnut). By simultaneously performing multiple tests (typically two) for each analyte, this magnetic bead-based assay offers built-in confirmatory analyses without the need for additional resources. Twenty-five of the assays were performed on buffer extracted samples, while five were conducted on samples extracted using reduced-denatured conditions. Thus, complete analysis for all 14 allergens and gluten requires only two wells of a 96-well microtiter plate. This makes it possible to include in a single analytical run up to 48 samples. All 30 bead sets in this multiplex assay detected 5 ng/mL of food allergen and gluten with responses greater than background. In addition, 26 of the bead sets displayed signal/noise ratios of five or greater. The bead-based design makes this 30-plex assay expandable to incorporate new antibodies and capture/detector methodologies by ascribing these new detectors to any of the unassigned bead sets that are commercially available. PMID

  14. Rapid detection and identification of Clostridium chauvoei by PCR based on flagellin gene sequence.

    PubMed

    Kojima, A; Uchida, I; Sekizaki, T; Sasaki, Y; Ogikubo, Y; Tamura, Y

    2001-02-26

    We developed a one-step polymerase chain reaction (PCR) system that specifically detects Clostridium chauvoei. Oligonucleotide primers were designed to amplify a 516-bp fragment of the structural flagellin gene. The specificity of the PCR was investigated by analyzing 59 strains of clostridia, and seven strain of other genera. A 516-bp fragment could be amplified from all the C. chauvoei strains tested, and no amplification was observed by using DNAs from the other strains tested, including Clostridium septicum. Similarly, this PCR-based method specifically detected C. chauvoei DNA sequences in samples of muscle and exudate of obtained from mice within 12h of inoculation. In tests using samples of muscle or liver, the limit of detection was about 200 organisms per reaction. These results suggest that the one-step PCR system may be useful for direct detection and identification of C. chauvoei in clinical specimens. PMID:11182502

  15. Northern and southern Croatian population data on seven PCR-based loci.

    PubMed

    Keys, K M; Budowle, B; Andelinovic, S; Definis-Gojanovic, M; Drmic, I; Mladen, M; Primorac, D

    1996-08-15

    Northern and southern Croatian sample populations were typed at seven PCR-based loci -LDLR, GYPA, HBGG, D7S8, Gc, HLA-DQA1 and D1S80. The results show that all loci meet Hardy-Weinberg expectations and that there is little evidence for association of alleles between loci. Allelic frequency distributions at all loci, except HLA-DQA1, show no differences between the northern and southern Croatian sample populations. Moreover, the population data for Croatians are similar to U.S. Caucasians; only HLA-DQA1 for southern Croatians was statistically different compared with U.S. Caucasians. A Croatian population database(s) has been created and can be used for forensic analyses to estimate the frequency of a multiple locus DNA profile. PMID:8837495

  16. PCR-Based Analysis of Mitochondrial DNA Copy Number, Mitochondrial DNA Damage, and Nuclear DNA Damage.

    PubMed

    Gonzalez-Hunt, Claudia P; Rooney, John P; Ryde, Ian T; Anbalagan, Charumathi; Joglekar, Rashmi; Meyer, Joel N

    2016-01-01

    Because of the role that DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR-based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA-QPCR) is used to detect DNA damage by measuring the number of polymerase-inhibiting lesions present based on the amount of PCR amplification; real-time PCR (RT-PCR) is used to calculate genome content. In this unit, we provide step-by-step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays. PMID:26828332

  17. A PCR-BASED DETECTION OF BURKHOLDERIA PSEUDOMALLEI DIVERSITY USING MYOVIRIDAE PROPHAGE TYPING.

    PubMed

    Nakornpakdee, Yaowarin; Sermswan, Rasana W; Tattawasart, Unchalee; Yordpratum, Umaporn; Wongratanacheewin, Surasakdi

    2015-01-01

    PCR-based detection of Myoviridae lysogenic phages in Burkholderia pseudomallei was developed using primers targeting K96243 prophage GI2, phiE12-2 and phi52237/phiX216. Investigation of 50 clinical and 50 environmental (soil) isolates revealed that K96243 prophage GI2 was the most common (48%) among the isolates, followed by phiE12-2 (38%) and phi52237/phiX216 (35%), with K96243 prophage GI2 being significantly more frequent in soil (64%) than clinical (32%) samples. Twenty-four percent of soil isolates contained all three prophage types, while clinical isolates harbored no more than two types. Although B. pseudomallei isolates from soil were found to be more diverse based on prophage typing, all isolates were equally susceptible to a battery of lytic phages (although to different extents), suggesting the possibility of using lytic phages to control environmental B. pseudomallei. PMID:26513903

  18. Rapid PCR-Based Method Which Can Determine Both Phenotype and Genotype of Lactococcus lactis Subspecies

    PubMed Central

    Nomura, Masaru; Kobayashi, Miho; Okamoto, Takashi

    2002-01-01

    A highly efficient, rapid, and reliable PCR-based method for distinguishing Lactococcus lactis subspecies (L. lactis subsp. lactis and L. lactis subsp. cremoris) is described. Primers complementary to positions in the glutamate decarboxylase gene have been constructed. PCR analysis with extracted DNA or with cells of different L. lactis strains resulted in specific fragments. The length polymorphism of the PCR fragments allowed a clear distinction of the L. lactis subspecies. The amplified fragment length polymorphism with the primers and the restriction fragment length polymorphism of the amplified products agreed perfectly with the identification based on genotypic and phenotypic analyses, respectively. Isolates from cheese starters were investigated by this method, and amplified fragments of genetic variants were found to be approximately 40 bp shorter than the typical L. lactis subsp. cremoris fragments. PMID:11976090

  19. PCR-based assessment of shellfish traceability and sustainability in international Mediterranean seafood markets.

    PubMed

    Galal-Khallaf, Asmaa; Ardura, Alba; Borrell, Yaisel J; Garcia-Vazquez, Eva

    2016-07-01

    Two mitochondrial markers (cytochrome oxidase COI and 16S rDNA) were employed for species identification of commercial shellfish from two Mediterranean countries. New COI Barcodes were generated for six species: Pleoticus robustus, Metapenaeopsis barbata, Parapenaeus fissuroides, Hymenopenaeus debilis, Metapenaeus affinis and Sepia aculeata. Biodiversity of the seafood species analyzed was greater in Egypt, with nine crustacean and two cephalopod species found compared with only three crustaceans and three cephalopods in Spain. In total, 17.2% and 15.2% products were mislabeled in Egypt and Spain, respectively. Population decline is a problem for some of the substitute species. Others were exotic and/or invasive in exporters' regions. This study offers the first comparable study of shellfish traceability in these Mediterranean markets. The PCR-based method used in this study proved to be reliable, effective and, therefore, could be employed for routine seafood analysis. PMID:26920298

  20. PCR-based analysis of mitochondrial DNA copy number, mitochondrial DNA damage, and nuclear DNA damage

    PubMed Central

    Gonzalez-Hunt, Claudia P.; Rooney, John P.; Ryde, Ian T.; Anbalagan, Charumathi; Joglekar, Rashmi

    2016-01-01

    Because of the role DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR-based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA-QPCR) is used to detect DNA damage by measuring the number of polymerase-inhibiting lesions present based on the amount of PCR amplification; real-time PCR (RT-PCR) is used to calculate genome content. In this unit we provide step-by-step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays. PMID:26828332

  1. Time-division SQUID multiplexers

    NASA Astrophysics Data System (ADS)

    Irwin, K. D.; Vale, L. R.; Bergren, N. E.; Deiker, S.; Grossman, E. N.; Hilton, G. C.; Nam, S. W.; Reintsema, C. D.; Rudman, D. A.; Huber, M. E.

    2002-02-01

    SQUID multiplexers make it possible to build arrays of thousands of low-temperature bolometers and microcalorimeters based on superconducting transition-edge sensors with a manageable number of readout channels. We discuss the technical tradeoffs between proposed time-division multiplexer and frequency-division multiplexer schemes and motivate our choice of time division. Our first-generation SQUID multiplexer is now in use in an astronomical instrument. We describe our second-generation SQUID multiplexer, which is based on a new architecture that significantly reduces the dissipation of power at the first stage, allowing thousands of SQUIDs to be operated at the base temperature of a cryostat. .

  2. The Microwave SQUID Multiplexer

    NASA Astrophysics Data System (ADS)

    Mates, John Arthur Benson

    2011-12-01

    This thesis describes a multiplexer of Superconducting Quantum Interference Devices (SQUIDs) with low-noise, ultra-low power dissipation, and great scalability. The multiplexer circuit measures the magnetic flux in a large number of unshunted rf SQUIDs by coupling each SQUID to a superconducting microwave resonator tuned to a unique resonance frequency and driving the resonators from a common feedline. A superposition of microwave tones measures each SQUID simultaneously using only two coaxial cables between the cryogenic device and room temperature. This multiplexer will enable the instrumentation of arrays with hundreds of thousands of low-temperature detectors for new applications in cosmology, materials analysis, and nuclear non-proliferation. The driving application of the Microwave SQUID Multiplexer is the readout of large arrays of superconducting transition-edge sensors, by some figures of merit the most sensitive detectors of electromagnetic signals over a span of more than nine orders of magnitude in energy, from 40 GHz microwaves to 200 keV gamma rays. Modern transition-edge sensors have noise-equivalent power as low as 10-20 W / Hz1/2 and energy resolution as good as 2 eV at 6 keV. These per-pixel sensitivities approach theoretical limits set by the underlying signals, motivating a rapid increase in pixel count to access new science. Compelling applications, like the non-destructive assay of nuclear material for treaty verification or the search for primordial gravity waves from inflation use arrays of these detectors to increase collection area or tile a focal plane. We developed three generations of SQUID multiplexers, optimizing the first for flux noise 0.17 muPhi0 / Hz1/2, the second for input current noise 19 pA / Hz1/2, and the last for practical multiplexing of large arrays of cosmic microwave background polarimeters based on transition-edge sensors. Using the last design we demonstrated multiplexed readout of prototype polarimeters with the

  3. Improved detection of episomal Banana streak viruses by multiplex immunocapture PCR.

    PubMed

    Le Provost, Grégoire; Iskra-Caruana, Marie-Line; Acina, Isabelle; Teycheney, Pierre-Yves

    2006-10-01

    Banana streak viruses (BSV) are currently the main viral constraint to Musa germplasm movement, genetic improvement and mass propagation. Therefore, it is necessary to develop and implement BSV detection strategies that are both reliable and sensitive, such as PCR-based techniques. Unfortunately, BSV endogenous pararetrovirus sequences (BSV EPRVs) are present in the genome of Musa balbisiana. They interfere with PCR-based detection of episomal BSV in infected banana and plantain, such as immunocapture PCR. Therefore, a multiplex, immunocapture PCR (M-IC-PCR) was developed for the detection of BSV. Musa sequence tagged microsatellite site (STMS) primers were selected and used in combination with BSV species-specific primers in order to monitor possible contamination by Musa genomic DNA, using multiplex PCR. Furthermore, immunocapture conditions were optimized in order to prevent Musa DNA from interfering with episomal BSV DNA during the PCR step. This improved detection method successfully allowed the accurate, specific and sensitive detection of episomal DNA only from distinct BSV species. Its implementation should benefit PCR-based detection of viruses for which homologous sequences are present in the genome of their hosts, including transgenic plants expressing viral sequences. PMID:16857272

  4. Extracting information from multiplex networks.

    PubMed

    Iacovacci, Jacopo; Bianconi, Ginestra

    2016-06-01

    Multiplex networks are generalized network structures that are able to describe networks in which the same set of nodes are connected by links that have different connotations. Multiplex networks are ubiquitous since they describe social, financial, engineering, and biological networks as well. Extending our ability to analyze complex networks to multiplex network structures increases greatly the level of information that is possible to extract from big data. For these reasons, characterizing the centrality of nodes in multiplex networks and finding new ways to solve challenging inference problems defined on multiplex networks are fundamental questions of network science. In this paper, we discuss the relevance of the Multiplex PageRank algorithm for measuring the centrality of nodes in multilayer networks and we characterize the utility of the recently introduced indicator function Θ̃(S) for describing their mesoscale organization and community structure. As working examples for studying these measures, we consider three multiplex network datasets coming for social science. PMID:27368796

  5. Extracting information from multiplex networks

    NASA Astrophysics Data System (ADS)

    Iacovacci, Jacopo; Bianconi, Ginestra

    2016-06-01

    Multiplex networks are generalized network structures that are able to describe networks in which the same set of nodes are connected by links that have different connotations. Multiplex networks are ubiquitous since they describe social, financial, engineering, and biological networks as well. Extending our ability to analyze complex networks to multiplex network structures increases greatly the level of information that is possible to extract from big data. For these reasons, characterizing the centrality of nodes in multiplex networks and finding new ways to solve challenging inference problems defined on multiplex networks are fundamental questions of network science. In this paper, we discuss the relevance of the Multiplex PageRank algorithm for measuring the centrality of nodes in multilayer networks and we characterize the utility of the recently introduced indicator function Θ ˜ S for describing their mesoscale organization and community structure. As working examples for studying these measures, we consider three multiplex network datasets coming for social science.

  6. Downlink Data Multiplexer

    NASA Technical Reports Server (NTRS)

    Holland, Douglas; Steele, Glen F.; Romero, Denise M.; Koudelka, Robert David

    2004-01-01

    A multiplexer/demultiplexer system has been developed to enable the transmission, over a single channel, of four data streams generated by a variety of sources at different (including variable) bit rates. In the original intended application, replicas of this multiplexer/demultiplexer system would be incorporated into the spacecraft-to-ground communication systems of the space shuttles. The multiplexer of each system would be installed in the spacecraft, where it would acquire and process data from such sources as commercial digital camcorders, video tape recorders, and the spacecraft telemetry system. The demultiplexer of each system would be installed in a ground station. Purely terrestrial systems of similar design could be attractive for use in situations in which there are requirements to transmit multiple streams of high-quality video data and possibly other data over single channels. The figure is a block diagram of the multiplexer as configured to process data received via three fiber-optic channels like those of the International Space Station and one electrical-cable channel that conforms to the Institute of Electrical and Electronic Engineers (IEEE) 1394 standard. (This standard consists of specifications of a high-speed serial data interface, the physical layer of which includes a cable known in the art as "FireWire." An IEEE 1394 interface can also transfer power between the components to which it is connected.) The fiber-optic channels carry packet and/or bit-stream signals that conform to the standards of the Consultative Committee for Space Data Systems (CCSDS). The IEEE 1394 interface accepts an isochronous signal like that from a digital camcorder or a video tape recorder. The processing of the four input data streams to combine them into one output stream is governed by a statistical multiplexing algorithm that features a flow-control capability and makes it possible to utilize the transmission channel with nearly 100-percent efficiency. This

  7. Single-step multiplex reverse transcription-polymerase chain reaction assay for detection and differentiation of the 2009 (H1N1) influenza A virus pandemic in Thai swine populations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A recently emerged H1N1 Influenza A virus (pandemic 1 H1N1: pH1N1) with a Swine influenza virus (SIV) genetic background spread globally from human-to-human causing the first influenza virus pandemic of the 21st century. In a short period reverse zoonotic cases in pigs followed by a wide spread of t...

  8. Multiplexing oscillatory biochemical signals.

    PubMed

    de Ronde, Wiet; ten Wolde, Pieter Rein

    2014-04-01

    In recent years it has been increasingly recognized that biochemical signals are not necessarily constant in time and that the temporal dynamics of a signal can be the information carrier. Moreover, it is now well established that the protein signaling network of living cells has a bow-tie structure and that components are often shared between different signaling pathways. Here we show by mathematical modeling that living cells can multiplex a constant and an oscillatory signal: they can transmit these two signals simultaneously through a common signaling pathway, and yet respond to them specifically and reliably. We find that information transmission is reduced not only by noise arising from the intrinsic stochasticity of biochemical reactions, but also by crosstalk between the different channels. Yet, under biologically relevant conditions more than 2 bits of information can be transmitted per channel, even when the two signals are transmitted simultaneously. These observations suggest that oscillatory signals are ideal for multiplexing signals. PMID:24685537

  9. The effect of sampling technique on PCR-based bacteriological results of bovine milk samples.

    PubMed

    Hiitiö, Heidi; Simojoki, Heli; Kalmus, Piret; Holopainen, Jani; Pyörälä, Satu; Taponen, Suvi

    2016-08-01

    The aim of the study was to evaluate the effect of sampling technique on the microbiological results of bovine milk samples using multiplex real-time PCR. Comparison was made between a technique where the milk sample was taken directly from the udder cistern of the udder quarter using a needle and vacuum tube and conventional sampling. The effect of different cycle threshold (Ct) cutoff limits on the results was also tested to estimate the amount of amplified DNA in the samples. A total of 113 quarters from 53 cows were tested pairwise using both techniques, and each sample was studied with real-time PCR. Sampling from the udder cistern reduced the number of species per sample compared with conventional sampling. In conventional samples, the number of positive Staphylococcus spp. results was over twice that of samples taken with the needle technique, indicating that most of the Staphylococcus spp. originated from the teat or environmental sources. The Ct values also showed that Staphylococcus spp. were present in most samples only in low numbers. Routine use of multiplex real-time PCR in mastitis diagnostics could benefit from critical evaluation of positive Staphylococcus spp. results with Ct values between 34.0 and 37.0. Our results emphasize the importance of a careful aseptic milk sampling technique and a microbiologically positive result for a milk sample should not be automatically interpreted as an intramammary infection or mastitis. PMID:27209134

  10. Multiplex data bus simulator

    SciTech Connect

    Garbo, D.L.

    1983-01-01

    A multiplex data-bus simulator for analyzing multiprocessor designs is presented. The simulator was designed to be user-friendly, thus allowing a multiprocessor designer to enter various configuration inputs in a concise and orderly fashion through the use of menus. The designer is also provided a method of visualizing a message traffic flow through the use of graphical representation of events. 3 references.

  11. Use of mitochondrial RNA genes for the differentiation of four Trichinella species by multiplex PCR amplification.

    PubMed

    Blaga, R; Fu, BaoQuan; Le Rhun, D; Le Naour, E; Heckman, A; Zocevic, A; Liu, MingYuan; Boireau, P

    2009-06-01

    Until now, four species of the Trichinella genus have been identified in Europe: Trichinella spiralis, T. nativa, T. britovi and T. pseudospiralis. The aim of this work was to establish a sound polymerase chain reaction (PCR)-based method to differentiate these four species using mitochondrial rDNA as a reliable genetic marker and to evaluate the sensitivity of this method. Full-length DNA sequences coding for the small and large mitochondrial rRNA (mt-rrnS and mt-rrnL) of the four species are described. A multiplex PCR was designed and successfully tested on 24 European isolates. As few as one larva, or 100 pg of genomic DNA was detected, providing equivalent sensitivity to previously described PCR methods. The PCR-based method of mitochondrial rDNA amplification was thereby established as a sensitive and reproductive diagnostic method for the four European Trichinella species. PMID:19389269

  12. Multiplex PCR for detection of acquired carbapenemase genes.

    PubMed

    Poirel, Laurent; Walsh, Timothy R; Cuvillier, Vincent; Nordmann, Patrice

    2011-05-01

    A rapid and reliable PCR-based technique was developed for detection of genes encoding carbapenemases belonging to different classes. Primers were designed to amplify the following 11 genes: bla(IMP), bla(VIM), bla(NDM), bla(SPM), bla(AIM), bla(DIM), bla(GIM), bla(SIM)bla(KPC), bla(BIC), and bla(OXA-48). Three different multiplex reaction mixtures were defined and evaluated for the detection of all these 11 genes. Using optimized conditions, each reaction mixture allowed to identify the respective genes, with PCR giving distinct amplicon sizes corresponding to the different genes for each mixture. We reported here a rapid and reliable technique for screening all clinically relevant carbapenemase genes. PMID:21398074

  13. Self-calibrating multiplexer circuit

    DOEpatents

    Wahl, Chris P.

    1997-01-01

    A time domain multiplexer system with automatic determination of acceptable multiplexer output limits, error determination, or correction is comprised of a time domain multiplexer, a computer, a constant current source capable of at least three distinct current levels, and two series resistances employed for calibration and testing. A two point linear calibration curve defining acceptable multiplexer voltage limits may be defined by the computer by determining the voltage output of the multiplexer to very accurately known input signals developed from predetermined current levels across the series resistances. Drift in the multiplexer may be detected by the computer when the output voltage limits, expected during normal operation, are exceeded, or the relationship defined by the calibration curve is invalidated.

  14. Self-calibrating multiplexer circuit

    SciTech Connect

    Wahl, C.P.

    1995-12-31

    A time domain multiplexer system with automatic determination of acceptable multiplexer output limits, error determination, or correction is comprised of a time domain multiplexer, a computer, a constant current source capable of at least three distinct current levels, and two series resistances employed for calibration and testing. A two point linear calibration curve defining acceptable multiplexer voltage limits may be defined by the computer by determining the voltage output of the multiplexer to very accurately known input signals developed from predetermined current levels across the series resistances. Drift in the multiplexer may be detected by the computer when the output voltage limits, expected during normal operation, are exceeded, or the relationship defined by the calibration curve is invalidated.

  15. Effective PCR-based detection of Naegleria fowleri from cultured sample and PAM-developed mouse.

    PubMed

    Kang, Heekyoung; Seong, Gi-Sang; Sohn, Hae-Jin; Kim, Jong-Hyun; Lee, Sang-Eun; Park, Mi Yeoun; Lee, Won-Ja; Shin, Ho-Joon

    2015-10-01

    Increasing numbers of Primary Amoebic Meningoencephalitis (PAM) cases due to Naegleria fowleri are becoming a serious issue in subtropical and tropical countries as a Neglected Tropical Disease (NTD). To establish a rapid and effective diagnostic tool, a PCR-based detection technique was developed based on previous PCR methods. Four kinds of primer pairs, Nfa1, Nae3, Nf-ITS, and Naegl, were employed in the cultured amoebic trophozoites and a mouse with PAM experimentally developed by N. fowleri inoculation (PAM-mouse). For the extraction of genomic DNA from N. fowleri trophozoites (1×10(6)), simple boiling with 10μl of PBS (pH 7.4) at 100°C for 30min was found to be the most rapid and efficient procedure, allowing amplification of 2.5×10(2) trophozoites using the Nfa-1 primer. The primers Nfa1 and Nae3 amplified only N. fowleri DNA, whereas the ITS primer detected N. fowleri and N. gruberi DNA. Using the PAM-mouse brain tissue, the Nfa1 primer was able to amplify the N. fowleri DNA 4 days post infection with 1ng/μl of genomic DNA being detectable. Using the PAM-mouse CSF, amplification of the N. fowleri DNA with the Nae3 primer was possible 5 days post infection showing a better performance than the Nfa1 primer at day 6. PMID:26322498

  16. A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells.

    PubMed

    Findlay, Scott D; Vincent, Krista M; Berman, Jennifer R; Postovit, Lynne-Marie

    2016-01-01

    The rapid adoption of gene editing tools such as CRISPRs and TALENs for research and eventually therapeutics necessitates assays that can rapidly detect and quantitate the desired alterations. Currently, the most commonly used assay employs "mismatch nucleases" T7E1 or "Surveyor" that recognize and cleave heteroduplexed DNA amplicons containing mismatched base-pairs. However, this assay is prone to false positives due to cancer-associated mutations and/or SNPs and requires large amounts of starting material. Here we describe a powerful alternative wherein droplet digital PCR (ddPCR) can be used to decipher homozygous from heterozygous mutations with superior levels of both precision and sensitivity. We use this assay to detect knockout inducing alterations to stem cell associated proteins, NODAL and SFRP1, generated using either TALENs or an "all-in-one" CRISPR/Cas plasmid that we have modified for one-step cloning and blue/white screening of transformants. Moreover, we highlight how ddPCR can be used to assess the efficiency of varying TALEN-based strategies. Collectively, this work highlights how ddPCR-based screening can be paired with CRISPR and TALEN technologies to enable sensitive, specific, and streamlined approaches to gene editing and validation. PMID:27089539

  17. Helicobacter pylori is not eradicated after triple therapy: a nested PCR based study.

    PubMed

    Patel, Saurabh Kumar; Mishra, Girish Narayan; Pratap, Chandra Bhan; Jain, Ashok Kumar; Nath, Gopal

    2014-01-01

    Detection of Helicobacter pylori after triple therapy is usually carried out by either rapid urease test (RUT), urea breath test (UBT), histology, bacterial isolation, and single round PCR or serological tests. In this study, antral biopsy specimens from 25 patients were tested for H. pylori by RUT, culture, histology, and nested PCR in their antral biopsy specimens before and after treatment. Three genes, namely, heat shock protein (hsp60), phosphoglucosamine mutase (ureC), and flagellar export ATP synthase (fliI) of H. pylori were targeted. Of the 25 antral biopsy specimens, the RUT, culture, histology, and nested PCR positivity dropped from 81.8% to 12%, 31% to 0%, 100 to 84%, and 100% to 92%, respectively, before and after therapy. Further, hsp60 specific amplicons from 23 out of 25 patients gave identical restriction pattern, while 6 fliI and 1 ureC specific amplicon produced different restriction pattern. Furthermore, variations in fliI gene sequences in H. pylori after treatment were also confirmed by sequencing and compared in silico. Nested PCR based detection of H. pylori is more sensitive method to detect H. pylori after therapy than culture, RUT, and histology. Further, this study suggests that H. pylori is not eradicated completely after triple therapy. PMID:25054141

  18. Rapid and Robust PCR-Based All-Recombinant Cloning Methodology

    PubMed Central

    Jain, Vikas

    2016-01-01

    We report here a PCR-based cloning methodology that requires no post-PCR modifications such as restriction digestion and phosphorylation of the amplified DNA. The advantage of the present method is that it yields only recombinant clones thus eliminating the need for screening. Two DNA amplification reactions by PCR are performed wherein the first reaction amplifies the gene of interest from a source template, and the second reaction fuses it with the designed expression vector fragments. These vector fragments carry the essential elements that are required for the fusion product selection. The entire process can be completed in less than 8 hours. Furthermore, ligation of the amplified DNA by a DNA ligase is not required before transformation, although the procedure yields more number of colonies upon transformation if ligation is carried out. As a proof-of-concept, we show the cloning and expression of GFP, adh, and rho genes. Using GFP production as an example, we further demonstrate that the E. coli T7 express strain can directly be used in our methodology for the protein expression immediately after PCR. The expressed protein is without or with 6xHistidine tag at either terminus, depending upon the chosen vector fragments. We believe that our method will find tremendous use in molecular and structural biology. PMID:27007922

  19. Rapid and Robust PCR-Based All-Recombinant Cloning Methodology.

    PubMed

    Dubey, Abhishek Anil; Singh, Manika Indrajit; Jain, Vikas

    2016-01-01

    We report here a PCR-based cloning methodology that requires no post-PCR modifications such as restriction digestion and phosphorylation of the amplified DNA. The advantage of the present method is that it yields only recombinant clones thus eliminating the need for screening. Two DNA amplification reactions by PCR are performed wherein the first reaction amplifies the gene of interest from a source template, and the second reaction fuses it with the designed expression vector fragments. These vector fragments carry the essential elements that are required for the fusion product selection. The entire process can be completed in less than 8 hours. Furthermore, ligation of the amplified DNA by a DNA ligase is not required before transformation, although the procedure yields more number of colonies upon transformation if ligation is carried out. As a proof-of-concept, we show the cloning and expression of GFP, adh, and rho genes. Using GFP production as an example, we further demonstrate that the E. coli T7 express strain can directly be used in our methodology for the protein expression immediately after PCR. The expressed protein is without or with 6xHistidine tag at either terminus, depending upon the chosen vector fragments. We believe that our method will find tremendous use in molecular and structural biology. PMID:27007922

  20. A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells

    PubMed Central

    Berman, Jennifer R.; Postovit, Lynne-Marie

    2016-01-01

    The rapid adoption of gene editing tools such as CRISPRs and TALENs for research and eventually therapeutics necessitates assays that can rapidly detect and quantitate the desired alterations. Currently, the most commonly used assay employs “mismatch nucleases” T7E1 or “Surveyor” that recognize and cleave heteroduplexed DNA amplicons containing mismatched base-pairs. However, this assay is prone to false positives due to cancer-associated mutations and/or SNPs and requires large amounts of starting material. Here we describe a powerful alternative wherein droplet digital PCR (ddPCR) can be used to decipher homozygous from heterozygous mutations with superior levels of both precision and sensitivity. We use this assay to detect knockout inducing alterations to stem cell associated proteins, NODAL and SFRP1, generated using either TALENs or an “all-in-one” CRISPR/Cas plasmid that we have modified for one-step cloning and blue/white screening of transformants. Moreover, we highlight how ddPCR can be used to assess the efficiency of varying TALEN-based strategies. Collectively, this work highlights how ddPCR-based screening can be paired with CRISPR and TALEN technologies to enable sensitive, specific, and streamlined approaches to gene editing and validation. PMID:27089539

  1. Development of rapid canine fecal source identification PCR-based assays.

    PubMed

    Green, Hyatt C; White, Karen M; Kelty, Cathy A; Shanks, Orin C

    2014-10-01

    The extent to which dogs contribute to aquatic fecal contamination is unknown despite the potential for zoonotic transfer of harmful human pathogens. We used genome fragment enrichment (GFE) to identify novel nonribosomal microbial genetic markers potentially useful for detecting dog fecal contamination with PCR-based methods in environmental samples. Of the 679 sequences obtained from GFE, we used 84 for the development of PCR assays targeting putative canine-associated genetic markers. Twelve genetic markers were shown to be prevalent among dog fecal samples and were rarely found in other animals. Three assays, DG3, DG37, and DG72, performed best in terms of specificity and sensitivity and were used for the development of SYBR Green and TaqMan quantitative PCR (qPCR) assays. qPCR analysis of 244 fecal samples collected from a wide geographic range indicated that marker concentrations were below limits of detection in noncanine hosts. As a proof-of-concept, these markers were detected in urban stormwater samples, suggesting a future application of newly developed methods for water quality monitoring. PMID:25203917

  2. PCR-based detection of bioluminescent microbial populations in Tyrrhenian Sea

    NASA Astrophysics Data System (ADS)

    Gentile, Gabriela; De Luca, Massimo; Denaro, Renata; La Cono, Violetta; Smedile, Francesco; Scarfì, Simona; De Domenico, Emilio; De Domenico, Maria; Yakimov, Michail M.

    2009-05-01

    The present study is focused on the development of a cultivation-independent molecular approach for specific detection of bioluminescent bacteria within microbial communities by direct amplification of luxA gene from environmental DNA. A new set of primers, specifically targeting free-living bioluminescent bacteria, was designed on the base of l uxA sequences available from the public database. Meso- and bathypelagic seawater samples were collected from two stations in Tyrrhenian Sea at the depths of 500 and 2750 m. The same seawater samples also were used to isolate bioluminescent bacteria that were further subjected to luxA and 16S rRNA gene sequencing. PCR products obtained by amplification with designed primers were cloned, and the phylogenetic affiliation of 40 clones was determined. All of them were clustered into three groups, only distantly related to the Photobacterium phosphoreum and Photobacterium kishitanii clades. The half of all clones formed a tight monophyletic clade, while the rest of clones were organized in "compartment"-specific, meso- and bathypelagic ecotypes. No matches with luxA gene sequences of four bioluminescent strains, isolated from the same seawater samples, were observed. These findings indicate that the PCR-based approach developed in present manuscript, allowed us to detect the novel, "yet to be cultivated" lineages of bioluminescent bacteria, which are likely specific for distinct warm bathypelagic realms of Mediterranean Sea.

  3. Effect of reference database on frequency estimates of polymerase chain reaction (PCR)-based DNA profiles.

    PubMed

    Monson, K L; Budowle, B

    1998-05-01

    A variety of general, regional, ancestral and ethnic databases is available for the polymerase chain reaction (PCR)-based loci LDLR, GYPA, HBGG, D7S8, Gc, DQA1, and D1S80. Generally, we observed greater differences in frequency estimations of DNA profiles between racial groups than between ethnic or geographic subgroups. Analysis revealed few forensically significant differences within ethnic subgroups, particularly within general United States groups, and multi-locus frequency estimates typically differ by less than a factor of ten. Using a database different from the one to which a target profile belongs tends to overestimate rarity. Implementation of the general correction of homozygote frequencies for a population substructure, advised by the 1996 National Research Council report, The Evaluation of Forensic DNA Evidence, has a minimal effect on profile frequencies. Even when it is known that both the suspect and all possible perpetrators must belong to the same isolated population, the special correction for inbreeding, which was proposed by the 1996 National Research Council report for this special case, has a relatively modest effect, typically a factor of two or less for 1% inbreeding. The effect becomes more substantial (exceeding a factor of ten) for inbreeding of 3% or more in multi-locus profiles rarer than about one in a million. PMID:9608687

  4. A versatile PCR-based tandem epitope tagging system for Streptomyces coelicolor genome.

    PubMed

    Kim, Ji-Nu; Yi, Jeong Sang; Lee, Bo-Rahm; Kim, Eun-Jung; Kim, Min Woo; Song, Yoseb; Cho, Byung-Kwan; Kim, Byung-Gee

    2012-07-20

    Epitope tagging approaches have been widely used for the analysis of functions, interactions and subcellular distributions of proteins. However, incorporating epitope sequence into protein loci in Streptomyces is time-consuming procedure due to the absence of the versatile tagging methods. Here, we developed a versatile PCR-based tandem epitope tagging tool for the Streptomyces genome engineering. We constructed a series of template plasmids that carry repeated sequence of c-myc epitope, Flp recombinase target (FRT) sites, and apramycin resistance marker to insert epitope tags into any desired spot of the chromosomal loci. A DNA module which includes the tandem epitope-encoding sequence and a selectable marker was amplified by PCR with primers that carry homologous extensions to the last portion and downstream region of the targeted gene. We fused the epitope tags at the 3' region of global transcription factors of Streptomyces coelicolor to test the validity of this system. The proper insertion of the epitope tag was confirmed by PCR and western blot analysis. The recombinants showed the identical phenotype to the wild-type that proved the conservation of in vivo function of the tagged proteins. Finally, the direct binding targets were successfully detected by chromatin immunoprecipitation with the increase in the signal-to-noise ratio. The epitope tagging system describes here would provide wide applications to study the protein functions in S. coelicolor. PMID:22704935

  5. PCR-Based Seamless Genome Editing with High Efficiency and Fidelity in Escherichia coli

    PubMed Central

    Liu, Yilan; Yang, Maohua; Chen, Jinjin; Yan, Daojiang; Cheng, Wanwan; Wang, Yanyan; Thygesen, Anders; Chen, Ruonan; Xing, Jianmin; Wang, Qinhong; Ma, Yanhe

    2016-01-01

    Efficiency and fidelity are the key obstacles for genome editing toolboxes. In the present study, a PCR-based tandem repeat assisted genome editing (TRAGE) method with high efficiency and fidelity was developed. The design of TRAGE is based on the mechanism of repair of spontaneous double-strand breakage (DSB) via replication fork reactivation. First, cat-sacB cassette flanked by tandem repeat sequence was integrated into target site in chromosome assisted by Red enzymes. Then, for the excision of the cat-sacB cassette, only subculturing is needed. The developed method was successfully applied for seamlessly deleting, substituting and inserting targeted genes using PCR products. The effects of different manipulations including sucrose addition time, subculture times in LB with sucrose and stages of inoculation on the efficiency were investigated. With our recommended procedure, seamless excision of cat-sacB cassette can be realized in 48 h efficiently. We believe that the developed method has great potential for seamless genome editing in E. coli. PMID:27019283

  6. PCR-based Approaches for the Detection of Clinical Methicillin-resistant Staphylococcus aureus

    PubMed Central

    Liu, Ying; Zhang, Jiang; Ji, Yinduo

    2016-01-01

    Staphylococcus aureus is an important pathogen that can cause a variety of infections, including superficial and systematic infections, in humans and animals. The persistent emergence of multidrug resistant S. aureus, particularly methicillin-resistant S. aureus, has caused dramatically economic burden and concerns in the public health due to limited options of treatment of MRSA infections. In order to make a correct choice of treatment for physicians and understand the prevalence of MRSA, it is extremely critical to precisely and timely diagnose the pathogen that induces a specific infection of patients and to reveal the antibiotic resistant profile of the pathogen. In this review, we outlined different PCR-based approaches that have been successfully utilized for the rapid detection of S. aureus, including MRSA and MSSA, directly from various clinical specimens. The sensitivity and specificity of detections were pointed out. Both advantages and disadvantages of listed approaches were discussed. Importantly, an alternative approach is necessary to further confirm the detection results from the molecular diagnostic assays. PMID:27335617

  7. PCR-based positive hybridization to detect genomic diversity associated with bacterial secondary metabolism

    PubMed Central

    Pomati, Francesco; Neilan, Brett A.

    2004-01-01

    A PCR-based positive hybridization (PPH) method was developed to explore toxic-specific genes in common between toxigenic strains of Anabaena circinalis, a cyanobacterium able to produce saxitoxin (STX). The PPH technique is based on the same principles of suppression subtractive hybridization (SSH), although with the former no driver DNA is required and two tester genomic DNAs are hybridized at high stringency. The aim was to obtain genes associated with cyanobacterial STX production. The genetic diversity within phylogenetically similar strains of A.circinalis was investigated by comparing the results of the standard SSH protocol to the PPH approach by DNA-microarray analysis. SSH allowed the recovery of DNA libraries that were mainly specific for each of the two STX-producing strains used. Several candidate sequences were found by PPH to be in common between both the STX-producing testers. The PPH technique performed using unsubtracted genomic libraries proved to be a powerful tool to identify DNA sequences possibly transferred laterally between two cyanobacterial strains that may be candidate(s) in STX biosynthesis. The approach presented in this study represents a novel and valid tool to study the genetic basis for secondary metabolite production in microorganisms. PMID:14718552

  8. PCR-based approach to distinguish group A human rotavirus genotype 1 vs. genotype 2 genes.

    PubMed

    McKell, Allison O; Nichols, Joshua C; McDonald, Sarah M

    2013-12-01

    Group A rotaviruses (RVs) are eleven-segmented, double-stranded RNA viruses and important causes of severe diarrhea in children. A full-genome classification system is readily used to describe the genetic makeup of individual RV strains. In this system, each viral gene is assigned a specific genotype based upon its nucleotide sequence and established percent identity cut-off values. However, a faster and more cost-effective approach to determine RV gene genotypes is to utilize specific oligonucleotide primer sets in RT-PCR/PCR. Such primer sets and PCR-based genotyping methods have already been developed for the VP7-, VP6-, VP4- and NSP4-coding gene segments. In this study, primers were developed for the remaining seven RV gene segments, which encode proteins VP1, VP2, VP3, NSP1, NSP2, NSP3, and NSP5/6. Specifically, primers were designed to distinguish the two most common human RV genotypes (1 vs. 2) for these genes and were validated on several cell culture-adapted human and animal RV strains, as well as on human RVs from clinical fecal specimens. As such, primer sets now exist for all eleven genes of common human RVs, allowing for the identification of reassortant strains with mixed constellations of both genotype 1 and 2 genes using a rapid and economical RT-PCR/PCR method. PMID:24012969

  9. PCR-based detection of gene transfer vectors: application to gene doping surveillance.

    PubMed

    Perez, Irene C; Le Guiner, Caroline; Ni, Weiyi; Lyles, Jennifer; Moullier, Philippe; Snyder, Richard O

    2013-12-01

    Athletes who illicitly use drugs to enhance their athletic performance are at risk of being banned from sports competitions. Consequently, some athletes may seek new doping methods that they expect to be capable of circumventing detection. With advances in gene transfer vector design and therapeutic gene transfer, and demonstrations of safety and therapeutic benefit in humans, there is an increased probability of the pursuit of gene doping by athletes. In anticipation of the potential for gene doping, assays have been established to directly detect complementary DNA of genes that are top candidates for use in doping, as well as vector control elements. The development of molecular assays that are capable of exposing gene doping in sports can serve as a deterrent and may also identify athletes who have illicitly used gene transfer for performance enhancement. PCR-based methods to detect foreign DNA with high reliability, sensitivity, and specificity include TaqMan real-time PCR, nested PCR, and internal threshold control PCR. PMID:23912835

  10. Performance Assessment PCR-Based Assays Targeting Bacteroidales Genetic Markers of Bovine Fecal Pollution▿

    PubMed Central

    Shanks, Orin C.; White, Karen; Kelty, Catherine A.; Hayes, Sam; Sivaganesan, Mano; Jenkins, Michael; Varma, Manju; Haugland, Richard A.

    2010-01-01

    There are numerous PCR-based assays available to characterize bovine fecal pollution in ambient waters. The determination of which approaches are most suitable for field applications can be difficult because each assay targets a different gene, in many cases from different microorganisms, leading to variation in assay performance. We describe a performance evaluation of seven end-point PCR and real-time quantitative PCR (qPCR) assays reported to be associated with either ruminant or bovine feces. Each assay was tested against a reference collection of DNA extracts from 247 individual bovine fecal samples representing 11 different populations and 175 fecal DNA extracts from 24 different animal species. Bovine-associated genetic markers were broadly distributed among individual bovine samples ranging from 39 to 93%. Specificity levels of the assays spanned 47.4% to 100%. End-point PCR sensitivity also varied between assays and among different bovine populations. For qPCR assays, the abundance of each host-associated genetic marker was measured within each bovine population and compared to results of a qPCR assay targeting 16S rRNA gene sequences from Bacteroidales. Experiments indicate large discrepancies in the performance of bovine-associated assays across different bovine populations. Variability in assay performance between host populations suggests that the use of bovine microbial source-tracking applications will require a priori characterization at each watershed of interest. PMID:20061457

  11. Development of PCR-based assays for detecting and differentiating three species of botrytis infecting broad bean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botrytis cinerea, B. fabae and B. fabiopsis are known to cause chocolate spot on broad bean. This study was conducted to develop PCR-based assays to detect and differentiate this three species. Two sets of primers, Bc-f/Bc-r for B. cinerea and Bfab-f/Bfab-r for B. fabiopsis, were designed based on t...

  12. A Reliable and Inexpensive Method of Nucleic Acid Extraction for the PCR-Based Detection of Diverse Plant Pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A reliable extraction method is described for the preparation of total nucleic acids from several plant genera for subsequent detection of plant pathogens by PCR-based techniques. By the combined use of a modified CTAB (cetyltrimethylammonium bromide) extraction protocol and a semi-automatic homogen...

  13. Combining watershed attributes with culture- and PCR-based methods for improved characterization and management of fecal pollution

    EPA Science Inventory

    Culture- and PCR-based methods for characterization of fecal pollution were evaluated in relation to physiographic, biotic, and chemical indicators of stream condition. Stream water samples (n = 235) were collected monthly over a two year period from ten channels draining subwat...

  14. Combining Watershed Variables with PCR-based Methods for Better Characterization and Management of Fecal Pollution in Small Streams

    EPA Science Inventory

    Ability to distinguish between human and animal fecal pollution is important for risk assessment and watershed management, particularly in bodies of water used as sources of drinking water or for recreation. PCR-based methods were used to determine the source of fecal pollution ...

  15. Combining Watershed Variables with PCR-based Methods for Better Characterization and Management of Fecal Pollution in Small Streams

    EPA Science Inventory

    Culture- and PCR-based measurements of fecal pollution were determined and compared to hydrologic and land use indicators. Stream water samples (n = 235) were collected monthly over a two year period from ten streams draining headwatersheds with different land use intensities ra...

  16. Evaluation of the repeatability and reproducibility of a suite of qPCR based microbial source tracking methods

    EPA Science Inventory

    Many PCR-based methods for microbial source tracking (MST) have been developed and validated within individual research laboratories. Inter-laboratory validation of these methods, however, has been minimal, and the effects of protocol standardization regimes have not been thor...

  17. Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study

    EPA Science Inventory

    A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing. Here, we evaluated ten of these methods (BacH, BacHum-UCD, B. thetaiotaomic...

  18. Evaluation of culture- and PCR-based detection methods for Escherichia coli O157:H7 in inoculated ground beeft.

    PubMed

    Arthur, Terrance M; Bosilevac, Joseph M; Nou, Xiangwu; Koohmaraie, Mohammad

    2005-08-01

    Currently, several beef processors employ test-and-hold systems for increased quality control of ground beef. In such programs, each lot of product must be tested and found negative for Escherichia coli O157:H7 prior to release of the product into commerce. Optimization of three testing attributes (detection time, specificity, and sensitivity) is critical to the success of such strategies. Because ground beef is a highly perishable product, the testing methodology used must be as rapid as possible. The test also must have a low false-positive result rate so product is not needlessly discarded. False-negative results cannot be tolerated because they would allow contaminated product to be released and potentially cause disease. In this study, two culture-based and three PCR-based methods for detecting E. coli O157:H7 in ground beef were compared for their abilities to meet the above criteria. Ground beef samples were individually spiked with five genetically distinct strains of E. coli O157: H7 at concentrations of 17 and 1.7 CFU/65 g and then subjected to the various testing methodologies. There was no difference (P > 0.05) in the abilities of the PCR-based methods to detect E. coli O157:H7 inoculated in ground beef at 1.7 CFU/65 g. The culture-based systems detected more positive samples than did the PCR-based systems, but the detection times (21 to 48 h) were at least 9 h longer than those for the PCR-based methods (7.5 to 12 h). Ground beef samples were also spiked with potentially cross-reactive strains. The PCR-based systems that employed an immunomagnetic separation step prior to detection produced fewer false-positive results. PMID:21132961

  19. PCR-Based Multiple Species Cell Counting for In Vitro Mixed Culture

    PubMed Central

    Huang, Ruijie; Zhang, Junjie; Yang, X. Frank; Gregory, Richard L.

    2015-01-01

    Changes of bacterial profiles in microbial communities are strongly associated with human health. There is an increasing need for multiple species research in vitro. To avoid high cost or measurement of a limited number of species, PCR-based multiple species cell counting (PCR-MSCC) has been conceived. Species-specific sequence is defined as a unique sequence of one species in a multiple species mixed culture. This sequence is identified by comparing a random 1000 bp genomic sequence of one species with the whole genome sequences of the other species in the same artificial mixed culture. If absent in the other genomes, it is the species-specific sequence. Species-specific primers were designed based on the species-specific sequences. In the present study, ten different oral bacterial species were mixed and grown in Brain Heart Infusion Yeast Extract with 1% sucrose for 24 hours. Biofilm was harvested and processed for DNA extraction and q-PCR amplification with the species-specific primers. By comparing the q-PCR data of each species in the unknown culture with reference cultures, in which the cell number of each species was determined by colony forming units on agar plate, the cell number of that strain in the unknown mixed culture was calculated. This technique is reliable to count microorganism numbers that are less than 100,000 fold different from other species within the same culture. Theoretically, it can be used in detecting a species in a mixed culture of over 200 species. Currently PCR-MSCC is one of the most economic methods for quantifying single species cell numbers, especially for the low abundant species, in a multiple artificial mixed culture in vitro. PMID:25970462

  20. Fluorescent detection of Southern blots and PCR-based genetic typing tests

    SciTech Connect

    Mansfield, E.S.; Worley, J.M.; Zimmerman, P.A.

    1994-09-01

    The Southern blot is used to study gene organization, to identify disease-causing genomic rearrangements, or for typing RFLP markers in forensic, paternity, or prenatal diagnostic testing. Fluorescence offers a much greater dynamic range and a more linear response than film used in radioactive or chemiluminescent detection of RFLPs. We therefore investigated using the Fluorimager{trademark} 575 (Molecular Dynamics, Inc.) for analyzing Southern blots. Using a single-locus probe to D2S44 (YNH24) (Promega Corp.), we detect as little as 100 ng (0.05 attomole) genomic DNA. The alkaline phosphatase-labeled probe is detected using AttoPhos (JBL Scientific), and the developed membrane is scanned with the Fluorimager. Biotinylated hybridization probes can also be developed using a streptavidin-alkaline phosphatase conjugate and AttoPhos. The instrument scan parameters can be adjusted to prevent overexposure and accompanying loss of resolution in images of blots, gels, or 96-well microplates. We have used these other sample formats in PCR-based genetic typing assays. We use FluorKit DQS (Molecular Dynamics) to accurately quantify PCR template DNA (1-500 ng) in 96-well microplates scanned using the same instrument. Mutation detection assays run include heteroduplex gels (5% polyacrylamide, 2.7 M urea), short tandem repeat (STR) markers, amplified fragment length polymorphisms (AmpFLP), competitive priming PCR, and allele-specific oligotyping. These assays are run using either 1- or 2-color labeling. We detect unlabeled PCR products, such as the AmpFLP marker D1S80 (Perkin-Elmer) by post-staining gels for 10 minutes with SYBR Green 1 (Molecular Probes) and scanning the wet gel. The Fluorimager scans a 20 x 25 cm sample within three minutes, allowing rapid optimization of fluorescent protocols and high sample throughput.

  1. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, Edward S.; Li, Qingbo; Lu, Xiandan

    1998-04-21

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification ("base calling") is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations.

  2. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, Edward S.; Chang, Huan-Tsang; Fung, Eliza N.; Li, Qingbo; Lu, Xiandan

    1996-12-10

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification ("base calling") is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations.

  3. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, E.S.; Li, Q.; Lu, X.

    1998-04-21

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification (``base calling``) is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations. 19 figs.

  4. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, E.S.; Chang, H.T.; Fung, E.N.; Li, Q.; Lu, X.

    1996-12-10

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification (``base calling``) is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations. 19 figs.

  5. Hardware Counter Multiplexing

    Energy Science and Technology Software Center (ESTSC)

    2000-10-13

    The Hardware Counter Multiplexer works with the built-in counter registers on computer processors. These counters record various low-level events as software runs, but they can not record all possible events at the same time. This software helps work around that limitation by counting a series of different events in sequence over a period of time. This in turn allows programmers to measure interesting combinations of events, rather than single events. The software is designed tomore » work with multithreaded or single-threaded programs.« less

  6. Quantitative multiplexed quantum dot immunohistochemistry

    SciTech Connect

    Sweeney, E.; Ward, T.H.; Gray, N.; Womack, C.; Jayson, G.; Hughes, A.; Dive, C.; Byers, R.

    2008-09-19

    Quantum dots are photostable fluorescent semiconductor nanocrystals possessing wide excitation and bright narrow, symmetrical, emission spectra. These characteristics have engendered considerable interest in their application in multiplex immunohistochemistry for biomarker quantification and co-localisation in clinical samples. Robust quantitation allows biomarker validation, and there is growing need for multiplex staining due to limited quantity of clinical samples. Most reported multiplexed quantum dot staining used sequential methods that are laborious and impractical in a high-throughput setting. Problems associated with sequential multiplex staining have been investigated and a method developed using QDs conjugated to biotinylated primary antibodies, enabling simultaneous multiplex staining with three antibodies. CD34, Cytokeratin 18 and cleaved Caspase 3 were triplexed in tonsillar tissue using an 8 h protocol, each localised to separate cellular compartments. This demonstrates utility of the method for biomarker measurement enabling rapid measurement of multiple co-localised biomarkers on single paraffin tissue sections, of importance for clinical trial studies.

  7. Highspeed multiplexed heterodyne interferometry.

    PubMed

    Isleif, Katharina-S; Gerberding, Oliver; Köhlenbeck, Sina; Sutton, Andrew; Sheard, Benjamin; Goßler, Stefan; Shaddock, Daniel; Heinzel, Gerhard; Danzmann, Karsten

    2014-10-01

    Digitally enhanced heterodyne interferometry is a metrology technique that uses pseudo-random noise codes for modulating the phase of the laser light. Multiple interferometric signals from the same beam path can thereby be isolated based on their propagation delay, allowing one to use advantageous optical layouts in comparison to classic laser interferometers. We present here a high speed version of this technique for measuring multiple targets spatially separated by only a few centimetres. This allows measurements of multiplexed signals using free beams, making the technique attractive for several applications requiring compact optical set-ups like for example space-based interferometers. In an experiment using a modulation and sampling rate of 1.25 GHz we are able to demonstrate multiplexing between targets only separated by 36 cm and we achieve a displacement measurement noise floor of <3 pm/√Hz at 10 Hz between them. We identify a limiting excess noise at low frequencies which is unique to this technique and is probably caused by the finite bandwidth in our measurement set-up. Utilising an active clock jitter correction scheme we are also able to reduce this noise in a null measurement configuration by one order of magnitude. PMID:25322043

  8. PCR-based method for the rapid identification of astaxanthin-accumulating yeasts (Phaffia spp.).

    PubMed

    Colabella, Fernando; Libkind, Diego

    2016-01-01

    It has been recently found that the natural distribution, habitat, and genetic diversity of astaxanthin-producing yeasts (i.e. Phaffia rhodozyma, synonym Xanthophyllomyces dendrorhous) is much greater than previously thought. P. rhodozyma is biotechnologically exploited due to its ability to produce the carotenoid pigment astaxanthin and thus, it is used as a natural source of this pigment for aquaculture. P. rhodozyma was also capable of synthesizing the potent UVB sunscreen mycosporine-glutaminol-glucoside (MGG). Therefore, further environmental studies are needed to elucidate its ecological aspects and detect new potential strains for the production of astaxanthin and MGG. However, obtaining new isolates of P. rhodozyma and related species is not always easy due to its low abundance and the presence of other sympatric and pigmented yeasts. In this work we report a successful development of a species-specific primer which has the ability to quickly and accurately detecting isolates representing all known lineages of the genus Phaffia (including novel species of the genus) and excluding closely related taxa. For this purpose, a primer of 20 nucleotides (called PhR) was designed to be used in combination with universal primers ITS3 and NL4 in a multiplex amplification. The proposed method has the sensitivity and specificity required for the precise detection of new isolates, and therefore represents an important tool for the environmental search for novel astaxanthin-producing yeasts. PMID:26922472

  9. PCR Based Microbial Monitor for Analysis of Recycled Water Aboard the ISSA: Issues and Prospects

    NASA Technical Reports Server (NTRS)

    Cassell, Gail H.; Lefkowitz, Elliot J.; Glass, John I.

    1995-01-01

    The monitoring of spacecraft life support systems for the presence of health threatening microorganisms is paramount for crew well being and successful completion of missions. Development of technology to monitor spacecraft recycled water based on detection and identification of the genetic material of contaminating microorganisms and viruses would be a substantial improvement over current NASA plans to monitor recycled water samples that call for the use of conventional microbiology techniques which are slow, insensitive, and labor intensive. The union of the molecular biology techniques of DNA probe hybridization and polymerase chain reaction (PCR) offers a powerful method for the detection, identification, and quantification of microorganisms and viruses. This technology is theoretically capable of assaying samples in as little as two hours with specificity and sensitivity unmatched by any other method. A major advance in probe-hybridization/PCR has come about in a technology called TaqMan(TM), which was invented by Perkin Elmer. Instrumentation using TaqMan concepts is evolving towards devices that could meet NASA's needs of size, low power use, and simplicity of operation. The chemistry and molecular biology needed to utilize these probe-hybridization/PCR instruments must evolve in parallel with the hardware. The following issues of chemistry and biology must be addressed in developing a monitor: Early in the development of a PCR-based microbial monitor it will be necessary to decide how many and which organisms does the system need the capacity to detect. We propose a set of 17 different tests that would detect groups of bacteria and fungus, as well as specific eukaryotic parasites and viruses; In order to use the great sensitivity of PCR it will be necessary to concentrate water samples using filtration. If a lower limit of detection of 1 microorganism per 100 ml is required then the microbes in a 100 ml sample must be concentrated into a volume that can be

  10. Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design

    PubMed Central

    Pilotte, Nils; Papaiakovou, Marina; Grant, Jessica R.; Bierwert, Lou Ann; Llewellyn, Stacey; McCarthy, James S.; Williams, Steven A.

    2016-01-01

    Background The soil transmitted helminths are a group of parasitic worms responsible for extensive morbidity in many of the world’s most economically depressed locations. With growing emphasis on disease mapping and eradication, the availability of accurate and cost-effective diagnostic measures is of paramount importance to global control and elimination efforts. While real-time PCR-based molecular detection assays have shown great promise, to date, these assays have utilized sub-optimal targets. By performing next-generation sequencing-based repeat analyses, we have identified high copy-number, non-coding DNA sequences from a series of soil transmitted pathogens. We have used these repetitive DNA elements as targets in the development of novel, multi-parallel, PCR-based diagnostic assays. Methodology/Principal Findings Utilizing next-generation sequencing and the Galaxy-based RepeatExplorer web server, we performed repeat DNA analysis on five species of soil transmitted helminths (Necator americanus, Ancylostoma duodenale, Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis). Employing high copy-number, non-coding repeat DNA sequences as targets, novel real-time PCR assays were designed, and assays were tested against established molecular detection methods. Each assay provided consistent detection of genomic DNA at quantities of 2 fg or less, demonstrated species-specificity, and showed an improved limit of detection over the existing, proven PCR-based assay. Conclusions/Significance The utilization of next-generation sequencing-based repeat DNA analysis methodologies for the identification of molecular diagnostic targets has the ability to improve assay species-specificity and limits of detection. By exploiting such high copy-number repeat sequences, the assays described here will facilitate soil transmitted helminth diagnostic efforts. We recommend similar analyses when designing PCR-based diagnostic tests for the detection of other

  11. Information multiplexing in ptychography.

    PubMed

    Batey, Darren J; Claus, Daniel; Rodenburg, John M

    2014-03-01

    We show for the first time that ptychography (a form of lensless diffractive imaging) can recover the spectral response of an object through simultaneous reconstruction of multiple images that represent the object's response to a particular mode present in the illumination. We solve the phase problem for each mode independently, even though the intensity arriving at every detector pixel is an incoherent superposition of several uncorrelated diffracted waves. Until recently, the addition of incoherent modes has been seen as a nuisance in diffractive imaging: here we show that not only can the difficulties they pose be removed, but that they can also be used to discover much more information about the object. If the illumination function is also mode-specific, we show that we can also solve simultaneously for a multiplicity of such illumination modes. The work opens exciting possibilities for information multiplexing in ptychography over all visible, X-ray and electron wavelengths. PMID:24413077

  12. A PCR-based genotyping method to distinguish between wild-type and ornamental varieties of Imperata cylindrica.

    PubMed

    Cseke, Leland J; Talley, Sharon M

    2012-01-01

    viable wind-dispersed seeds that spread cogongrass over wide distances(5-7). JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass(8-10), and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential(3,7,11). The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert. PMID:22370715

  13. Development of three specific PCR-based tools to determine quantity, cellulolytic transcriptional activity and phylogeny of anaerobic fungi.

    PubMed

    Dollhofer, Veronika; Callaghan, Tony Martin; Dorn-In, Samart; Bauer, Johann; Lebuhn, Michael

    2016-08-01

    Anaerobic fungi (AF) decompose plant material with their rhizoid and multiple cellulolytic enzymes. They disintegrate the complex structure of lignocellulosic substrates, making them more accessible and suitable for further microbial degradation. There is also much interest in their use as biocatalysts for biotechnological applications. Here, three novel polymerase chain reaction (PCR)-based methods for detecting AF and their transcriptional activity in in vitro cultures and environmental samples were developed. Two real-time quantitative PCR (qPCR)-based methods targeting AF were developed: AF-SSU, was designed to quantify the 18S rRNA genes of AF. AF-Endo, measuring transcripts of an endoglucanase gene from the glycoside hydrolase family 5 (GH5), was developed to quantify their transcriptional cellulolytic activity. The third PCR based approach was designed for phylogenetical analysis. It targets the 28S rRNA gene (LSU) of AF revealing their phylogenetic affiliation. The in silico-designed primer/probe combinations were successfully tested for the specific amplification of AF from animal and biogas plant derived samples. In combination, these three methods represent useful tools for the analysis of AF transcriptional cellulolytic activity, their abundance and their phylogenetic placement. PMID:27220661

  14. Portable Multiplex Pathogen Detector

    SciTech Connect

    Visuri, S; McBride, M T; Matthews, D; Rao, R

    2002-07-15

    Tumor marker concentrations in serum provide useful information regarding clinical stage and prognosis of cancer and can thus be used for presymptomatic diagnostic purposes. Currently, detection and identification of soluble analytes in biological fluids is conducted by methods including bioassays, ELISA, PCR, DNA chip or strip tests. While these technologies are generally sensitive and specific, they are time consuming, labor intensive and cannot be multiplexed. Our goal is to develop a simple, point-of-care, portable, liquid array-based immunoassay device capable of simultaneous detection of a variety of cancer markers. Here we describe the development of assays for the detection of Serum Prostate Specific Antigen, and Ovalbumin from a single sample. The multiplexed immunoassays utilize polystyrene microbeads. The beads are imbedded with precise ratios of red and orange fluorescent dyes yielding an array of 100 beads, each with a unique spectral address (Figure 1). Each bead can be coated with capture antibodies specific for a given antigen. After antigen capture, secondary antibodies sandwich the bound antigen and are indirectly labeled by the fluorescent reporter phycoerythrin (PE). Each optically encoded and fluorescently-labeled microbead is then individually interrogated. A red laser excites the dye molecules imbedded inside the bead and classifies the bead to its unique bead set, and a green laser quantifies the assay at the bead surface. This technology has been proven to be comparable to the ELISA in terms of sensitivity and specificity. We also describe the laser-based instrumentation used to acquire fluorescent bead images Following the assay, droplets of bead suspension containing a mixture of bead classes were deposited onto filters held in place by a disposable plexiglass device and the resultant arrays viewed under the fluorescent imaging setup. Using the appropriate filter sets to extract the necessary red, orange and green fluorescence from the

  15. High throughput multiplex PCR and probe-based detection with Luminex beads for seven intestinal parasites.

    PubMed

    Taniuchi, Mami; Verweij, Jaco J; Noor, Zannatun; Sobuz, Shihab U; Lieshout, Lisette van; Petri, William A; Haque, Rashidul; Houpt, Eric R

    2011-02-01

    Polymerase chain reaction (PCR) assays for intestinal parasites are increasingly being used on fecal DNA samples for enhanced specificity and sensitivity of detection. Comparison of these tests against microscopy and copro-antigen detection has been favorable, and substitution of PCR-based assays for the ova and parasite stool examination is a foreseeable goal for the near future. One challenge is the diverse list of protozoan and helminth parasites. Several existing real-time PCR assays for the major intestinal parasites-Cryptosporidium spp., Giardia intestinalis, Entamoeba histolytica, Ancylostoma duodenale, Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis-were adapted into a high throughput protocol. The assay involves two multiplex PCR reactions, one with specific primers for the protozoa and one with specific primers for the helminths, after which PCR products are hybridized to beads linked to internal oligonucleotide probes and detected on a Luminex platform. When compared with the parent multiplex real-time PCR assays, this multiplex PCR-bead assay afforded between 83% and 100% sensitivity and specificity on a total of 319 clinical specimens. In conclusion, this multiplex PCR-bead protocol provides a sensitive diagnostic screen for a large panel of intestinal parasites. PMID:21292910

  16. Multiplexer and time duration measuring circuit

    SciTech Connect

    Gray, Jr., James

    1980-01-01

    A multiplexer device is provided for multiplexing data in the form of randomly developed, variable width pulses from a plurality of pulse sources to a master storage. The device includes a first multiplexer unit which includes a plurality of input circuits each coupled to one of the pulse sources, with all input circuits being disabled when one input circuit receives an input pulse so that only one input pulse is multiplexed by the multiplexer unit at any one time.

  17. A 128 Multiplexing Factor Time-Domain SQUID Multiplexer

    NASA Astrophysics Data System (ADS)

    Prêle, D.; Voisin, F.; Piat, M.; Decourcelle, T.; Perbost, C.; Chapron, C.; Rambaud, D.; Maestre, S.; Marty, W.; Montier, L.

    2016-01-01

    A cryogenic 128:1 Time-Domain Multiplexer (TDM) has been developed for the readout of kilo-pixel Transition Edge Sensor (TES) arrays dedicated to the Q&U Bolometric Interferometer for Cosmology (QUBIC) instrument which aims to measure the B-mode polarization of the Cosmic Microwave Background. Superconducting QUantum Interference Devices (SQUIDs) are usually used to read out TESs. Moreover, SQUIDs are used to build TDM by biasing sequentially the SQUIDs connected together—one for each TES. In addition to this common technique which allows a typical 32 multiplexing factor, a cryogenic integrated circuit provides a 4:1 second multiplexing stage. This cryogenic integrated circuit is one of the original part of our TDM achieving an unprecedented 128 multiplexing factor. We present these two dimension TDM stages: topology of the SQUID multiplexer, operation of the cryogenic integrated circuit, and integration of the full system to read out a TES array dedicated to the QUBIC instrument. Flux-locked loop operation in multiplexed mode is also discussed.

  18. A 128 Multiplexing Factor Time-Domain SQUID Multiplexer

    NASA Astrophysics Data System (ADS)

    Prêle, D.; Voisin, F.; Piat, M.; Decourcelle, T.; Perbost, C.; Chapron, C.; Rambaud, D.; Maestre, S.; Marty, W.; Montier, L.

    2016-07-01

    A cryogenic 128:1 Time-Domain Multiplexer (TDM) has been developed for the readout of kilo-pixel Transition Edge Sensor (TES) arrays dedicated to the Q&U Bolometric Interferometer for Cosmology (QUBIC) instrument which aims to measure the B-mode polarization of the Cosmic Microwave Background. Superconducting QUantum Interference Devices (SQUIDs) are usually used to read out TESs. Moreover, SQUIDs are used to build TDM by biasing sequentially the SQUIDs connected together—one for each TES. In addition to this common technique which allows a typical 32 multiplexing factor, a cryogenic integrated circuit provides a 4:1 second multiplexing stage. This cryogenic integrated circuit is one of the original part of our TDM achieving an unprecedented 128 multiplexing factor. We present these two dimension TDM stages: topology of the SQUID multiplexer, operation of the cryogenic integrated circuit, and integration of the full system to read out a TES array dedicated to the QUBIC instrument. Flux-locked loop operation in multiplexed mode is also discussed.

  19. MPprimer: a program for reliable multiplex PCR primer design

    PubMed Central

    2010-01-01

    Background Multiplex PCR, defined as the simultaneous amplification of multiple regions of a DNA template or multiple DNA templates using more than one primer set (comprising a forward primer and a reverse primer) in one tube, has been widely used in diagnostic applications of clinical and environmental microbiology studies. However, primer design for multiplex PCR is still a challenging problem and several factors need to be considered. These problems include mis-priming due to nonspecific binding to non-target DNA templates, primer dimerization, and the inability to separate and purify DNA amplicons with similar electrophoretic mobility. Results A program named MPprimer was developed to help users for reliable multiplex PCR primer design. It employs the widely used primer design program Primer3 and the primer specificity evaluation program MFEprimer to design and evaluate the candidate primers based on genomic or transcript DNA database, followed by careful examination to avoid primer dimerization. The graph-expanding algorithm derived from the greedy algorithm was used to determine the optimal primer set combinations (PSCs) for multiplex PCR assay. In addition, MPprimer provides a virtual electrophotogram to help users choose the best PSC. The experimental validation from 2× to 5× plex PCR demonstrates the reliability of MPprimer. As another example, MPprimer is able to design the multiplex PCR primers for DMD (dystrophin gene which caused Duchenne Muscular Dystrophy), which has 79 exons, for 20×, 20×, 20×, 14×, and 5× plex PCR reactions in five tubes to detect underlying exon deletions. Conclusions MPprimer is a valuable tool for designing specific, non-dimerizing primer set combinations with constrained amplicons size for multiplex PCR assays. PMID:20298595

  20. ROVER variant caller: read-pair overlap considerate variant-calling software applied to PCR-based massively parallel sequencing datasets

    PubMed Central

    2014-01-01

    Background We recently described Hi-Plex, a highly multiplexed PCR-based target-enrichment system for massively parallel sequencing (MPS), which allows the uniform definition of library size so that subsequent paired-end sequencing can achieve complete overlap of read pairs. Variant calling from Hi-Plex-derived datasets can thus rely on the identification of variants appearing in both reads of read-pairs, permitting stringent filtering of sequencing chemistry-induced errors. These principles underly ROVER software (derived from Read Overlap PCR-MPS variant caller), which we have recently used to report the screening for genetic mutations in the breast cancer predisposition gene PALB2. Here, we describe the algorithms underlying ROVER and its usage. Results ROVER enables users to quickly and accurately identify genetic variants from PCR-targeted, overlapping paired-end MPS datasets. The open-source availability of the software and threshold tailorability enables broad access for a range of PCR-MPS users. Methods ROVER is implemented in Python and runs on all popular POSIX-like operating systems (Linux, OS X). The software accepts a tab-delimited text file listing the coordinates of the target-specific primers used for targeted enrichment based on a specified genome-build. It also accepts aligned sequence files resulting from mapping to the same genome-build. ROVER identifies the amplicon a given read-pair represents and removes the primer sequences by using the mapping co-ordinates and primer co-ordinates. It considers overlapping read-pairs with respect to primer-intervening sequence. Only when a variant is observed in both reads of a read-pair does the signal contribute to a tally of read-pairs containing or not containing the variant. A user-defined threshold informs the minimum number of, and proportion of, read-pairs a variant must be observed in for a ‘call’ to be made. ROVER also reports the depth of coverage across amplicons to facilitate the

  1. Clinical performance of multiplex high-risk e6 mrna expression in comparison with hpv dna subtypes for the identification of women at risk of cervical cancer.

    PubMed

    Ho, Chih-Ming; Pan, Kui-You; Chen, Yun-Yuan; Huang, Chia-Yen; Chen, Yu-Li; Chang, Shwu-Fen

    2015-08-01

    We compared multiplex E6 messenger ribonucleic acid (mRNA) tests using real-time quantitative reverse transcriptase polymerase chain reactions (PCR) with human papillomavirus (HPV) DNA subtypes using a MY11/GP6+ PCR-based reverse-blot assay to identify cervical intraepithelial neoplasias of grade 2 or worse (CIN2+). In total, 684 women were studied, of whom 377 (55%) were diagnosed with CIN2+ histologically. The specificity of HPV mRNA to predict histological CIN2+ was higher than that of HPV DNA (81.3% vs. 44.2%). The odds ratios (ORs) to predict histological CIN2+ in women with positive for type 16, 18, 31, and 45 E6 mRNA or by HPV DNA detection were 7.1 (95% confidence interval [CI] 3.9-13.1) and 2.5 (95%CI 1.9-3.5), respectively, compared to those with negative for E6 mRNA or HPV DNA. The OR to predict histological CIN2+ in women with a cytological grade multiplex HPV E6 mRNA detection can be used as a triage for women with cytological grade

  2. A multiplex nested PCR for the detection and identification of Candida species in blood samples of critically ill paediatric patients

    PubMed Central

    2014-01-01

    Background Nosocomial candidaemia is associated with high mortality rates in critically ill paediatric patients; thus, the early detection and identification of the infectious agent is crucial for successful medical intervention. The PCR-based techniques have significantly increased the detection of Candida species in bloodstream infections. In this study, a multiplex nested PCR approach was developed for candidaemia detection in neonatal and paediatric intensive care patients. Methods DNA samples from the blood of 54 neonates and children hospitalised in intensive care units with suspected candidaemia were evaluated by multiplex nested PCR with specific primers designed to identify seven Candida species, and the results were compared with those obtained from blood cultures. Results The multiplex nested PCR had a detection limit of four Candida genomes/mL of blood for all Candida species. Blood cultures were positive in 14.8% of patients, whereas the multiplex nested PCR was positive in 24.0% of patients, including all culture-positive patients. The results obtained with the molecular technique were available within 24 hours, and the assay was able to identify Candida species with 100% of concordance with blood cultures. Additionally, the multiplex nested PCR detected dual candidaemia in three patients. Conclusions Our proposed PCR method may represent an effective tool for the detection and identification of Candida species in the context of candidaemia diagnosis in children, showing highly sensitive detection and the ability to identify the major species involved in this infection. PMID:25047415

  3. Development of the 19 X-STR loci multiplex system and genetic analysis of a Zhejiang Han population in China.

    PubMed

    Yang, XingYi; Wu, WeiWei; Chen, LinLi; Liu, ChangHui; Zhang, XiaoFang; Chen, Ling; Feng, XingLin; Wang, HuiJun; Liu, Chao

    2016-08-01

    The 19 X-STRs multiplex system is a PCR-based amplification kit that facilitates simultaneous amplification of 19 X-chromosomal STR loci (i.e. DXS7423, DXS10148, DXS10159, DXS6809, DXS7424, DXS8378, DXS10164, DXS10162, DXS7132, DXS10079, DXS6789, DXS101, DXS10103,DXS10101, HPTRB, DXS10075, DXS10074, DXS10135, and DXS10134). Eleven loci were extensively used in an Investigator Qiagen Argus X-12 (DXS7423, DXS10148, DXS8378, DXS10162, DXS7132, DXS10079, DXS10103, DXS10101, HPTRB, DXS10074, and DXS10135). In this research, the multiplex system was tested for detection sensitivity, DNA mixtures, inhibitor tolerance and species specificity; SWGDAM Validation Guidelines - Approved December 2012 were followed for the human fluorescent STR multiplex PCR reagent. Samples from 181 unrelated Zhejiang Han individuals (121 males and 60 females) were typed using this multiplex system. The results show that this 19X-STRs multiplex system is a robust and reliable amplification means to facilitate forensic and human identification testing. PMID:27184937

  4. Comparison of cell-based and PCR-based assays as methods for measuring infectivity of Tulane virus.

    PubMed

    Shan, Lei; Yang, David; Wang, Dapeng; Tian, Peng

    2016-05-01

    In this study, we used Tulane virus (TV) as a surrogate for HuNoV to evaluate for correlation between two cell-based assays and three PCR-based assays. Specifically, the cell-based plaque and TCID50 assays measure for infectious virus particles, while the PCR-based RNase exposure, porcine gastric mucin in-situ-capture qRT-PCR (PGM-ISC-qRT-PCR), and antibody in-situ-capture qRT-PCR (Ab-ISC-qRT-PCR) assays measure for an amplicon within encapsidated viral genome. Ten batches of viral stocks ranging from 3.41 × 10(5) to 6.67 × 10(6) plaque forming units (PFUs) were used for side by side comparison with PFU as a reference. The results indicate that one PFU was equivalent to 6.69 ± 2.34 TCID50 units, 9.75 ± 10.87 RNase-untreated genomic copies (GCs), 2.87 ± 3.05 RNase-treated GCs, 0.07 ± 0.07 PGM-ISC-qRT-PCR GCs, and 0.52 ± 0.39 Ab-ISC-qRT-PCR GCs. We observed that while the cell-based assays were consistent with each other, the TCID50 assay was more sensitive than the plaque assay. In contrast, the PCR-based assays were not always consistent with the cell-based assays. The very high variations in GCs as measured by both ISC-RT-qPCR assays made them difficult to correlate against the relatively small variations (<20-fold) in the PFUs or TCID50 units as measured by the cell-based assays. PMID:26875997

  5. European validation of a real-time PCR-based method for detection of Listeria monocytogenes in soft cheese.

    PubMed

    Gianfranceschi, Monica Virginia; Rodriguez-Lazaro, David; Hernandez, Marta; González-García, Patricia; Comin, Damiano; Gattuso, Antonietta; Delibato, Elisabetta; Sonnessa, Michele; Pasquali, Frederique; Prencipe, Vincenza; Sreter-Lancz, Zuzsanna; Saiz-Abajo, María-José; Pérez-De-Juan, Javier; Butrón, Javier; Kozačinski, Lidija; Tomic, Danijela Horvatek; Zdolec, Nevijo; Johannessen, Gro S; Jakočiūnė, Džiuginta; Olsen, John Elmerdahl; De Santis, Paola; Lovari, Sarah; Bertasi, Barbara; Pavoni, Enrico; Paiusco, Antonella; De Cesare, Alessandra; Manfreda, Gerardo; De Medici, Dario

    2014-08-01

    The classical microbiological method for detection of Listeria monocytogenes requires around 7 days for final confirmation, and due to perishable nature of RTE food products, there is a clear need for an alternative methodology for detection of this pathogen. This study presents an international (at European level) ISO 16140-based validation trial of a non-proprietary real-time PCR-based methodology that can generate final results in the following day of the analysis. This methodology is based on an ISO compatible enrichment coupled to a bacterial DNA extraction and a consolidated real-time PCR assay. Twelve laboratories from six European countries participated in this trial, and soft cheese was selected as food model since it can represent a difficult matrix for the bacterial DNA extraction and real-time PCR amplification. The limit of detection observed was down to 10 CFU per 25 of sample, showing excellent concordance and accordance values between samples and laboratories (>75%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (82.75%, 96.70% and 97.62%, respectively) when the results obtained for the real-time PCR-based methods were compared to those of the ISO 11290-1 standard method. An interesting observation was that the L. monocytogenes detection by the real-time PCR method was less affected in the presence of Listeria innocua in the contaminated samples, proving therefore to be more reliable than the reference method. The results of this international trial demonstrate that the evaluated real-time PCR-based method represents an excellent alterative to the ISO standard since it shows a higher performance as well as reduce the extent of the analytical process, and can be easily implemented routinely by the competent authorities and food industry laboratories. PMID:24468028

  6. COMPARISON BETWEEN AUTOMATED SYSTEM AND PCR-BASED METHOD FOR IDENTIFICATION AND ANTIMICROBIAL SUSCEPTIBILITY PROFILE OF CLINICAL Enterococcus spp

    PubMed Central

    Furlaneto-Maia, Luciana; Rocha, Kátia Real; Siqueira, Vera Lúcia Dias; Furlaneto, Márcia Cristina

    2014-01-01

    Enterococci are increasingly responsible for nosocomial infections worldwide. This study was undertaken to compare the identification and susceptibility profile using an automated MicrosScan system, PCR-based assay and disk diffusion assay of Enterococcus spp. We evaluated 30 clinical isolates of Enterococcus spp. Isolates were identified by MicrosScan system and PCR-based assay. The detection of antibiotic resistance genes (vancomycin, gentamicin, tetracycline and erythromycin) was also determined by PCR. Antimicrobial susceptibilities to vancomycin (30 µg), gentamicin (120 µg), tetracycline (30 µg) and erythromycin (15 µg) were tested by the automated system and disk diffusion method, and were interpreted according to the criteria recommended in CLSI guidelines. Concerning Enterococcus identification the general agreement between data obtained by the PCR method and by the automatic system was 90.0% (27/30). For all isolates of E. faecium and E. faecalis we observed 100% agreement. Resistance frequencies were higher in E. faecium than E. faecalis. The resistance rates obtained were higher for erythromycin (86.7%), vancomycin (80.0%), tetracycline (43.35) and gentamicin (33.3%). The correlation between disk diffusion and automation revealed an agreement for the majority of the antibiotics with category agreement rates of > 80%. The PCR-based assay, the van(A) gene was detected in 100% of vancomycin resistant enterococci. This assay is simple to conduct and reliable in the identification of clinically relevant enterococci. The data obtained reinforced the need for an improvement of the automated system to identify some enterococci. PMID:24626409

  7. Efficient exploration of multiplex networks

    NASA Astrophysics Data System (ADS)

    Battiston, Federico; Nicosia, Vincenzo; Latora, Vito

    2016-04-01

    Efficient techniques to navigate networks with local information are fundamental to sample large-scale online social systems and to retrieve resources in peer-to-peer systems. Biased random walks, i.e. walks whose motion is biased on properties of neighbouring nodes, have been largely exploited to design smart local strategies to explore a network, for instance by constructing maximally mixing trajectories or by allowing an almost uniform sampling of the nodes. Here we introduce and study biased random walks on multiplex networks, graphs where the nodes are related through different types of links organised in distinct and interacting layers, and we provide analytical solutions for their long-time properties, including the stationary occupation probability distribution and the entropy rate. We focus on degree-biased random walks and distinguish between two classes of walks, namely those whose transition probability depends on a number of parameters which is extensive in the number of layers, and those whose motion depends on intrinsically multiplex properties of the neighbouring nodes. We analyse the effect of the structure of the multiplex network on the steady-state behaviour of the walkers, and we find that heterogeneous degree distributions as well as the presence of inter-layer degree correlations and edge overlap determine the extent to which a multiplex can be efficiently explored by a biased walk. Finally we show that, in real-world multiplex transportation networks, the trade-off between efficient navigation and resilience to link failure has resulted into systems whose diffusion properties are qualitatively different from those of appropriately randomised multiplex graphs. This fact suggests that multiplexity is an important ingredient to include in the modelling of real-world systems.

  8. Evaluation of a novel PCR-based diagnostic assay for detection of Mycobacterium tuberculosis in sputum samples.

    PubMed Central

    Maher, M; Glennon, M; Martinazzo, G; Turchetti, E; Marcolini, S; Smith, T; Dawson, M T

    1996-01-01

    We report on a PCR-based assay we have developed for the detection of Mycobacterium tuberculosis in sputum samples. One hundred sputum specimens, which included 34 culture-positive and 66 culture-negative specimens, were evaluated with this system. Of the 34 culture-positive specimens, 31 were PCR positive, and 60 of the culture-negative specimens were PCR negative. An internal standard has been included in the assay system to monitor PCR inhibition and to confirm the reliability of the PCR assay. PMID:8862607

  9. Long-PCR based next generation sequencing of the whole mitochondrial genome of the peacock skate Pavoraja nitida (Elasmobranchii: Arhynchobatidae).

    PubMed

    Yang, Lei; Naylor, Gavin J P

    2016-01-01

    We determined the complete mitochondrial genome sequence (16,760 bp) of the peacock skate Pavoraja nitida using a long-PCR based next generation sequencing method. It has 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control region in the typical vertebrate arrangement. Primers, protocols, and procedures used to obtain this mitogenome are provided. We anticipate that this approach will facilitate rapid collection of mitogenome sequences for studies on phylogenetic relationships, population genetics, and conservation of cartilaginous fishes. PMID:24938110

  10. Evaluation of independence assumptions for PCR-based and protein-based genetic markers in New Jersey Caucasians.

    PubMed

    Budowle, B; Jankowski, L B; Corey, H W; Swec, N T; Freck-Tootell, S; Pino, J A; Schwartz, R; Kelley, C A; Tarver, M L

    1997-03-01

    Allele frequencies for six PCR-based loci and three protein-based (i.e., enzyme systems) loci were determined in a Caucasian sample population from New Jersey. The loci are LDLR, GYPA, HBGG, D7S8, Gc, HLA-DQA1, PGM1, ESD, and EAP. All loci meet Hardy-Weinberg expectations. In addition, there is little evidence for association of alleles among the nine loci. The allelic frequency data generally are similar to another Caucasian population database. PMID:9068180

  11. Survey for protozoan parasites in Eastern oysters (Crassostrea virginica) from the Gulf of Maine using PCR-based assays.

    PubMed

    Marquis, Nicholas D; Record, Nicholas R; Robledo, José A Fernández

    2015-10-01

    Protozoan pathogens represent a serious threat to oyster aquaculture, since they can lead to significant production loses. Moreover, oysters can concentrate human pathogens through filter feeding, thus putting at risk raw oyster consumers' health. Using PCR-based assays in oysters (Crassostrea virginica) from Maine, we expand the Northeast range in the USA for the protozoans Perkinsus marinus, Perkinsus chesapeaki, and Haplosporidium nelsoni, and report for the first time the detection of the human pathogens Toxoplasma gondii and Cryptosporidium parvum. Oysters hosting both P. marinus and P. chesapeaki were more than three times as likely to be infected by a non-Perkinsus than those free of Perkinsus infections. PMID:25889457

  12. Thermally multiplexed polymerase chain reaction

    PubMed Central

    Phaneuf, Christopher R.; Pak, Nikita; Saunders, D. Curtis; Holst, Gregory L.; Birjiniuk, Joav; Nagpal, Nikita; Culpepper, Stephen; Popler, Emily; Shane, Andi L.; Jerris, Robert; Forest, Craig R.

    2015-01-01

    Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously—each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel. PMID:26339317

  13. Structural measures for multiplex networks

    NASA Astrophysics Data System (ADS)

    Battiston, Federico; Nicosia, Vincenzo; Latora, Vito

    2014-03-01

    Many real-world complex systems consist of a set of elementary units connected by relationships of different kinds. All such systems are better described in terms of multiplex networks, where the links at each layer represent a different type of interaction between the same set of nodes rather than in terms of (single-layer) networks. In this paper we present a general framework to describe and study multiplex networks, whose links are either unweighted or weighted. In particular, we propose a series of measures to characterize the multiplexicity of the systems in terms of (i) basic node and link properties such as the node degree, and the edge overlap and reinforcement, (ii) local properties such as the clustering coefficient and the transitivity, and (iii) global properties related to the navigability of the multiplex across the different layers. The measures we introduce are validated on a genuinely multiplex data set of Indonesian terrorists, where information among 78 individuals are recorded with respect to mutual trust, common operations, exchanged communications, and business relationships.

  14. Structural measures for multiplex networks.

    PubMed

    Battiston, Federico; Nicosia, Vincenzo; Latora, Vito

    2014-03-01

    Many real-world complex systems consist of a set of elementary units connected by relationships of different kinds. All such systems are better described in terms of multiplex networks, where the links at each layer represent a different type of interaction between the same set of nodes rather than in terms of (single-layer) networks. In this paper we present a general framework to describe and study multiplex networks, whose links are either unweighted or weighted. In particular, we propose a series of measures to characterize the multiplexicity of the systems in terms of (i) basic node and link properties such as the node degree, and the edge overlap and reinforcement, (ii) local properties such as the clustering coefficient and the transitivity, and (iii) global properties related to the navigability of the multiplex across the different layers. The measures we introduce are validated on a genuinely multiplex data set of Indonesian terrorists, where information among 78 individuals are recorded with respect to mutual trust, common operations, exchanged communications, and business relationships. PMID:24730896

  15. Bond Percolation on Multiplex Networks

    NASA Astrophysics Data System (ADS)

    Hackett, A.; Cellai, D.; Gómez, S.; Arenas, A.; Gleeson, J. P.

    2016-04-01

    We present an analytical approach for bond percolation on multiplex networks and use it to determine the expected size of the giant connected component and the value of the critical bond occupation probability in these networks. We advocate the relevance of these tools to the modeling of multilayer robustness and contribute to the debate on whether any benefit is to be yielded from studying a full multiplex structure as opposed to its monoplex projection, especially in the seemingly irrelevant case of a bond occupation probability that does not depend on the layer. Although we find that in many cases the predictions of our theory for multiplex networks coincide with previously derived results for monoplex networks, we also uncover the remarkable result that for a certain class of multiplex networks, well described by our theory, new critical phenomena occur as multiple percolation phase transitions are present. We provide an instance of this phenomenon in a multiplex network constructed from London rail and European air transportation data sets.

  16. Survey and RT-PCR Based Detection of Cardamom mosaic virus Affecting Small Cardamom in India.

    PubMed

    Biju, C N; Siljo, A; Bhat, A I

    2010-10-01

    Mosaic or marble or katte disease caused by Cardamom mosaic virus (CdMV) is an important production constraint in all cardamom growing regions of the world. In the present study, 84 cardamom plantations in 44 locations of Karnataka and Kerala were surveyed. The incidence of the disease ranged from 0 to 85%. The incidence was highest in Madikeri (Karnataka) while no incidence was recorded in Peermade (Kerala). In general, incidence and severity of the disease was higher in cardamom plantations of Karnataka. A procedure for total RNA isolation from cardamom and detection of CdMV through reverse transcription-polymerase chain reaction (RT-PCR) using primers targeting the conserved region of coat protein was standardized and subsequently validated by testing more than 50 field cardamom samples originating from Karnataka and Kerala states. The method can be used for indexing the planting material and identifying resistant lines/cultivars before either they are further multiplied in large scale or incorporated in breeding. PMID:23637495

  17. A PCR based method for the identification of equine influenza virus from clinical samples.

    PubMed

    Oxburgh, L; Hagström, A

    1999-06-30

    In this paper we describe the development of a nested RT-PCR assay for the rapid diagnosis and characterisation of influenza virus directly from clinical specimens. Viral RNA is extracted from nasal swabs by the guanidine thiocyanate extraction method, and subsequently reverse transcribed. The complementary DNA is then used as template in a nested PCR reaction. Primers designed for use in this assay are specific for three templates; (1) the nucleoprotein (NP) gene, (2) the haemagglutinin gene of the H7N7 equine influenza virus (A1), and (3) the haemagglutinin gene of the H3N8 equine influenza virus (A2). We show that the assays are specific for the target genes chosen, and display sensitivity similar to virus isolation. The NP assay detects a variety of different influenza subtypes, whereas A1 and A2 assays are specific for influenza subtypes H7N7 and H3N8, respectively. Sequencing of amplicons obtained in the A2 assay yields information on antigenic regions of the haemagglutinin molecule, and use of this procedure in the routine surveillance of equine influenza will enable tentative characterisation of circulating viruses despite difficulties in isolating field strains of the H3N8 subtype. The A1 assay will be useful in ascertaining whether viruses of the H7N7 subtype still circulate amongst horses, or whether these are extinct. PMID:10418871

  18. Two RT-PCR based assays to detect human metapneumovirus in nasopharyngeal aspirates.

    PubMed

    López-Huertas, María Rosa; Casas, Inmaculada; Acosta-Herrera, Belsy; García, María Luz; Coiras, María Teresa; Pérez-Breña, Pilar

    2005-10-01

    Two sensitive and specific RT-PCR assays were standardised for testing the presence of human metapneumovirus. A total of 300 nasopharyngeal aspirates collected from infants suffering from bronchiolitis since October 2000 to June 2003 and shown previously as negative to common respiratory viruses were examined. Matrix and polymerase viral genes, which show a low rate of variation, were chosen to design amplification assays to ensure that any genotype of the human metapneumovirus could be detected. A RT-PCR followed by a reverse line blotting hybridisation was developed for viral polymerase gene. For the matrix gene, after the RT-PCR assay, a subsequent nested PCR was carried out. Both assays had similar sensitivity, equivalent to 0.1 TCID50 of human metapneumovirus strain NL/1/99 which was used as the positive control. The human metapneumovirus was present in 16.6% of the specimens studied. The approaches described below are not only a robust method for rapid diagnosis of the human metapneumovirus, but also to establish an etiological surveillance tool for epidemiological studies. Based on the results obtained, human metapneumovirus infections in Madrid followed a seasonal pattern, with most of the infections occurring between February and April. PMID:15961167

  19. Evaluation of a Modular Multiplex-PCR Methicillin-Resistant Staphylococcus aureus Detection Assay Adapted for mecC Detection

    PubMed Central

    Larsen, Anders R.; Skov, Robert L.; Paterson, Gavin K.; Holmes, Mark A.; Sabat, Artur J.; Friedrich, Alexander W.; Köck, Robin; Peters, Georg; Kriegeskorte, André

    2013-01-01

    A mecC (mecALGA251)-adapted multiplex PCR-based methicillin-resistant Staphylococcus aureus (MRSA) detection assay was evaluated using an international, spa-typed Staphylococcus aureus collection comprising 51 mecC-positive MRSA, 240 mecA-positive MRSA, and 50 mecA- and mecC-negative methicillin-susceptible S. aureus (MSSA) isolates. The assay showed 100% sensitivity and specificity for S. aureus species identification as well as for mecA and mecC detection. PMID:23515551

  20. Universal multiplexable matK primers for DNA barcoding of angiosperms1

    PubMed Central

    Heckenhauer, Jacqueline; Barfuss, Michael H. J.; Samuel, Rosabelle

    2016-01-01

    Premise of the study: PCR amplification of the matK barcoding region is often difficult when dealing with multiple angiosperm families. We developed a primer cocktail to amplify this region efficiently across angiosperm diversity. Methods and Results: We developed 14 matK primers (seven forward, seven reverse) for multiplex PCR, using sequences available in GenBank for 178 taxa belonging to 123 genera in 41 families and 18 orders. Universality of these new multiplexed primers was tested with 53 specimens from 44 representative angiosperm families in 23 different orders. Our primers showed high PCR amplification and sequencing success. Conclusions: These results show that our newly developed primers are highly effective for multiplex PCR and can be employed in future barcode projects involving taxonomically diverse samples across angiosperms. Using multiplex primers for barcoding will reduce the cost and time needed for PCR amplification. PMID:27347449

  1. Real-time PCR based analysis of metal resistance genes in metal resistant Pseudomonas aeruginosa strain J007.

    PubMed

    Choudhary, Sangeeta; Sar, Pinaki

    2016-07-01

    A uranium (U)-resistant and -accumulating Pseudomonas aeruginosa strain was characterized to assess the response of toxic metals toward its growth and expression of metal resistance determinants. The bacterium showed MIC (minimum inhibitory concentration) values of 6, 3, and 2 mM for Zn, Cu, and Cd, respectively; with resistance phenotype conferred by periplasmic Cu sequestering copA and RND type heavy metal efflux czcA genes. Real-time PCR-based expression analysis revealed significant upregulation of both these genes upon exposure to low concentrations of metals for short duration, whereas the global stress response gene sodA encoding superoxide dismutase enzyme was upregulated only at higher metal concentrations or longer exposure time. It could also be inferred that copA and czcA are involved in providing resistance only at low metal concentrations, whereas involvement of "global stress response" phenomenon (expression of sodA) at higher metal concentration or increased exposure was evident. This study provides significant understanding of the adaptive response of bacteria surviving in metal and radionuclide contaminated environments along with the development of real-time PCR-based quantification method of using metal resistance genes as biomarker for monitoring relevant bacteria in such habitats. PMID:26662317

  2. PCR-based method for sex identification of Eastern sarus crane (Grus antigone sharpii): implications for reintroduction programs in Thailand.

    PubMed

    Insee, Jiranan; Kamolnorranath, Sumate; Baicharoen, Sudarat; Chumpadang, Sriphapai; Sawasu, Wanchai; Wajjwalku, Worawidh

    2014-02-01

    Due to human activity and a reduction in the size and quality of wetland habitats, populations of the Eastern sarus crane (Grus antigone sharpii) have declined dramatically across their range in Southeast Asia. Conservation efforts in Thailand have focused on reintroduction of the founders harboring the highest genetic diversity. One of the most important requirements to ensure the persistence of the reintroduced populations is a balanced sex ratio. In this study we tested three simple PCR-based methods which may be used for reliable sex identification in G. a. sharpii. The first method employs two combined primer sets based on a 0.6 kb EcoRI fragment (EE0.6). The second method is based on the intronic length polymorphism of the chromo-helicase DNA binding protein (CHD). The last technique relies on PCR-RFLP technique. The sex of six known and 24 unknown cranes were successfully identified by all three methods. These PCR-based sex identification methods are also useful for captive breeding management of G. a. sharpii. PMID:24521319

  3. Multiplex quantification of 12 European Union authorized genetically modified maize lines with droplet digital polymerase chain reaction.

    PubMed

    Dobnik, David; Spilsberg, Bjørn; Bogožalec Košir, Alexandra; Holst-Jensen, Arne; Žel, Jana

    2015-08-18

    Presence of genetically modified organisms (GMO) in food and feed products is regulated in many countries. The European Union (EU) has implemented a threshold for labeling of products containing more than 0.9% of authorized GMOs per ingredient. As the number of GMOs has increased over time, standard-curve based simplex quantitative polymerase chain reaction (qPCR) analyses are no longer sufficiently cost-effective, despite widespread use of initial PCR based screenings. Newly developed GMO detection methods, also multiplex methods, are mostly focused on screening and detection but not quantification. On the basis of droplet digital PCR (ddPCR) technology, multiplex assays for quantification of all 12 EU authorized GM maize lines (per April first 2015) were developed. Because of high sequence similarity of some of the 12 GM targets, two separate multiplex assays were needed. In both assays (4-plex and 10-plex), the transgenes were labeled with one fluorescence reporter and the endogene with another (GMO concentration = transgene/endogene ratio). It was shown that both multiplex assays produce specific results and that performance parameters such as limit of quantification, repeatability, and trueness comply with international recommendations for GMO quantification methods. Moreover, for samples containing GMOs, the throughput and cost-effectiveness is significantly improved compared to qPCR. Thus, it was concluded that the multiplex ddPCR assays could be applied for routine quantification of 12 EU authorized GM maize lines. In case of new authorizations, the events can easily be added to the existing multiplex assays. The presented principle of quantitative multiplexing can be applied to any other domain. PMID:26169291

  4. Helicity multiplexed broadband metasurface holograms

    PubMed Central

    Wen, Dandan; Yue, Fuyong; Li, Guixin; Zheng, Guoxing; Chan, Kinlong; Chen, Shumei; Chen, Ming; Li, King Fai; Wong, Polis Wing Han; Cheah, Kok Wai; Yue Bun Pun, Edwin; Zhang, Shuang; Chen, Xianzhong

    2015-01-01

    Metasurfaces are engineered interfaces that contain a thin layer of plasmonic or dielectric nanostructures capable of manipulating light in a desirable manner. Advances in metasurfaces have led to various practical applications ranging from lensing to holography. Metasurface holograms that can be switched by the polarization state of incident light have been demonstrated for achieving polarization multiplexed functionalities. However, practical application of these devices has been limited by their capability for achieving high efficiency and high image quality. Here we experimentally demonstrate a helicity multiplexed metasurface hologram with high efficiency and good image fidelity over a broad range of frequencies. The metasurface hologram features the combination of two sets of hologram patterns operating with opposite incident helicities. Two symmetrically distributed off-axis images are interchangeable by controlling the helicity of the input light. The demonstrated helicity multiplexed metasurface hologram with its high performance opens avenues for future applications with functionality switchable optical devices. PMID:26354497

  5. Helicity multiplexed broadband metasurface holograms.

    PubMed

    Wen, Dandan; Yue, Fuyong; Li, Guixin; Zheng, Guoxing; Chan, Kinlong; Chen, Shumei; Chen, Ming; Li, King Fai; Wong, Polis Wing Han; Cheah, Kok Wai; Pun, Edwin Yue Bun; Zhang, Shuang; Chen, Xianzhong

    2015-01-01

    Metasurfaces are engineered interfaces that contain a thin layer of plasmonic or dielectric nanostructures capable of manipulating light in a desirable manner. Advances in metasurfaces have led to various practical applications ranging from lensing to holography. Metasurface holograms that can be switched by the polarization state of incident light have been demonstrated for achieving polarization multiplexed functionalities. However, practical application of these devices has been limited by their capability for achieving high efficiency and high image quality. Here we experimentally demonstrate a helicity multiplexed metasurface hologram with high efficiency and good image fidelity over a broad range of frequencies. The metasurface hologram features the combination of two sets of hologram patterns operating with opposite incident helicities. Two symmetrically distributed off-axis images are interchangeable by controlling the helicity of the input light. The demonstrated helicity multiplexed metasurface hologram with its high performance opens avenues for future applications with functionality switchable optical devices. PMID:26354497

  6. Development of a PCR-Based Line Probe Assay for Identification of Fungal Pathogens

    PubMed Central

    Martin, Cara; Roberts, David; van der Weide, Marjo; Rossau, Rudi; Jannes, Geert; Smith, Terry; Maher, Majella

    2000-01-01

    We report on a reverse-hybridization line probe assay (LiPA) which when combined with PCR amplification detects and identifies clinically significant fungal pathogens including Candida, Aspergillus, and Cryptococcus species. DNA probes have been designed from the internal transcribed-spacer (ITS) regions of Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida krusei, Candida dubliniensis, Cryptococcus neoformans, Aspergillus fumigatus, Aspergillus versicolor, Aspergillus nidulans and Aspergillus flavus. The probes were incorporated into a LiPA for detection of biotinylated ITS PCR products, and the specificity of the probes was evaluated. We established LiPA detection limits for ITS 1 and for full ITS amplicons for genomic DNA from C. albicans, A. fumigatus, and C. neoformans. Further evaluation of the LiPA was carried out on clinical fungal isolates. One hundred twenty-seven isolates consisting of dimorphic yeasts and dermatophytic and filamentous fungi were tested by the LiPA, which correctly identified 77 dimorphic yeasts and 23 of the filamentous isolates; the remaining 27 isolates represented species of fungi for which probes were not included in the LiPA. The fungal-PCR-LiPA technology was applied to blood samples inoculated with Candida cells which were pretreated by minibead beating to mechanically disrupt the cells, with the DNA extracted by either a previously described guanidium thiocyanate-silica method or the commercially available QIAmp tissue kit. PCR amplification of the extracted DNA and subsequent DNA probe hybridization in the LiPA assay yielded detection limits of 2 to 10 cells/ml. An internal standard control was included in the PCR amplification to monitor for PCR inhibition. This fungal PCR-LiPA assay is robust and sensitive and can easily be integrated into a clinical-testing laboratory with the potential for same-day diagnosis of fungal infection. PMID:11015393

  7. Integrated multiplexed capillary electrophoresis system

    SciTech Connect

    Yeung, Edward S.; Tan, Hongdong

    2002-05-14

    The present invention provides an integrated multiplexed capillary electrophoresis system for the analysis of sample analytes. The system integrates and automates multiple components, such as chromatographic columns and separation capillaries, and further provides a detector for the detection of analytes eluting from the separation capillaries. The system employs multiplexed freeze/thaw valves to manage fluid flow and sample movement. The system is computer controlled and is capable of processing samples through reaction, purification, denaturation, pre-concentration, injection, separation and detection in parallel fashion. Methods employing the system of the invention are also provided.

  8. Turing patterns in multiplex networks

    NASA Astrophysics Data System (ADS)

    Asllani, Malbor; Busiello, Daniel M.; Carletti, Timoteo; Fanelli, Duccio; Planchon, Gwendoline

    2014-10-01

    The theory of patterns formation for a reaction-diffusion system defined on a multiplex is developed by means of a perturbative approach. The interlayer diffusion constants act as a small parameter in the expansion and the unperturbed state coincides with the limiting setting where the multiplex layers are decoupled. The interaction between adjacent layers can seed the instability of a homogeneous fixed point, yielding self-organized patterns which are instead impeded in the limit of decoupled layers. Patterns on individual layers can also fade away due to cross-talking between layers. Analytical results are compared to direct simulations.

  9. On-chip multiplexing conversion between wavelength division multiplexing-polarization division multiplexing and wavelength division multiplexing-mode division multiplexing.

    PubMed

    Ye, Mengyuan; Yu, Yu; Zou, Jinghui; Yang, Weili; Zhang, Xinliang

    2014-02-15

    A compact silicon-on-insulator device used for conversions between polarization division multiplexing (PDM) and mode division multiplexing (MDM) signals is proposed and experimentally demonstrated by utilizing a structure combining the improved two-dimensional grating coupler and two-mode multiplexer. The detailed design of the proposed device is presented and the results show the extinction ratio of 16 and 20 dB for X- and Y-pol input, respectively. The processing of 40  Gb/s signal is achieved within the C-band with good performance. The proposed converter is capable of handling multiple wavelengths in wavelength division multiplexing (WDM) networks, enabling the conversions between WDM-PDM and WDM-MDM, which is promising to further increase the throughput at the network interface. PMID:24562199

  10. Holographic data storage system combining shift-multiplexing with peristrophic-multiplexing

    NASA Astrophysics Data System (ADS)

    Yoshikawa, Kengo; Tsukamoto, Yu; Okubo, Kaito; Yamamoto, Manabu

    2014-02-01

    Holographic data storage (HDS) is a next-generation optical storage that uses the principles of holography. The multiplex holographic recording method is an important factor that affects the recording capacity of this storage. Various multiplex recording methods have been proposed so far. In this study, we focus on shift multiplexing with spherical waves and propose a method of shift multiplex recording that combines the peristrophic multiplexed recording. Simulation and experimental verification shows that the proposed method is effective in principle.

  11. Simultaneous Detection of Genetically Modified Organisms in a Mixture by Multiplex PCR-Chip Capillary Electrophoresis.

    PubMed

    Patwardhan, Supriya; Dasari, Srikanth; Bhagavatula, Krishna; Mueller, Steffen; Deepak, Saligrama Adavigowda; Ghosh, Sudip; Basak, Sanjay

    2015-01-01

    An efficient PCR-based method to trace genetically modified food and feed products is in demand due to regulatory requirements and contaminant issues in India. However, post-PCR detection with conventional methods has limited sensitivity in amplicon separation that is crucial in multiplexing. The study aimed to develop a sensitive post-PCR detection method by using PCR-chip capillary electrophoresis (PCR-CCE) to detect and identify specific genetically modified organisms in their genomic DNA mixture by targeting event-specific nucleotide sequences. Using the PCR-CCE approach, novel multiplex methods were developed to detect MON531 cotton, EH 92-527-1 potato, Bt176 maize, GT73 canola, or GA21 maize simultaneously when their genomic DNAs in mixtures were amplified using their primer mixture. The repeatability RSD (RSDr) of the peak migration time was 0.06 and 3.88% for the MON531 and Bt176, respectively. The RSD (RSDR) of the Cry1Ac peak ranged from 0.12 to 0.40% in multiplex methods. The method was sensitive in resolving amplicon of size difference up to 4 bp. The PCR-CCE method is suitable to detect multiple genetically modified events in a composite DNA sample by tagging their event specific sequences. PMID:26525256

  12. mPSQed: a software for the design of multiplex pyrosequencing assays.

    PubMed

    Dabrowski, Piotr Wojtek; Nitsche, Andreas

    2012-01-01

    Molecular-based diagnostic assays are the gold standard for infectious diseases today, since they allow a rapid and sensitive identification and typing of various pathogens. While PCR can be designed to be specific for a certain pathogen, a subsequent sequence analysis is frequently required for confirmation or typing. The design of appropriate PCR-based assays is a complex task, especially when conserved discriminating polymorphisms are rare or if the number of types which need to be differentiated is high. One extremely useful but underused method for this purpose is the multiplex pyrosequencing technique. Unfortunately there is no software available to aid researchers in designing multiplex pyrosequencing assays. Here, we present mPSQed (Multiplex PyroSeQuencing EDitor), a program targeted at closing this gap. We also present the design of an exemplarily theoretical assay for the differentiation of human adenovirus types A-F using two pyrosequencing primers on two distinct PCR products, designed quickly and easily using our software. PMID:22675516

  13. mPSQed: A Software for the Design of Multiplex Pyrosequencing Assays

    PubMed Central

    Dabrowski, Piotr Wojtek; Nitsche, Andreas

    2012-01-01

    Molecular-based diagnostic assays are the gold standard for infectious diseases today, since they allow a rapid and sensitive identification and typing of various pathogens. While PCR can be designed to be specific for a certain pathogen, a subsequent sequence analysis is frequently required for confirmation or typing. The design of appropriate PCR-based assays is a complex task, especially when conserved discriminating polymorphisms are rare or if the number of types which need to be differentiated is high. One extremely useful but underused method for this purpose is the multiplex pyrosequencing technique. Unfortunately there is no software available to aid researchers in designing multiplex pyrosequencing assays. Here, we present mPSQed (Multiplex PyroSeQuencing EDitor), a program targeted at closing this gap. We also present the design of an exemplarily theoretical assay for the differentiation of human adenovirus types A–F using two pyrosequencing primers on two distinct PCR products, designed quickly and easily using our software. PMID:22675516

  14. Fluorescent multiplex linkage analysis and carrier detection for Duchenne/Becker muscular dystrophy

    SciTech Connect

    Schwartz, L.S.; Hoffman, E.P. ); Tarleton, J. ); Popovich, B. ); Seltzer, W.K. )

    1992-10-01

    The authors have developed a fast and accurate PCR-based linkage and carrier detection protocol for families of Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) patients with or without detectable deletions of the dystrophin gene, using fluorescent PCR products analyzed on an automated sequencer. When a deletion is found in the affected male DMD/BMD patient by standard multiplex PCR, fluorescently labeled primers specific for the deleted and nondeleted exon(s) are used to amplify the DNA of at-risk female relatives by using multiplex PCR at low cycle number (20 cycles). The products are then quantitatively analyzed on an automatic sequencer to determine whether they are heterozygous for the deletion and thus are carriers. As a confirmation of the deletion data, and in cases in which a deletion is not found in the proband, fluorescent multiplex PCR linkage is done by using four previously described polymorphic dinucleotide sequences. The four (CA)[sub n] repeats are located throughout the dystrophin gene, making the analysis highly informative and accurate. The authors present the successful application of this protocol in families who proved refractory to more traditional analyses. 22 refs., 3 figs.

  15. Weak percolation on multiplex networks.

    PubMed

    Baxter, Gareth J; Dorogovtsev, Sergey N; Mendes, José F F; Cellai, Davide

    2014-04-01

    Bootstrap percolation is a simple but nontrivial model. It has applications in many areas of science and has been explored on random networks for several decades. In single-layer (simplex) networks, it has been recently observed that bootstrap percolation, which is defined as an incremental process, can be seen as the opposite of pruning percolation, where nodes are removed according to a connectivity rule. Here we propose models of both bootstrap and pruning percolation for multiplex networks. We collectively refer to these two models with the concept of "weak" percolation, to distinguish them from the somewhat classical concept of ordinary ("strong") percolation. While the two models coincide in simplex networks, we show that they decouple when considering multiplexes, giving rise to a wealth of critical phenomena. Our bootstrap model constitutes the simplest example of a contagion process on a multiplex network and has potential applications in critical infrastructure recovery and information security. Moreover, we show that our pruning percolation model may provide a way to diagnose missing layers in a multiplex network. Finally, our analytical approach allows us to calculate critical behavior and characterize critical clusters. PMID:24827287

  16. Code retrieval via undercover multiplexing

    NASA Astrophysics Data System (ADS)

    Barrera, John Fredy; Henao, Rodrigo; Tebaldi, Myrian; Torroba, Roberto; Bolognini, Nestor

    2008-02-01

    The purpose of this research is to develop an undercover multiplexing technique to give additional protection for optical information encryption. We employ the double random phase mask as our basic optical encryption system. The holographic storage medium of choice is a photorefractive crystal. To achieve the multiplexing we use the aperture size of the pupil in the optical system, as it governs the speckle size. We introduce such variation in order to produce a decorrelation between two consecutively stored speckle patterns. Each stored speckle pattern is associated to an input encrypted image, thus producing a multiplexing of the encrypted information. We implement this operation without altering the setup architecture and the random phase masks. This multiplexing is our undercover operation to encipher a true code behind a fake code. Under this approach, the user can only recover the bulk information stored in the volume hologram. However, he cannot recover the true code without the additional information on the pupil size key, even if accessed in position of the original decoding mask.

  17. Weak percolation on multiplex networks

    NASA Astrophysics Data System (ADS)

    Baxter, Gareth J.; Dorogovtsev, Sergey N.; Mendes, José F. F.; Cellai, Davide

    2014-04-01

    Bootstrap percolation is a simple but nontrivial model. It has applications in many areas of science and has been explored on random networks for several decades. In single-layer (simplex) networks, it has been recently observed that bootstrap percolation, which is defined as an incremental process, can be seen as the opposite of pruning percolation, where nodes are removed according to a connectivity rule. Here we propose models of both bootstrap and pruning percolation for multiplex networks. We collectively refer to these two models with the concept of "weak" percolation, to distinguish them from the somewhat classical concept of ordinary ("strong") percolation. While the two models coincide in simplex networks, we show that they decouple when considering multiplexes, giving rise to a wealth of critical phenomena. Our bootstrap model constitutes the simplest example of a contagion process on a multiplex network and has potential applications in critical infrastructure recovery and information security. Moreover, we show that our pruning percolation model may provide a way to diagnose missing layers in a multiplex network. Finally, our analytical approach allows us to calculate critical behavior and characterize critical clusters.

  18. Quantitative and qualitative validations of a sonication-based DNA extraction approach for PCR-based molecular biological analyses.

    PubMed

    Dai, Xiaohu; Chen, Sisi; Li, Ning; Yan, Han

    2016-05-15

    The aim of this study was to comprehensively validate the sonication-based DNA extraction method, in hope of the replacement of the so-called 'standard DNA extraction method' - the commercial kit method. Microbial cells in the digested sludge sample, containing relatively high amount of PCR-inhibitory substances, such as humic acid and protein, were applied as the experimental alternatives. The procedure involving solid/liquid separation of sludge sample and dilution of both DNA templates and inhibitors, the minimum templates for PCR-based analyses, and the in-depth understanding from the bias analysis by pyrosequencing technology were obtained and confirmed the availability of the sonication-based DNA extraction method. PMID:26774955

  19. Real-Time PCR-Based Quantitation Method for the Genetically Modified Soybean Line GTS 40-3-2.

    PubMed

    Kitta, Kazumi; Takabatake, Reona; Mano, Junichi

    2016-01-01

    This chapter describes a real-time PCR-based method for quantitation of the relative amount of genetically modified (GM) soybean line GTS 40-3-2 [Roundup Ready(®) soybean (RRS)] contained in a batch. The method targets a taxon-specific soybean gene (lectin gene, Le1) and the specific DNA construct junction region between the Petunia hybrida chloroplast transit peptide sequence and the Agrobacterium 5-enolpyruvylshikimate-3-phosphate synthase gene (epsps) sequence present in GTS 40-3-2. The method employs plasmid pMulSL2 as a reference material in order to quantify the relative amount of GTS 40-3-2 in soybean samples using a conversion factor (Cf) equal to the ratio of the RRS-specific DNA to the taxon-specific DNA in representative genuine GTS 40-3-2 seeds. PMID:26614294

  20. qPCR based mRNA quality score show intact mRNA after heat stabilization

    PubMed Central

    Karlsson, Oskar; Segerström, Lova; Sjöback, Robert; Nylander, Ingrid; Borén, Mats

    2016-01-01

    Analysis of multiple analytes from biological samples can be challenging as different analytes require different preservation measures. Heat induced enzymatic inactivation is an efficient way to preserve proteins and their modifications in biological samples but RNA quality, as measured by RIN value, has been a concern in such samples. Here, we investigate the effect of heat stabilization compared with standard snap freezing on RNA quality using two RNA extraction protocols, QiaZol with and without urea pre-solubilization, and two RNA quality measurements: RIN value, as defined by the Agilent Bioanalyzer, and an alternative qPCR based method. DNA extraction from heat stabilized brain samples was also examined. The snap frozen samples had RIN values about 1 unit higher than heat stabilized samples for the direct QiaZol extraction but equal with stabilized samples using urea pre-solubilization. qPCR based RNA quality measurement showed no difference in quality between snap frozen and heat inactivated samples. The probable explanation for this discrepancy is that the RIN value is an indirect measure based on rRNA, while the qPCR score is based on actual measurement of mRNA quality. The DNA yield from heat stabilized brain tissue samples was significantly increased, compared to the snap frozen tissue, without any effects on purity or quality. Hence, heat stabilization of tissues opens up the possibility for a two step preservation protocol, where proteins and their modifications can be preserved in the first heat based step, while in a second step, using standard RNA preservation strategies, mRNA be preserved. This collection strategy will enable biobanking of samples where the ultimate analysis is not determined without loss of sample quality. PMID:27077049

  1. qPCR based mRNA quality score show intact mRNA after heat stabilization.

    PubMed

    Karlsson, Oskar; Segerström, Lova; Sjöback, Robert; Nylander, Ingrid; Borén, Mats

    2016-03-01

    Analysis of multiple analytes from biological samples can be challenging as different analytes require different preservation measures. Heat induced enzymatic inactivation is an efficient way to preserve proteins and their modifications in biological samples but RNA quality, as measured by RIN value, has been a concern in such samples. Here, we investigate the effect of heat stabilization compared with standard snap freezing on RNA quality using two RNA extraction protocols, QiaZol with and without urea pre-solubilization, and two RNA quality measurements: RIN value, as defined by the Agilent Bioanalyzer, and an alternative qPCR based method. DNA extraction from heat stabilized brain samples was also examined. The snap frozen samples had RIN values about 1 unit higher than heat stabilized samples for the direct QiaZol extraction but equal with stabilized samples using urea pre-solubilization. qPCR based RNA quality measurement showed no difference in quality between snap frozen and heat inactivated samples. The probable explanation for this discrepancy is that the RIN value is an indirect measure based on rRNA, while the qPCR score is based on actual measurement of mRNA quality. The DNA yield from heat stabilized brain tissue samples was significantly increased, compared to the snap frozen tissue, without any effects on purity or quality. Hence, heat stabilization of tissues opens up the possibility for a two step preservation protocol, where proteins and their modifications can be preserved in the first heat based step, while in a second step, using standard RNA preservation strategies, mRNA be preserved. This collection strategy will enable biobanking of samples where the ultimate analysis is not determined without loss of sample quality. PMID:27077049

  2. Development of one-step real-time reverse transcriptase-PCR-based assays for the rapid and simultaneous detection of four viruses causing porcine diarrhea.

    PubMed

    Masuda, Tsuneyuki; Tsuchiaka, Shinobu; Ashiba, Tomoko; Yamasato, Hiroshi; Fukunari, Kazuhiro; Omatsu, Tsutomu; Furuya, Tetsuya; Shirai, Junsuke; Mizutani, Tetsuya; Nagai, Makoto

    2016-02-01

    Porcine diarrhea caused by viruses is a major problem of the pig farming industry and can result in substantial losses of revenue. Thus, diagnosing the infectious agents is important to prevent and control diseases in pigs. We developed novel one-step real-time quantitative RT-PCR (qPCR) assays that can detect four porcine diarrheal viruses simultaneously: porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine group A rotavirus (PRVA). The qPCR analysis takes only 75 minutes to detect the presence of the four viruses. The limits of detection of our new assays for PEDV, TGEV, PDCoV, and PRVA were 100, 10, 10 and 10 copies per reaction, respectively. The sensitivity of qPCR was 1-1000 times higher than that of published gel-based RT-PCR. We used our qPCR method to successfully diagnose clinical samples from infected pigs, and no false positive results were obtained. In conclusion, qPCR can drastically reduce the diagnostic time to detect viruses compared to currently employed methods. We predict that the qPCR assays will become a useful tool for detecting viral infections that cause diarrhea and other complications in pigs. PMID:27348884

  3. In-vitro Cell Culture and Real-time Reverse Transcriptase PCR-based Assays to Detect Infective Toxoplas gondii Oocysts

    EPA Science Inventory

    Toxoplasma gondii is an obligate intracellular, apicomplexan parasite that infects humans. It is ubiquitous in nature and seroprevalence in the United States and in Europe ranges from 25->70%. Although typically associated with causing foodborne outbreaks, recent studies in Canad...

  4. Catch and Release: Integrated system for multiplexed detection of bacteria

    PubMed Central

    Verbarg, Jasenka; Plath, William D.; Shriver-Lake, Lisa C.; Howell, Peter B.; Erickson, Jeffrey S.; Golden, Joel P.; Ligler, Frances S.

    2013-01-01

    An integrated system with automated immunomagnetic separation and processing of fluidic samples was demonstrated for multiplexed optical detection of bacterial targets. Mixtures of target-specific magnetic bead sets were processed in the NRL MagTrap with the aid of rotating magnet arrays that entrapped and moved the beads within the channel during reagent processing. Processing was performed in buffer and human serum matrices with 10-fold dilutions in the range of 102 – 106 cells/mL of target bacteria. Reversal of magnets’ rotation post processing released the beads back into the flow and moved them into the Microflow Cytometer for optical interrogation. Identification of the beads and the detection of PE fluorescence were performed simultaneously for multiplexed detection. Multiplexing was performed with specifically targeted bead sets to detect E. coli 0157.H7, Salmonella Common Structural Antigen, Listeria sp. and Shigella sp. Dose-response curves were obtained, and limits of detection were calculated for each target in the buffer and clinical matrix. Additional tests demonstrated the potential for using the MagTrap to concentrate target from larger volumes of sample prior to the addition of assay reagents. PMID:23631439

  5. Catch and release: integrated system for multiplexed detection of bacteria.

    PubMed

    Verbarg, Jasenka; Plath, William D; Shriver-Lake, Lisa C; Howell, Peter B; Erickson, Jeffrey S; Golden, Joel P; Ligler, Frances S

    2013-05-21

    An integrated system with automated immunomagnetic separation and processing of fluidic samples was demonstrated for multiplexed optical detection of bacterial targets. Mixtures of target-specific magnetic bead sets were processed in the NRL MagTrap with the aid of rotating magnet arrays that entrapped and moved the beads within the channel during reagent processing. Processing was performed in buffer and human serum matrixes with 10-fold dilutions in the range of 10(2)-10(6) cells/mL of target bacteria. Reversal of magnets' rotation post-processing released the beads back into the flow and moved them into the microflow cytometer for optical interrogation. Identification of the beads and the detection of PE fluorescence were performed simultaneously for multiplexed detection. Multiplexing was performed with specifically targeted bead sets to detect E. coli 0157.H7, Salmonella Common Structural Antigen, Listeria sp., and Shigella sp., dose-response curves were obtained, and limits of detection were calculated for each target in the buffer and clinical matrix. Additional tests demonstrated the potential for using the MagTrap to concentrate target from larger volumes of sample prior to the addition of assay reagents. PMID:23631439

  6. Parallel multiplex laser feedback interferometry

    SciTech Connect

    Zhang, Song; Tan, Yidong; Zhang, Shulian

    2013-12-15

    We present a parallel multiplex laser feedback interferometer based on spatial multiplexing which avoids the signal crosstalk in the former feedback interferometer. The interferometer outputs two close parallel laser beams, whose frequencies are shifted by two acousto-optic modulators by 2Ω simultaneously. A static reference mirror is inserted into one of the optical paths as the reference optical path. The other beam impinges on the target as the measurement optical path. Phase variations of the two feedback laser beams are simultaneously measured through heterodyne demodulation with two different detectors. Their subtraction accurately reflects the target displacement. Under typical room conditions, experimental results show a resolution of 1.6 nm and accuracy of 7.8 nm within the range of 100 μm.

  7. Pattern formation in multiplex networks

    PubMed Central

    Kouvaris, Nikos E.; Hata, Shigefumi; Guilera, Albert Díaz-

    2015-01-01

    The advances in understanding complex networks have generated increasing interest in dynamical processes occurring on them. Pattern formation in activator-inhibitor systems has been studied in networks, revealing differences from the classical continuous media. Here we study pattern formation in a new framework, namely multiplex networks. These are systems where activator and inhibitor species occupy separate nodes in different layers. Species react across layers but diffuse only within their own layer of distinct network topology. This multiplicity generates heterogeneous patterns with significant differences from those observed in single-layer networks. Remarkably, diffusion-induced instability can occur even if the two species have the same mobility rates; condition which can never destabilize single-layer networks. The instability condition is revealed using perturbation theory and expressed by a combination of degrees in the different layers. Our theory demonstrates that the existence of such topology-driven instabilities is generic in multiplex networks, providing a new mechanism of pattern formation. PMID:26042606

  8. Lifetime-based tomographic multiplexing

    NASA Astrophysics Data System (ADS)

    Raymond, Scott B.; Boas, David A.; Bacskai, Brian J.; Kumar, Anand T. N.

    2010-07-01

    Near-infrared (NIR) fluorescence tomography of multiple fluorophores has previously been limited by the bandwidth of the NIR spectral regime and the broad emission spectra of most NIR fluorophores. We describe in vivo tomography of three spectrally overlapping fluorophores using fluorescence lifetime-based separation. Time-domain images are acquired using a voltage-gated, intensified charge-coupled device (CCD) in free-space transmission geometry with 750 nm Ti:sapphire laser excitation. Lifetime components are fit from the asymptotic portion of fluorescence decay curve and reconstructed separately with a lifetime-adjusted forward model. We use this system to test the in vivo lifetime multiplexing suitability of commercially available fluorophores, and demonstrate lifetime multiplexing in solution mixtures and in nude mice. All of the fluorophores tested exhibit nearly monoexponential decays, with narrow in vivo lifetime distributions suitable for lifetime multiplexing. Quantitative separation of two fluorophores with lifetimes of 1.1 and 1.37 ns is demonstrated for relative concentrations of 1:5. Finally, we demonstrate tomographic imaging of two and three fluorophores in nude mice with fluorophores that localize to distinct organ systems. This technique should be widely applicable to imaging multiple NIR fluorophores in 3-D.

  9. Evaluation of the repeatability and reproducibility of a suite of qPCR-based microbial source tracking methods.

    PubMed

    Ebentier, Darcy L; Hanley, Kaitlyn T; Cao, Yiping; Badgley, Brian D; Boehm, Alexandria B; Ervin, Jared S; Goodwin, Kelly D; Gourmelon, Michèle; Griffith, John F; Holden, Patricia A; Kelty, Catherine A; Lozach, Solen; McGee, Charles; Peed, Lindsay A; Raith, Meredith; Ryu, Hodon; Sadowsky, Michael J; Scott, Elizabeth A; Santo Domingo, Jorge; Schriewer, Alexander; Sinigalliano, Christopher D; Shanks, Orin C; Van De Werfhorst, Laurie C; Wang, Dan; Wuertz, Stefan; Jay, Jennifer A

    2013-11-15

    Many PCR-based methods for microbial source tracking (MST) have been developed and validated within individual research laboratories. Inter-laboratory validation of these methods, however, has been minimal, and the effects of protocol standardization regimes have not been thoroughly evaluated. Knowledge of factors influencing PCR in different laboratories is vital to future technology transfer for use of MST methods as a tool for water quality management. In this study, a blinded set of 64 filters (containing 32 duplicate samples generated from 12 composite fecal sources) were analyzed by three to five core laboratories with a suite of PCR-based methods utilizing standardized reagents and protocols. Repeatability (intra-laboratory variability) and reproducibility (inter-laboratory variability) of observed results were assessed. When standardized methodologies were used, intra- and inter-laboratory %CVs were generally low (median %CV 0.1-3.3% and 1.9-7.1%, respectively) and comparable to those observed in similar inter-laboratory validation studies performed on other methods of quantifying fecal indicator bacteria (FIB) in environmental samples. ANOVA of %CV values found three human-associated methods (BsteriF1, BacHum, and HF183Taqman) to be similarly reproducible (p > 0.05) and significantly more reproducible (p < 0.05) than HumM2. This was attributed to the increased variability associated with low target concentrations detected by HumM2 (approximately 1-2 log10copies/filter lower) compared to other human-associated methods. Cow-associated methods (BacCow and CowM2) were similarly reproducible (p > 0.05). When using standardized protocols, variance component analysis indicated sample type (fecal source and concentration) to be the major contributor to total variability with that from replicate filters and inter-laboratory analysis to be within the same order of magnitude but larger than inherent intra-laboratory variability. However, when reagents and

  10. Rotation spacing and multiplexing number in angle-peristrophic multiplexing holographic memory

    NASA Astrophysics Data System (ADS)

    Sawada, Masamitsu; Kinoshita, Nobuhiro; Muroi, Tetsuhiko; Motohashi, Mitsuya; Saito, Nobuo

    2015-09-01

    Holographic memory is expected to be the next-generation optical memory with several advantages including high data transfer rate and high recording density. Holographic memory enables the storage of holograms in the same location in a holographic medium typically using the angle multiplexing method. The multiplexing number is an important factor that determines the recording density when using this method. To increase the multiplexing number, it is known as an effective method to combine peristrophic (or rotation) multiplexing with angle multiplexing. We use the k-sphere to describe that the rotation spacing for peristrophic multiplexing depends on both the numerical aperture in the signal beam path and the angle between the reference and signal beams. We then formulate the rotation spacing and compare the results obtained using the theoretical formula with the measured results. Finally, we estimate the maximum multiplexing number for our experimental system using the angle-peristrophic multiplexing method on the basis of the measured results.

  11. Cells on chip for multiplex screening.

    PubMed

    Berthuy, Ophélie I; Blum, Loïc J; Marquette, Christophe A

    2016-02-15

    Microarray technology was developed in the early 1990s to measure the transcription levels of thousands of genes in parallel. The basic premise of high-density arraying has since been expanded to create cells microarrays. Cells on chip are powerful experimental tools for high-throughput and multiplex screening of samples or cellular functions. Miniaturization increases assay throughput while reducing both reagent consumption and cell population heterogeneity effect, making these systems attractive for a wide range of assays, from drug discovery to toxicology, stem cell research and therapy. One form of cell microarrays, the transfected cell microarray, wherein plasmid DNA or siRNA, spotted on the surface of a substrate, is reverse-transfected locally into adherent cells, has become a standard tool for parallel cell-based analysis. With the advent of technologies, cells can also be directly spotted onto functionalized surfaces using robotic fluid-dispensing devices, or printed directly in bio-ink material. We are providing herein an overview of the last developments in optical cell microarrays allowing high-throughput and high-content analysis. PMID:25892543

  12. Multiplexing of encrypted data using fractal masks.

    PubMed

    Barrera, John F; Tebaldi, Myrian; Amaya, Dafne; Furlan, Walter D; Monsoriu, Juan A; Bolognini, Néstor; Torroba, Roberto

    2012-07-15

    In this Letter, we present to the best of our knowledge a new all-optical technique for multiple-image encryption and multiplexing, based on fractal encrypting masks. The optical architecture is a joint transform correlator. The multiplexed encrypted data are stored in a photorefractive crystal. The fractal parameters of the key can be easily tuned to lead to a multiplexing operation without cross talk effects. Experimental results that support the potential of the method are presented. PMID:22825170

  13. Multiplex Holograms And Their Applications In Medicine

    NASA Astrophysics Data System (ADS)

    Tsujiuchi, Jumpei

    1988-01-01

    Fundamental properties of reconstructed images from a multiplex hologram are studied, and conditions for compensating distortions and for designing a reconstructing source are proposed. Applications of multiplex hologram to medical objects are reviewed, and a computer-aided hologram synthesizing system is proposed for obtaining better images and wider applications. An example of multiplex holograms synthesized from a series of CT images is also presented.

  14. Measuring and modeling correlations in multiplex networks.

    PubMed

    Nicosia, Vincenzo; Latora, Vito

    2015-09-01

    The interactions among the elementary components of many complex systems can be qualitatively different. Such systems are therefore naturally described in terms of multiplex or multilayer networks, i.e., networks where each layer stands for a different type of interaction between the same set of nodes. There is today a growing interest in understanding when and why a description in terms of a multiplex network is necessary and more informative than a single-layer projection. Here we contribute to this debate by presenting a comprehensive study of correlations in multiplex networks. Correlations in node properties, especially degree-degree correlations, have been thoroughly studied in single-layer networks. Here we extend this idea to investigate and characterize correlations between the different layers of a multiplex network. Such correlations are intrinsically multiplex, and we first study them empirically by constructing and analyzing several multiplex networks from the real world. In particular, we introduce various measures to characterize correlations in the activity of the nodes and in their degree at the different layers and between activities and degrees. We show that real-world networks exhibit indeed nontrivial multiplex correlations. For instance, we find cases where two layers of the same multiplex network are positively correlated in terms of node degrees, while other two layers are negatively correlated. We then focus on constructing synthetic multiplex networks, proposing a series of models to reproduce the correlations observed empirically and/or to assess their relevance. PMID:26465526

  15. Measuring and modeling correlations in multiplex networks

    NASA Astrophysics Data System (ADS)

    Nicosia, Vincenzo; Latora, Vito

    2015-09-01

    The interactions among the elementary components of many complex systems can be qualitatively different. Such systems are therefore naturally described in terms of multiplex or multilayer networks, i.e., networks where each layer stands for a different type of interaction between the same set of nodes. There is today a growing interest in understanding when and why a description in terms of a multiplex network is necessary and more informative than a single-layer projection. Here we contribute to this debate by presenting a comprehensive study of correlations in multiplex networks. Correlations in node properties, especially degree-degree correlations, have been thoroughly studied in single-layer networks. Here we extend this idea to investigate and characterize correlations between the different layers of a multiplex network. Such correlations are intrinsically multiplex, and we first study them empirically by constructing and analyzing several multiplex networks from the real world. In particular, we introduce various measures to characterize correlations in the activity of the nodes and in their degree at the different layers and between activities and degrees. We show that real-world networks exhibit indeed nontrivial multiplex correlations. For instance, we find cases where two layers of the same multiplex network are positively correlated in terms of node degrees, while other two layers are negatively correlated. We then focus on constructing synthetic multiplex networks, proposing a series of models to reproduce the correlations observed empirically and/or to assess their relevance.

  16. A PCR based protocol for detecting indel mutations induced by TALENs and CRISPR/Cas9 in zebrafish.

    PubMed

    Yu, Chuan; Zhang, Yaguang; Yao, Shaohua; Wei, Yuquan

    2014-01-01

    Genome editing techniques such as the zinc-finger nucleases (ZFNs), transcription activator-like effecter nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system Cas9 can induce efficient DNA double strand breaks (DSBs) at the target genomic sequence and result in indel mutations by the error-prone non-homologous end joining (NHEJ) DNA repair system. Several methods including sequence specific endonuclease assay, T7E1 assay and high resolution melting curve assay (HRM) etc have been developed to detect the efficiency of the induced mutations. However, these assays have some limitations in that they either require specific sequences in the target sites or are unable to generate sequencing-ready mutant DNA fragments or unable to distinguish induced mutations from natural nucleotide polymorphism. Here, we developed a simple PCR-based protocol for detecting indel mutations induced by TALEN and Cas9 in zebrafish. We designed 2 pairs of primers for each target locus, with one putative amplicon extending beyond the putative indel site and the other overlapping it. With these primers, we performed a qPCR assay to efficiently detect the frequencies of newly induced mutations, which was accompanied with a T-vector-based colony analysis to generate single-copy mutant fragment clones for subsequent DNA sequencing. Thus, our work has provided a very simple, efficient and fast assay for detecting induced mutations, which we anticipate will be widely used in the area of genome editing. PMID:24901507

  17. PCR-based detection of the CYP21 deletion and TNXA/TNXB hybrid in the RCCX module.

    PubMed

    Lee, Hsien-Hsiung; Lee, Yann-Jinn; Lin, Ching-Yu

    2004-05-01

    Detection of the CYP21 deletion in congenital adrenal hyperplasia (CAH) in the RCCX module has been previously done by Southern blot analysis with multiple probes and separate digestions with the restriction endonucleases TaqI and BglII, which is laborious and indirect. Here, we describe an established PCR-based amplification method to analyze directly a CAH patient with a single CYP21 deletion, followed by RFLP analysis to characterize the interconversion region between tenascin A (TNXA) and tenascin B (TNXB). Data indicate that TaqI digestion of the defective CYP21 gene in the CAH patient produced 3.2-kb fragments. The CYP21 allele carried mutations in the CYP21P gene as determined by analysis with the amplification-created restriction site method. In addition, RFLP analysis indicated that the TNXB gene in the defective allele was replaced by TNXA to produce a TNXA/TNXB hybrid. We conclude that deletion of the RCCX module in this CAH patient included the RP2, C4B, and CYP21 genes and part of the TNXB gene. The junction of the recombination of the TNXA/TNXB hybrid may be located between IVS44 and exon 44 of the TNXB gene. This rapid, nonradioactive detection method will be beneficial for diagnostic purposes that are limited to the population originally studied. PMID:15081125

  18. A PCR-Based Method to Construct Lentiviral Vector Expressing Double Tough Decoy for miRNA Inhibition.

    PubMed

    Qiu, Huiling; Zhong, Jiasheng; Luo, Lan; Liu, Nian; Kang, Kang; Qu, Junle; Peng, Wenda; Gou, Deming

    2015-01-01

    DNA vector-encoded Tough Decoy (TuD) miRNA inhibitor is attracting increased attention due to its high efficiency in miRNA suppression. The current methods used to construct TuD vectors are based on synthesizing long oligonucleotides (~90 mer), which have been costly and problematic because of mutations during synthesis. In this study, we report a PCR-based method for the generation of double Tough Decoy (dTuD) vector in which only two sets of shorter oligonucleotides (< 60 mer) were used. Different approaches were employed to test the inhibitory potency of dTuDs. We demonstrated that dTuD is the most efficient method in miRNA inhibition in vitro and in vivo. Using this method, a mini dTuD library against 88 human miRNAs was constructed and used for a high-throughput screening (HTS) of AP-1 pathway-related miRNAs. Seven miRNAs (miR-18b-5p, -101-3p, -148b-3p, -130b-3p, -186-3p, -187-3p and -1324) were identified as candidates involved in AP-1 pathway regulation. This novel method allows for an accurate and cost-effective generation of dTuD miRNA inhibitor, providing a powerful tool for efficient miRNA suppression in vitro and in vivo. PMID:26624995

  19. Performance of PCR-based assays targeting Bacteroidales genetic markers of human fecal pollution in sewage and fecal samples.

    PubMed

    Shanks, Orin C; White, Karen; Kelty, Catherine A; Sivaganesan, Mano; Blannon, Janet; Meckes, Mark; Varma, Manju; Haugland, Richard A

    2010-08-15

    There are numerous PCR-based assays available to characterize human fecal pollution in ambient waters. Each assay employs distinct oligonucleotides and many target different genes and microorganisms leading to potential variations in assay performance. Performance comparisons utilizing feces and raw sewage samples are needed to determine which assays are best suited for expensive and time-consuming field validation, fate, transport, and epidemiology studies. We report the assessment of five end-point PCR and 10 real-time quantitative PCR (qPCR) assays that target genes from presumptive Bacteroidales microorganisms reported to be associated with human feces. Each assay was tested against a reference collection of 54 primary influent sewage samples collected from different geographical locations across the United States and 174 fecal DNA extracts from 23 different animal sources. Experiments indicate that human-associated genetic markers are distributed across a broad range of human populations but show substantial differences in specificity for human feces suggesting that particular assays may be more suitable than others depending on the abundance of genetic marker required for detection and the animal sources impacting a particular watershed or beach of interest. PMID:20704227

  20. A dual PCR-based sequencing approach for the identification and discrimination of Echinococcus and Taenia taxa.

    PubMed

    Boubaker, Ghalia; Marinova, Irina; Gori, Francesca; Hizem, Amani; Müller, Norbert; Casulli, Adriano; Jerez Puebla, Luis Enrique; Babba, Hamouda; Gottstein, Bruno; Spiliotis, Markus

    2016-08-01

    Reliable and rapid molecular tools for the genetic identification and differentiation of Echinococcus species and/or genotypes are crucial for studying spatial and temporal transmission dynamics. Here, we describe a novel dual PCR targeting regions in the small (rrnS) and large (rrnL) subunits of mitochondrial ribosomal RNA (rRNA) genes, which enables (i) the specific identification of species and genotypes of Echinococcus (rrnS + L-PCR) and/or (ii) the identification of a range of taeniid cestodes, including different species of Echinococcus, Taenia and some others (17 species of diphyllidean helminths). This dual PCR approach was highly sensitive, with an analytical detection limit of 1 pg for genomic DNA of Echinococcus. Using concatenated sequence data derived from the two gene markers (1225 bp), we identified five unique and geographically informative single nucleotide polymorphisms (SNPs) that allowed genotypes (G1 and G3) of Echinococcus granulosus sensu stricto to be distinguished, and 25 SNPs that allowed differentiation within Echinococcus canadensis (G6/7/8/10). In conclusion, we propose that this dual PCR-based sequencing approach can be used for molecular epidemiological studies of Echinococcus and other taeniid cestodes. PMID:27242008

  1. Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study

    USGS Publications Warehouse

    Layton, Blythe A.; Cao, Yiping; Ebentier, Darcy L.; Hanley, Kaitlyn; Ballesté, Elisenda; Brandão, João; Byappanahalli, Muruleedhara N.; Converse, Reagan; Farnleitner, Andreas H.; Gentry-Shields, Jennifer, Gidley, Maribeth L.; Gourmelon, Michèle; Lee, Chang Soo; Lee, Jiyoung; Lozach, Solen; Madi, Tania; Meijer, Wim G.; Noble, Rachel; Peed, Lindsay; Reischer, Georg H.; Rodrigues, Raquel; Rose, Joan B.; Schriewer, Alexander; Sinigalliano, Chris; Srinivasan, Sangeetha; Stewart, Jill; Van De Werfhorst; Laurie, C.; Wang, Dan; Whitman, Richard; Wuertz, Stefan; Jay, Jenny; Holden, Patricia A.; Boehm, Alexandria B.; Shanks, Orin; Griffith, John F.

    2013-01-01

    A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing in large multi-laboratory studies. Here, we evaluated ten of these methods (BacH, BacHum-UCD, Bacteroides thetaiotaomicron (BtH), BsteriF1, gyrB, HF183 endpoint, HF183 SYBR, HF183 Taqman®, HumM2, and Methanobrevibacter smithii nifH (Mnif)) using 64 blind samples prepared in one laboratory. The blind samples contained either one or two fecal sources from human, wastewater or non-human sources. The assay results were assessed for presence/absence of the human markers and also quantitatively while varying the following: 1) classification of samples that were detected but not quantifiable (DNQ) as positive or negative; 2) reference fecal sample concentration unit of measure (such as culturable indicator bacteria, wet mass, total DNA, etc); and 3) human fecal source type (stool, sewage or septage). Assay performance using presence/absence metrics was found to depend on the classification of DNQ samples. The assays that performed best quantitatively varied based on the fecal concentration unit of measure and laboratory protocol. All methods were consistently more sensitive to human stools compared to sewage or septage in both the presence/absence and quantitative analysis. Overall, HF183 Taqman® was found to be the most effective marker of human fecal contamination in this California-based study.

  2. An improved method of DNA isolation suitable for PCR-based detection of begomoviruses from jute and other mucilaginous plants.

    PubMed

    Ghosh, Raju; Paul, Sujay; Ghosh, Subrata Kumar; Roy, Anirban

    2009-07-01

    A relatively quick and inexpensive modified cetyl trimethylammonium bromide method for extraction of DNA from leaf materials containing large quantities of mucilage is described. The modification including use of more volume of extraction buffer and dissolving crude nucleic acid pellet in 1 M NaCl, reduced markedly the viscosity of the mucilage and thus in the final purification step yielded a larger quantity of mucilage-free DNA suitable for subsequent PCR-based detection of begomoviruses. The method was standardized with jute samples with yellow mosaic disease and validated with different other mucilaginous-hosts with low titre of begomoviruses. DNA isolated using this method showed consistency in yield and compatibility with PCR for detection of begomoviruses from different mucilaginous plant species. The method was compared for efficacy with other reported methods and it was found to be superior over the existing methods described for isolation of DNA from mucilaginous hosts. Thus the method described could be used on a wider scale for reliable and consistent detection of begomoviruses from mucilaginous hosts for characterization and variability study. PMID:19442842

  3. Differential diagnosis of Brucella abortus by real-time PCR based on a single-nucleotide polymorphisms

    PubMed Central

    KIM, Ji-Yeon; KANG, Sung-Il; LEE, Jin Ju; LEE, Kichan; SUNG, So-Ra; ERDENEBAATAAR, Janchivdorj; VANAABAATAR, Batbaatar; JUNG, Suk Chan; PARK, Yong Ho; YOO, Han-Sang; HER, Moon

    2015-01-01

    To diagnose brucellosis effectively, many genus- and species-specific detection methods based on PCR have been developed. With conventional PCR assays, real-time PCR techniques have been developed as rapid diagnostic tools. Among them, real-time PCR using hybridization probe (hybprobe) has been recommended for bacteria with high DNA homology among species, with which it is possible to make an accurate diagnosis by means of an amplification curve and melting peak analysis. A hybprobe for B. abortus was designed from a specific single-nucleotide polymorphism (SNP) on the fbaA gene. This probe only showed specific amplification of B. abortus from approximately the 14th cycle, given a melting peak at 69°C. The sensitivity of real-time PCR was revealed to be 20 fg/µl by 10-fold DNA dilution, and the detection limit was 4 CFU in clinical samples. This real-time PCR showed greater sensitivity than that of conventional PCR and previous real-time PCR based on Taqman probe. Therefore, this new real-time PCR assay could be helpful for differentiating B. abortus infection with rapidity and accuracy. PMID:26666176

  4. Ultrasensitive detection of protease activity of anthrax and botulinum toxins by a new PCR-based assay.

    PubMed

    Kolesnikov, Alexander V; Kozyr, Arina V; Ryabko, Alyona K; Shemyakin, Igor G

    2016-02-01

    Anthrax and botulism are dangerous infectious diseases that can be fatal unless detected and treated quickly. Fatalities from these diseases are primarily due to endopeptidase toxins secreted by the pathogens. Rapid and sensitive detection of the presence of active toxins is the key element for protection from natural outbreaks of anthrax and botulism, as well as from the threat of bioterrorism. We describe an ultrasensitive polymerase chain reaction (PCR)-based assay for detecting proteolytic activity of anthrax and botulinum toxins using composite probes consisting of covalent peptide-DNA conjugate for the detection of anthrax, and noncovalent protein-aptamer assembly to assay botulinum toxin activity. Probes immobilized on the solid-phase support are cleaved by toxins to release DNA, which is detected by real-time PCR. Both assays can detect subpicogram quantities of active toxins isolated from composite matrices. Special procedures were developed to isolate intact toxins from the matrices under mild conditions. The assay is rapid, uses proven technologies, and can be modified to detect other proteolytic and biopolymer-degrading enzymes. PMID:26620058

  5. Differential diagnosis of Brucella abortus by real-time PCR based on a single-nucleotide polymorphisms.

    PubMed

    Kim, Ji-Yeon; Kang, Sung-Il; Lee, Jin Ju; Lee, Kichan; Sung, So-Ra; Erdenebaataar, Janchivdorj; Vanaabaatar, Batbaatar; Jung, Suk Chan; Park, Yong Ho; Yoo, Han-Sang; Her, Moon

    2016-05-01

    To diagnose brucellosis effectively, many genus- and species-specific detection methods based on PCR have been developed. With conventional PCR assays, real-time PCR techniques have been developed as rapid diagnostic tools. Among them, real-time PCR using hybridization probe (hybprobe) has been recommended for bacteria with high DNA homology among species, with which it is possible to make an accurate diagnosis by means of an amplification curve and melting peak analysis. A hybprobe for B. abortus was designed from a specific single-nucleotide polymorphism (SNP) on the fbaA gene. This probe only showed specific amplification of B. abortus from approximately the 14th cycle, given a melting peak at 69°C. The sensitivity of real-time PCR was revealed to be 20 fg/µl by 10-fold DNA dilution, and the detection limit was 4 CFU in clinical samples. This real-time PCR showed greater sensitivity than that of conventional PCR and previous real-time PCR based on Taqman probe. Therefore, this new real-time PCR assay could be helpful for differentiating B. abortus infection with rapidity and accuracy. PMID:26666176

  6. An improved colorimetric PCR-based method for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in feces.

    PubMed Central

    Britten, D; Wilson, S M; McNerney, R; Moody, A H; Chiodini, P L; Ackers, J P

    1997-01-01

    The epidemiological implications of the recent separation of "Entamoeba histolytica" into two separate species, pathogenic E. histolytica sensu stricto and commensal E. dispar, will not become apparent without methods of distinguishing between them which are applicable to large numbers of specimens. We have modified a PCR-based method to produce such a technique which may be completed in 1 day while still identifying 10(-1) E. histolytica and 1 to 10 E. dispar trophozoites per g of feces when present separately and 10 E. histolytica and 100 E. dispar trophozoites per g in the presence of 10(6) trophozoites per g of the other species. Applied to fecal specimens from 18 patients from which E. histolytica or E. dispar had been grown and identified to the species level by hexokinase isoenzyme analysis, the method in every case yielded the correct result. Positive and negative results are easily distinguished by eye, and we are now applying this technique to a large-scale epidemiological study of amebiasis in the eastern Mediterranean region. PMID:9114390

  7. A sensitive PCR-based assay to detect Neotyphodium fungi in seed and plant tissue of tall fescue and ryegrass spp.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A polymerase chain reaction (PCR)-based method for detection of Neotyphodium endophytes in seed and plant tissue from tall fescue (Festuca arundinacea Schreb.), Italian (Lolium multiflorum Lam.) and perennial (Lolium perenne L.) ryegrasses was developed. Based on DNA mixture tests and bulk seed anal...

  8. Identification and structure of the mating-type locus and development of PCR-based markers for mating type in powdery mildew fungi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In fungi, mating compatibility is regulated by mating-type loci. The objectives of this study were to identify and sequence mating-type genes at the MAT1 locus in the grape powdery mildew fungus, Erysiphe necator, to develop a PCR-based marker for determining mating type in E. necator, and to devel...

  9. Reversible Sterilization

    ERIC Educational Resources Information Center

    Largey, Gale

    1977-01-01

    Notes that difficult questions arise concerning the use of sterilization for alleged eugenic and euthenic purposes. Thus, how reversible sterilization will be used with relation to the poor, mentally ill, mentally retarded, criminals, and minors, is questioned. (Author/AM)

  10. A fully sealed plastic chip for multiplex PCR and its application in bacteria identification.

    PubMed

    Xu, Youchun; Yan, He; Zhang, Yan; Jiang, Kewei; Lu, Ying; Ren, Yonghong; Wang, Hui; Wang, Shan; Xing, Wanli

    2015-07-01

    Multiplex PCR is an effective tool for simultaneous multiple target detection but is limited by the intrinsic interference and competition among primer pairs when it is performed in one reaction tube. Dividing a multiplex PCR into many single PCRs is a simple strategy to overcome this issue. Here, we constructed a plastic, easy-to-use, fully sealed multiplex PCR chip based on reversible centrifugation for the simultaneous detection of 63 target DNA sequences. The structure of the chip is quite simple, which contains sine-shaped infusing channels and a number of reaction chambers connecting to one side of these channels. Primer pairs for multiplex PCR were sequentially preloaded in the different reaction chambers, and the chip was enclosed with PCR-compatible adhesive tape. For usage, the PCR master mix containing a DNA template is pipetted into the infusing channels and centrifuged into the reaction chambers, leaving the infusing channels filled with air to avoid cross-contamination of the different chambers. Then, the chip is sealed and placed on a flat thermal cycler for PCR. Finally, amplification products can be detected in situ using a fluorescence scanner or recovered by reverse centrifugation for further analyses. Therefore, our chip possesses two functions: 1) it can be used for multi-target detection based on end-point in situ fluorescence detection; and 2) it can work as a sample preparation unit for analyses that need multiplex PCR such as hybridization and target sequencing. The performance of this chip was carefully examined and further illustrated in the identification of 8 pathogenic bacterial genomic DNA samples and 13 drug-resistance genes. Due to simplicity of its structure and operation, accuracy and generality, high-throughput capacity, and versatile functions (i.e., for in situ detection and sample preparation), our multiplex PCR chip has great potential in clinical diagnostics and nucleic acid-based point-of-care testing. PMID:26016439

  11. Evaluation of PCR Based Assays for the Improvement of Proportion Estimation of Bacterial and Viral Pathogens in Diarrheal Surveillance

    PubMed Central

    Guan, Hongxia; Zhang, Jingyun; Xiao, Yong; Sha, Dan; Ling, Xia; Kan, Biao

    2016-01-01

    Diarrhea can be caused by a variety of bacterial, viral and parasitic organisms. Laboratory diagnosis is essential in the pathogen-specific burden assessment. In the pathogen spectrum monitoring in the diarrheal surveillance, culture methods are commonly used for the bacterial pathogens' detection whereas nucleic acid based amplification, the non-cultural methods are used for the viral pathogens. Different methodology may cause the inaccurate pathogen spectrum for the bacterial pathogens because of their different culture abilities with the different media, and for the comparison of bacterial vs. viral pathogens. The application of nucleic acid-based methods in the detection of viral and bacterial pathogens will likely increase the number of confirmed positive diagnoses, and will be comparable since all pathogens will be detected based on the same nucleic acid extracts from the same sample. In this study, bacterial pathogens, including diarrheagenic Escherichia coli (DEC), Salmonella spp., Shigella spp., Vibrio parahaemolyticus and V. cholerae, were detected in 334 diarrheal samples by PCR-based methods using nucleic acid extracted from stool samples and associated enrichment cultures. A protocol was established to facilitate the consistent identification of bacterial pathogens in diarrheal patients. Five common enteric viruses were also detected by RT-PCR, including rotavirus, sapovirus, norovirus (I and II), human astrovirus, and enteric adenovirus. Higher positive rates were found for the bacterial pathogens, showing the lower proportion estimation if only using culture methods. This application will improve the quality of bacterial diarrheagenic pathogen survey, providing more accurate information pertaining to the pathogen spectrum associated with finding of food safety problems and disease burden evaluation. PMID:27065958

  12. Real-time PCR-based assay for quantitative detection of Hematodinium sp. in the blue crab Callinectes sapidus.

    PubMed

    Nagle, L; Place, A R; Schott, E J; Jagus, R; Messick, G; Pitula, J S

    2009-03-01

    Hematodinium sp. is a parasitic dinoflagellate infecting the blue crab Callinectes sapidus and other crustaceans. PCR-based assays are currently being used to identify infections in crabs that would have been undetectable by traditional microscopic examination. We therefore sought to define the limits of quantitative PCR (qPCR) detection within the context of field collection protocols. We present a qPCR assay based on the Hematodinium sp. 18S rRNA gene that can detect 10 copies of the gene per reaction. Analysis of a cell dilution series vs. defined numbers of a cloned Hematodinium sp. 18S rRNA gene suggests a copy number of 10,000 per parasite and predicts a sensitivity of 0.001 cell equivalents. In practice, the assays are based on analysis of 1% of the DNA extracted from 200 microl of serum, yielding a theoretical detection limit of 5 cells ml(-1) hemolymph, assuming that 1 cell is present per sample. When applied to a limited field survey of blue crabs collected in Maryland coastal bays from May to August 2005, 24 of 128 crabs (18.8%) were identified as positive for Hematodinium sp. infection using qPCR. In comparison, only 6 of 128 crabs (4.7%) were identified as positive using traditional hemolymph microscopic examination. The qPCR method also detected the parasite in gill, muscle, heart and hepatopancreas tissues, with 17.2% of the crabs showing infection in at least one of these tissues. Importantly, it is now possible to enumerate parasites within defined quantities of crab tissue, which permits collection of more detailed information on the epizootiology of the pathogen. PMID:19419009

  13. Multicenter study using standardized protocols and reagents for evaluation of reproducibility of PCR-based fingerprinting of Acinetobacter spp.

    PubMed Central

    Grundmann, H J; Towner, K J; Dijkshoorn, L; Gerner-Smidt, P; Maher, M; Seifert, H; Vaneechoutte, M

    1997-01-01

    Seven laboratories in six European countries examined 40 isolates belonging to the Acinetobacter calcoaceticus-Acinetobacter baumannii complex to investigate whether standardized protocols and quality-controlled reagents could produce reliable, discriminatory, and reproducible PCR-based fingerprinting results. Four PCR protocols with different primers (primers DAF4, ERIC-2, M13, and REP1 + REP2) were used. The epidemiological conclusions reached by the participating laboratories were substantially correct, with 96.4% of the total isolate grouping allocations agreeing with the consensus view. All laboratories identified the main epidemiological clusters, and each laboratory also identified two non-outbreak-related isolates. There were no significant differences between the isolate grouping results obtained by the different protocols and with the different primers. Visual comparison indicated that the standardized protocols and reagents yielded reproducible fingerprint patterns, but with some variations in particular band intensities. Minor variations in fingerprint profiles were detected, but computer-assisted analysis of PCR fingerprints obtained on agarose gels demonstrated that 88.3 to 91.6% (depending on the source of DNA) of the patterns clustered correctly, while 96.4 to 98.9% of the patterns clustered correctly following automated high-resolution laser fluorescence analysis. Correlation of the patterns for isogenic isolates ranged from 83.3 to 86.6% but was slightly better (mean correlation, 87.1%) for centrally prepared DNA extracts than for DNA extracts prepared by individual laboratories (mean correlation, 84.7%). It was concluded that independently produced PCR fingerprint patterns can be obtained reproducibly for Acinetobacter spp. at the practical level if (i) quality-controlled reagents, (ii) standardized extraction of DNA, and (iii) standardized amplification conditions are used. PMID:9399496

  14. Evaluation of PCR Based Assays for the Improvement of Proportion Estimation of Bacterial and Viral Pathogens in Diarrheal Surveillance.

    PubMed

    Guan, Hongxia; Zhang, Jingyun; Xiao, Yong; Sha, Dan; Ling, Xia; Kan, Biao

    2016-01-01

    Diarrhea can be caused by a variety of bacterial, viral and parasitic organisms. Laboratory diagnosis is essential in the pathogen-specific burden assessment. In the pathogen spectrum monitoring in the diarrheal surveillance, culture methods are commonly used for the bacterial pathogens' detection whereas nucleic acid based amplification, the non-cultural methods are used for the viral pathogens. Different methodology may cause the inaccurate pathogen spectrum for the bacterial pathogens because of their different culture abilities with the different media, and for the comparison of bacterial vs. viral pathogens. The application of nucleic acid-based methods in the detection of viral and bacterial pathogens will likely increase the number of confirmed positive diagnoses, and will be comparable since all pathogens will be detected based on the same nucleic acid extracts from the same sample. In this study, bacterial pathogens, including diarrheagenic Escherichia coli (DEC), Salmonella spp., Shigella spp., Vibrio parahaemolyticus and V. cholerae, were detected in 334 diarrheal samples by PCR-based methods using nucleic acid extracted from stool samples and associated enrichment cultures. A protocol was established to facilitate the consistent identification of bacterial pathogens in diarrheal patients. Five common enteric viruses were also detected by RT-PCR, including rotavirus, sapovirus, norovirus (I and II), human astrovirus, and enteric adenovirus. Higher positive rates were found for the bacterial pathogens, showing the lower proportion estimation if only using culture methods. This application will improve the quality of bacterial diarrheagenic pathogen survey, providing more accurate information pertaining to the pathogen spectrum associated with finding of food safety problems and disease burden evaluation. PMID:27065958

  15. Evaluation of a Novel PCR-Based Assay for Detection and Identification of Chlamydia trachomatis Serovars in Cervical Specimens▿

    PubMed Central

    Quint, Koen; Porras, Carolina; Safaeian, Mahboobeh; González, Paula; Hildesheim, Allan; Quint, Wim; van Doorn, Leen-Jan; Silva, Sandra; Melchers, Willem; Schiffman, Mark; Rodríguez, Ana Cecilia; Wacholder, Sholom; Freer, Enrique; Cortes, Bernal; Herrero, Rolando

    2007-01-01

    The aims of this study were to compare a novel PCR-based Chlamydia trachomatis detection and genotyping (Ct-DT) assay with the FDA-approved, commercially available C. trachomatis detection Hybrid Capture 2 (HC2) assay and to investigate the C. trachomatis serovar distribution among young women in a rural Costa Rican study population. A total of 5,828 sexually active women participating in a community-based trial in Costa Rica were tested for C. trachomatis by HC2. A sample of 1,229 specimens consisting of 100% HC2 C. trachomatis-positive specimens (n = 827) and a random sample of 8% HC2 C. trachomatis-negative specimens (n = 402) were tested with the Ct-DT assay. Agreement between the two assays was determined by the unweighted kappa statistic. Discrepant specimens were tested with a second commercially available test (COBAS TaqMan). The Ct-DT-positive specimens were further analyzed with the Ct-DT genotyping step to investigate the distribution of 14 different C. trachomatis serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a, and L3). After accounting for the sampling fraction selected for Ct-DT testing, crude agreement with the HC2 assay was 98% and the kappa was 0.92 (95% confidence interval [CI], 0.89 to 0.97). The 33 discordant samples that were further analyzed with the COBAS TaqMan test showed better agreement with the Ct-DT assay (31/33, P < 0.001). Among the 806 Ct-DT-positive samples, serovar E was the most common serovar (31%), followed by serovars F and D (both 21%) and serovar I (15%). In conclusion, the novel Ct-DT assay permits reliable detection and identification of C. trachomatis serovars. PMID:17959760

  16. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences.

    PubMed

    Ågren, Joakim; Hamidjaja, Raditijo A; Hansen, Trine; Ruuls, Robin; Thierry, Simon; Vigre, Håkan; Janse, Ingmar; Sundström, Anders; Segerman, Bo; Koene, Miriam; Löfström, Charlotta; Van Rotterdam, Bart; Derzelle, Sylviane

    2013-11-15

    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S-layer, and prophage-lambda. Following a review of the literature, an in silico analysis of all signature sequences reported for identification of B. anthracis was conducted. Published primer and probe sequences were compared for specificity against 134 available Bacillus spp. genomes. Although many of the chromosomal targets evaluated are claimed to be specific to B. anthracis, cross-reactions with closely related B. cereus and B. thuringiensis strains were often observed. Of the 35 investigated PCR assays, only 4 were 100% specific for the B. anthracis chromosome. An interlaboratory ring trial among five European laboratories was then performed to evaluate six assays, including the WHO recommended procedures, using a collection of 90 Bacillus strains. Three assays performed adequately, yielding no false positive or negative results. All three assays target chromosomal markers located within the lambdaBa03 prophage region (PL3, BA5345, and BA5357). Detection limit was further assessed for one of these highly specific assays. PMID:24005110

  17. Aspergillus Collagen-Like Genes (acl): Identification, Sequence Polymorphism, and Assessment for PCR-Based Pathogen Detection

    PubMed Central

    Tuntevski, Kiril; Durney, Brandon C.; Snyder, Anna K.; LaSala, P. Rocco; Nayak, Ajay P.; Green, Brett J.; Beezhold, Donald H.; Rio, Rita V. M.; Holland, Lisa A.

    2013-01-01

    The genus Aspergillus is a burden to public health due to its ubiquitous presence in the environment, its production of allergens, and wide demographic susceptibility among cystic fibrosis, asthmatic, and immunosuppressed patients. Current methods of detection of Aspergillus colonization and infection rely on lengthy morphological characterization or nonstandardized serological assays that are restricted to identifying a fungal etiology. Collagen-like genes have been shown to exhibit species-specific conservation across the noncollagenous regions as well as strain-specific polymorphism in the collagen-like regions. Here we assess the conserved region of the Aspergillus collagen-like (acl) genes and explore the application of PCR amplicon size-based discrimination among the five most common etiologic species of the Aspergillus genus, including Aspergillus fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus. Genetic polymorphism and phylogenetic analysis of the aclF1 gene were additionally examined among the available strains. Furthermore, the applicability of the PCR-based assay to identification of these five species in cultures derived from sputum and bronchoalveolar fluid from 19 clinical samples was explored. Application of capillary electrophoresis on nanogels was additionally demonstrated to improve the discrimination between Aspergillus species. Overall, this study demonstrated that Aspergillus acl genes could be used as PCR targets to discriminate between clinically relevant Aspergillus species. Future studies aim to utilize the detection of Aspergillus acl genes in PCR and microfluidic applications to determine the sensitivity and specificity for the identification of Aspergillus colonization and invasive aspergillosis in immunocompromised subjects. PMID:24123732

  18. PCR-Based Method To Differentiate the Subspecies of the Mycobacterium tuberculosis Complex on the Basis of Genomic Deletions

    PubMed Central

    Huard, Richard C.; de Oliveira Lazzarini, Luiz Claudio; Butler, W. Ray; van Soolingen, Dick; Ho, John L.

    2003-01-01

    The classical Mycobacterium tuberculosis complex (MtbC) subspecies include Mycobacterium tuberculosis, Mycobacterium africanum (subtypes I and II), Mycobacterium bovis (along with the attenuated M. bovis bacillus Calmette-Guérin [BCG]), and Mycobacterium microti; increasingly recognized MtbC groupings include Mycobacterium bovis subsp. caprae and “Mycobacterium tuberculosis subsp. canettii.” Previous investigations have documented each MtbC subspecies as a source of animal and/or human tuberculosis. However, study of these organisms is hindered by the lack of a single protocol that quickly and easily differentiates all of the MtbC groupings. Towards this end we have developed a rapid, simple, and reliable PCR-based MtbC typing method that makes use of MtbC chromosomal region-of-difference deletion loci. Here, seven primer pairs (which amplify within the loci 16S rRNA, Rv0577, IS1561′, Rv1510, Rv1970, Rv3877/8, and Rv3120) were run in separate but simultaneous reactions. Each primer pair either specifically amplified a DNA fragment of a unique size or failed, depending upon the source mycobacterial DNA. The pattern of amplification products from all of the reactions, visualized by agarose gel electrophoresis, allowed immediate identification either as MtbC composed of M. tuberculosis (or M. africanum subtype II), M. africanum subtype I, M. bovis, M. bovis BCG, M. caprae, M. microti, or “M. canettii” or as a Mycobacterium other than MtbC (MOTT). This MtbC PCR typing panel provides an advanced approach to determine the subspecies of MtbC isolates and to differentiate them from clinically important MOTT species. It has proven beneficial in the management of Mycobacterium collections and may be applied for practical clinical and epidemiological use. PMID:12682155

  19. System for Multiplexing Acoustic Emission (AE) Instrumentation

    NASA Technical Reports Server (NTRS)

    Prosser, William H. (Inventor); Perey, Daniel F. (Inventor); Gorman, Michael R. (Inventor); Scales, Edgar F. (Inventor)

    2003-01-01

    An acoustic monitoring device has at least two acoustic sensors with a triggering mechanism and a multiplexing circuit. After the occurrence of a triggering event at a sensor, the multiplexing circuit allows a recording component to record acoustic emissions at adjacent sensors. The acoustic monitoring device is attached to a solid medium to detect the occurrence of damage.

  20. Reversible Cardiomyopathies

    PubMed Central

    Patel, Harsh; Madanieh, Raef; Kosmas, Constantine E; Vatti, Satya K; Vittorio, Timothy J

    2015-01-01

    Cardiomyopathies (CMs) have many etiological factors that can result in severe structural and functional dysregulation. Fortunately, there are several potentially reversible CMs that are known to improve when the root etiological factor is addressed. In this article, we discuss several of these reversible CMs, including tachycardia-induced, peripartum, inflammatory, hyperthyroidism, Takotsubo, and chronic illness–induced CMs. Our discussion also includes a review on their respective pathophysiology, as well as possible management solutions. PMID:26052233

  1. SQUID Multiplexers for Cryogenic Detector Arrays

    NASA Technical Reports Server (NTRS)

    Irwin, Kent; Beall, James; Deiker, Steve; Doriese, Randy; Duncan, William; Hilton, Gene; Moseley, S. Harvey; Reintsema, Carl; Stahle, Caroline; Ullom, Joel; Vale, Leila

    2004-01-01

    SQUID multiplexers make it possible to build arrays of thousands of cryogenic detectors with a manageable number of readout channels. We are developing time-division SQUID multiplexers based on Nb trilayer SQUIDs to read arrays of superconducting transition-edge sensors. Our first-generation, 8-channel SQUID multiplexer was used in FIBRE, a one-dimensional TES array for submillimeter astronomy. Our second-generation 32-pixel multiplexer, based on an improved architecture, has been developed for instruments including Constellation-X, SCUBA-2, and solar x-ray astronomy missions. SCUBA-2, which is being developed for the James Clerk Maxwell Telescope, will have more than 10,000 pixels. We are now developing a third-generation architecture based on superconducting hot-electron switches. The use of SQUID multiplexers in instruments operating at above 2 K will also be discussed.

  2. High-throughput SNP scoring with GAMMArrays: genomic analysis using multiplexed microsphere arrays

    NASA Astrophysics Data System (ADS)

    Green, Lance D.; Cai, Hong; Torney, David C.; Wood, Diane J.; Uribe-Romeo, Francisco J.; Kaderali, Lars; Nolan, John P.; White, P. S.

    2002-06-01

    We have developed a SNP scoring platform, yielding high throughput, inexpensive assays. The basic platform uses fluorescently labeled DNA fragments bound to microspheres, which are analyzed using flow cytometry. SNP scoring is performed using minisequencing primers and fluorescently labeled dideoxynucleotides. Furthermore, multiplexed microspheres make it possible to score hundreds of SNPs simultaneously. Multiplexing, coupled with high throughput rates allow inexpensive scoring of several million SNPs/day. GAMMArrays use universal tags that consist of computer designed, unique DNA tails. These are incorporated into each primer, and the reverse-component is attached to a discrete population of microspheres in a multiplexed set. This enables simultaneous minisequencing of many SNPs in solution, followed by capture onto the appropriate microsphere for multiplexed analysis by flow cytometry. We present results from multiplexed SNP analyses of bacterial pathogens, and human mtDNA variation. Analytes are performed on PCR amplicons, each containing numerous SNPs scored simultaneously. In addition, these assays easily integrate into conventional liquid handling automation, and require no unique instrumentation for setup and analysis. Very high signal-to-noise ratios, ease of setup, flexibility in format and scale, and low cost make these assays extremely versatile and valuable tools for a wide variety of SNP scoring applications.

  3. Information transport in multiplex networks

    NASA Astrophysics Data System (ADS)

    Pu, Cunlai; Li, Siyuan; Yang, Xianxia; Yang, Jian; Wang, Kai

    2016-04-01

    In this paper, we study information transport in multiplex networks comprised of two coupled subnetworks. The upper subnetwork, called the logical layer, employs the shortest paths protocol to determine the logical paths for packets transmission, while the lower subnetwork acts as the physical layer, in which packets are delivered by the biased random walk mechanism characterized with a parameter α. Through simulation, we obtain the optimal α corresponding to the maximum network lifetime and the maximum number of the arrival packets. Assortative coupling is better than random coupling and disassortative coupling, since it achieves better transmission performance. Generally, the more homogeneous the lower subnetwork is, the better the transmission performance, which is the opposite for the upper subnetwork. Finally, we propose an attack centrality for nodes based on the topological information of both subnetworks, and investigate the transmission performance under targeted attacks. Our work aids in understanding the spread and robustness issues of multiplex networks and provides some clues about the design of more efficient and robust routing architectures in communication systems.

  4. Development of multiplex PCR assay for authentication of Cornu Cervi Pantotrichum in traditional Chinese medicine based on cytochrome b and C oxidase subunit 1 genes.

    PubMed

    Gao, Lijun; Xia, Wei; Ai, Jinxia; Li, Mingcheng; Yuan, Guanxin; Niu, Jiamu; Fu, Guilian; Zhang, Lihua

    2016-07-01

    This study describes a method for discriminating the true Cervus antlers from its counterfeits using multiplex PCR. Bioinformatics were carried out to design the specific alleles primers for mitochondrial (mt) cytochrome b (Cyt b) and cytochrome C oxidase subunit 1 (Cox 1) genes. The mt DNA and genomic DNA were extracted from Cervi Cornu Pantotrichum through the modified alkaline and the salt-extracting method in addition to its counterfeits, respectively. Sufficient DNA templates were extracted from all samples used in two methods, and joint fragments of 354 bp and 543 bp that were specifically amplified from both of true Cervus antlers served as a standard control. The data revealed that the multiplex PCR-based assays using two primer sets can be used for forensic and quantitative identification of original Cervus deer products from counterfeit antlers in a single step. PMID:26287950

  5. 3D multiplexed immunoplasmonics microscopy

    NASA Astrophysics Data System (ADS)

    Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

    2016-07-01

    Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K+ channel subunit KV1.1) on human cancer CD44+ EGFR+ KV1.1+ MDA-MB-231 cells and reference CD44- EGFR- KV1.1+ 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed

  6. Demonstration of Array-Based Analysis for Highly Multiplexed PCR Assays

    PubMed Central

    Spence, Janice M.; Rothberg, Paul G.; Wang, Nancy; Burack, W. Richard

    2011-01-01

    We demonstrate an approach that allowed rapid development of a robust assay for the detection of chromosomal translocations. The method includes highly multiplexed PCR with analysis of the PCR products performed by array detection. As proof of principle, we applied this approach to the detection of IGH@-BCL2 translocations in DNA prepared from FFPE specimens. This translocation and specimen type were chosen because of the known difficulties associated with PCR-based detection of this lesion and the additional loss of sensitivity associated with FFPE samples. The multiplex PCR with array detection method detected the IGH@-BCL2 translocation in 26 of 36 FFPE follicular lymphoma specimens, whereas the BIOMED-2 assay detected 13 of 36 specimens. This increased sensitivity was the result of both the increased density of BCL2 primers and identification of PCR products by low-density array. The method was specific and allowed mapping of the BCL2 break point in all cases. The method detected the IGH@-BCL2 lesion when the tumor DNA was diluted more than 1:20 in normal DNA but not when it was diluted more than 1:100. This sensitivity allows detection of diagnostically relevant levels of IGH@-BCL2 but will not detect the rare cells with IGH@-BCL2 translocations in healthy individuals. PMID:21497287

  7. Antimicrobial resistance of Enterococcus species from meat and fermented meat products isolated by a PCR-based rapid screening method.

    PubMed

    Jahan, Musarrat; Krause, Denis O; Holley, Richard A

    2013-05-15

    Enterococci are predominantly found in the gastrointestinal tract of humans and animals, but species commonly resident on vegetation are known. Their presence in large numbers in foods may indicate a lapse in sanitation and their ability to serve as a genetic reservoir of transferable antibiotic resistance is of concern. Conventional culture methods for identification of enterococci are slow and sometimes give false results because of the biochemical diversity of the organisms in this genus. This work reports the development of a PCR-based assay to detect enterococci at the genus level by targeting a 16S rRNA sequence. Published 16S rRNA sequences were aligned and used to design genus specific primers (EntF and EntR). The primers were able to amplify a 678 bp target region from Enterococcus faecalis ATCC 7080 and 20 other strains of enterococci from 11 different species, but there was no amplification by 32 species from closely related genera (Pediococcus, Lactobacillus, Streptococcus and Listeria) or species of Escherichia coli and Salmonella. The PCR positive samples were plated, screened by a colony patch technique and their identities were confirmed by API 20 Strep panels and sequencing. When dry fermented sausage and ham as well as fresh meat batter for dry cured sausage manufacture were tested for enterococci by the method, 29 Enterococcus strains (15 E. faecalis, 13 E. faecium, and one E. gallinarum) were identified. When susceptibility of these enterococci to 12 antibiotics was tested, the highest incidence of resistance was to clindamycin (89.6%), followed by tetracycline hydrochloride (65.5%), tylosin (62%), erythromycin (45%), streptomycin and neomycin (17%), chloramphenicol (10.3%), penicillin (10.3%), ciprofloxacin (10.3%) and gentamicin (3.4%). None was resistant to the clinically important drugs vancomycin or ampicillin. Most strains (27/29) were resistant to more than one antibiotic while 17 of 29 strains were resistant to three to 8 antibiotics

  8. A comparative evaluation of PCR- based methods for species- specific determination of African animal trypanosomes in Ugandan cattle

    PubMed Central

    2013-01-01

    Background In recent years, PCR has been become widely applied for the detection of trypanosomes overcoming many of the constraints of parasitological and serological techniques, being highly sensitive and specific for trypanosome detection. Individual species-specific multi-copy trypanosome DNA sequences can be targeted to identify parasites. Highly conserved ribosomal RNA (rRNA) genes are also useful for comparisons between closely related species. The internal transcribed spacer regions (ITS) in particular are relatively small, show variability among related species and are flanked by highly conserved segments to which PCR primers can be designed. Individual variations in inter-species length makes the ITS region a useful marker for identification of multiple trypanosome species within a sample. Methods Six hundred blood samples from cattle collected in Uganda on FTA cards were screened using individual species-specific primers for Trypanosoma congolense, Trypanosoma brucei and Trypanosoma vivax and compared to a modified (using eluate extracted using chelex) ITS-PCR reaction. Results The comparative analysis showed that the species-specific primer sets showed poor agreement with the ITS primer set. Using species-specific PCR for Trypanozoon, a prevalence of 10.5% was observed as compared to 0.2% using ITS PCR (Kappa = 0.03). For Trypanosoma congolense, the species-specific PCR reaction indicated a prevalence of 0% compared to 2.2% using ITS PCR (Kappa = 0). For T. vivax, species-specific PCR detected prevalence of 5.7% compared to 2.8% for ITS PCR (Kappa = 0.29). Conclusions When selecting PCR based tools to apply to epidemiological surveys for generation of prevalence data for animal trypanosomiasis, it is recommended that species-specific primers are used, being the most sensitive diagnostic tool for screening samples to identify members of Trypanozoon (T. b. brucei s.l). While ITS primers are useful for studying the prevalence of trypanosomes

  9. 3D multiplexed immunoplasmonics microscopy.

    PubMed

    Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

    2016-07-21

    Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K(+) channel subunit KV1.1) on human cancer CD44(+) EGFR(+) KV1.1(+) MDA-MB-231 cells and reference CD44(-) EGFR(-) KV1.1(+) 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third

  10. Simultaneous detection of four causal agents of tobacco bushy top disease by a multiplex one-step RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tobacco bushy top disease is a complex disease caused by mixed infection of Tobacco bushy top virus (TBTV), Tobacco vein distorting virus (TVDV), satellite RNA of TBTV (Sat-TBTV) and Tobacco vein distorting virus associate RNA (TVDVaRNA). A one-tube multiplex reverse transcription-PCR (RT-PCR) assay...

  11. Structure of triadic relations in multiplex networks

    NASA Astrophysics Data System (ADS)

    Cozzo, Emanuele; Kivelä, Mikko; De Domenico, Manlio; Solé-Ribalta, Albert; Arenas, Alex; Gómez, Sergio; Porter, Mason A.; Moreno, Yamir

    2015-07-01

    Recent advances in the study of networked systems have highlighted that our interconnected world is composed of networks that are coupled to each other through different ‘layers’ that each represent one of many possible subsystems or types of interactions. Nevertheless, it is traditional to aggregate multilayer networks into a single weighted network in order to take advantage of existing tools. This is admittedly convenient, but it is also extremely problematic, as important information can be lost as a result. It is therefore important to develop multilayer generalizations of network concepts. In this paper, we analyze triadic relations and generalize the idea of transitivity to multiplex networks. By focusing on triadic relations, which yield the simplest type of transitivity, we generalize the concept and computation of clustering coefficients to multiplex networks. We show how the layered structure of such networks introduces a new degree of freedom that has a fundamental effect on transitivity. We compute multiplex clustering coefficients for several real multiplex networks and illustrate why one must take great care when generalizing standard network concepts to multiplex networks. We also derive analytical expressions for our clustering coefficients for ensemble averages of networks in a family of random multiplex networks. Our analysis illustrates that social networks have a strong tendency to promote redundancy by closing triads at every layer and that they thereby have a different type of multiplex transitivity from transportation networks, which do not exhibit such a tendency. These insights are invisible if one only studies aggregated networks.

  12. A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing.

    PubMed

    Parameswaran, Poornima; Jalili, Roxana; Tao, Li; Shokralla, Shadi; Gharizadeh, Baback; Ronaghi, Mostafa; Fire, Andrew Z

    2007-01-01

    Multiplexed high-throughput pyrosequencing is currently limited in complexity (number of samples sequenced in parallel), and in capacity (number of sequences obtained per sample). Physical-space segregation of the sequencing platform into a fixed number of channels allows limited multiplexing, but obscures available sequencing space. To overcome these limitations, we have devised a novel barcoding approach to allow for pooling and sequencing of DNA from independent samples, and to facilitate subsequent segregation of sequencing capacity. Forty-eight forward-reverse barcode pairs are described: each forward and each reverse barcode unique with respect to at least 4 nt positions. With improved read lengths of pyrosequencers, combinations of forward and reverse barcodes may be used to sequence from as many as n(2) independent libraries for each set of 'n' forward and 'n' reverse barcodes, for each defined set of cloning-linkers. In two pilot series of barcoded sequencing using the GS20 Sequencer (454/Roche), we found that over 99.8% of obtained sequences could be assigned to 25 independent, uniquely barcoded libraries based on the presence of either a perfect forward or a perfect reverse barcode. The false-discovery rate, as measured by the percentage of sequences with unexpected perfect pairings of unmatched forward and reverse barcodes, was estimated to be <0.005%. PMID:17932070

  13. Multiwavelength metasurfaces through spatial multiplexing

    PubMed Central

    Arbabi, Ehsan; Arbabi, Amir; Kamali, Seyedeh Mahsa; Horie, Yu; Faraon, Andrei

    2016-01-01

    Metasurfaces are two-dimensional arrangements of optical scatterers rationally arranged to control optical wavefronts. Despite the significant advances made in wavefront engineering through metasurfaces, most of these devices are designed for and operate at a single wavelength. Here we show that spatial multiplexing schemes can be applied to increase the number of operation wavelengths. We use a high contrast dielectric transmittarray platform with amorphous silicon nano-posts to demonstrate polarization insensitive metasurface lenses with a numerical aperture of 0.46, that focus light at 915 and 1550 nm to the same focal distance. We investigate two different methods, one based on large scale segmentation and one on meta-atom interleaving, and compare their performances. An important feature of this method is its simple generalization to adding more wavelengths or new functionalities to a device. Therefore, it provides a relatively straightforward method for achieving multi-functional and multiwavelength metasurface devices. PMID:27597568

  14. Code-multiplexed optical scanner

    NASA Astrophysics Data System (ADS)

    Riza, Nabeel A.; Arain, Muzammil A.

    2003-03-01

    A three-dimensional (3-D) optical-scanning technique is proposed based on spatial optical phase code activation on an input beam. This code-multiplexed optical scanner (C-MOS) relies on holographically stored 3-D beam-forming information. Proof-of-concept C-MOS experimental results by use of a photorefractive crystal as a holographic medium generates eight beams representing a basic 3-D voxel element generated via a binary-code matrix of the Hadamard type. The experiment demonstrates the C-MOS features of no moving parts, beam-forming flexibility, and large centimeter-size apertures. A novel application of the C-MOS as an optical security lock is highlighted.

  15. Cooperative epidemics on multiplex networks

    NASA Astrophysics Data System (ADS)

    Azimi-Tafreshi, N.

    2016-04-01

    The spread of one disease, in some cases, can stimulate the spreading of another infectious disease. Here, we treat analytically a symmetric coinfection model for spreading of two diseases on a two-layer multiplex network. We allow layer overlapping, but we assume that each layer is random and locally loopless. Infection with one of the diseases increases the probability of getting infected with the other. Using the generating function method, we calculate exactly the fraction of individuals infected with both diseases (so-called coinfected clusters) in the stationary state, as well as the epidemic spreading thresholds and the phase diagram of the model. With increasing cooperation, we observe a tricritical point and the type of transition changes from continuous to hybrid. Finally, we compare the coinfected clusters in the case of cooperating diseases with the so-called "viable" clusters in networks with dependencies.

  16. Multiplexed Primer Prediction for PCR

    Energy Science and Technology Software Center (ESTSC)

    2007-07-23

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

  17. Analog bus driver and multiplexer

    NASA Technical Reports Server (NTRS)

    Pain, Bedabrata (Inventor); Hancock, Bruce (Inventor); Cunningham, Thomas J. (Inventor)

    2012-01-01

    For a source-follower signal chain, the ohmic drop in the selection switch causes unacceptable voltage offset, non-linearity, and reduced small signal gain. For an op amp signal chain, the required bias current and the output noise rises rapidly with increasing the array format due to a rapid increase in the effective capacitance caused by the Miller effect boosting up the contribution of the bus capacitance. A new switched source-follower signal chain circuit overcomes limitations of existing op-amp based or source follower based circuits used in column multiplexers and data readout. This will improve performance of CMOS imagers, and focal plane read-out integrated circuits for detectors of infrared or ultraviolet light.

  18. Multiwavelength metasurfaces through spatial multiplexing.

    PubMed

    Arbabi, Ehsan; Arbabi, Amir; Kamali, Seyedeh Mahsa; Horie, Yu; Faraon, Andrei

    2016-01-01

    Metasurfaces are two-dimensional arrangements of optical scatterers rationally arranged to control optical wavefronts. Despite the significant advances made in wavefront engineering through metasurfaces, most of these devices are designed for and operate at a single wavelength. Here we show that spatial multiplexing schemes can be applied to increase the number of operation wavelengths. We use a high contrast dielectric transmittarray platform with amorphous silicon nano-posts to demonstrate polarization insensitive metasurface lenses with a numerical aperture of 0.46, that focus light at 915 and 1550 nm to the same focal distance. We investigate two different methods, one based on large scale segmentation and one on meta-atom interleaving, and compare their performances. An important feature of this method is its simple generalization to adding more wavelengths or new functionalities to a device. Therefore, it provides a relatively straightforward method for achieving multi-functional and multiwavelength metasurface devices. PMID:27597568

  19. Primers to block the amplification of symbiotic apostome ciliate 18S rRNA gene in a PCR-based copepod diet study

    NASA Astrophysics Data System (ADS)

    Yi, Xiaoyan; Zhang, Huan; Liu, Guangxing

    2014-05-01

    Pelagic copepods play an important role in the marine food web. However, a full understanding of the ecological status of this zooplankton group depends on the careful study of their natural diets. In previous PCR-based copepod diet studies, we found many apostome ciliates that live symbiotically under the exoskeleton of the copepods, and their sequences were often over-represented in the 18S rRNA gene (18S rDNA) libraries. As a first step to address this issue, we designed three apostome ciliate 18S rDNA blocking primers, and tested their blocking efficiency against apostome ciliate 18s rDNA under various PCR conditions. Using a semi-quantitative PCR method, we optimized the conditions to efficiently amplify the 18S rDNA of the prey while simultaneously excluding the symbiotic apostome ciliates. This technique will facilitate PCR-based diet studies of copepods and other zooplankton in their natural environments.

  20. A novel reversible carry-selected adder with low latency

    NASA Astrophysics Data System (ADS)

    Li, Ming-Cui; Zhou, Ri-Gui

    2016-07-01

    Reversible logic is getting more and more attention in quantum computing, optical computing, nanotechnology and low-power complementary metal oxide semiconductor designs since reversible circuits do not loose information during computation and have only small energy dissipation. In this paper, a novel carry-selected reversible adder is proposed primarily optimised for low latency. A 4-bit reversible full adder with two kinds of outputs, minimum delay and optimal quantum cost is presented as the building block for ?-bit reversible adder. Three new reversible gates NPG (new Peres gate), TEPG (triple extension of Peres gate) and RMUX21 (reversible 2-to-1 multiplexer) are proposed and utilised to design efficient adder units. The secondary carry propagation chain is carefully designed to reduce the time consumption. The novelty of the proposed design is the consideration of low latency. The comparative study shows that the proposed adder achieves the improvement from 61.46% to 95.29% in delay over the existing designs.

  1. Focus issue introduction: space-division multiplexing.

    PubMed

    Li, Guifang; Karlsson, Magnus; Liu, Xiang; Quiquempois, Yves

    2014-12-29

    Since the publication of the first focus issue [Opt. Express 19(11), 2011], single-fiber transmission capacity has eclipsed the 1 Pb/s mark. All aspects related to space-division multiplexing including fiber, passive components [(de)multiplexer, couplers], active components (EDFA and Raman amplifiers), switching and routing elements (ROADM and WSS), as well as transmission and networking have progressed rapidly. This focus issue is intended to bring together the most up-to-date research in space-division multiplexing, including fibers, passive and active components, transmission systems and networking. PMID:25607215

  2. 3D multiplexed immunoplasmonics microscopy

    NASA Astrophysics Data System (ADS)

    Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

    2016-07-01

    Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K+ channel subunit KV1.1) on human cancer CD44+ EGFR+ KV1.1+ MDA-MB-231 cells and reference CD44- EGFR- KV1.1+ 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed

  3. A method of multiplex PCR for detection of field released Beauveria bassiana, a fungal entomopathogen applied for pest management in jute (Corchorus olitorius).

    PubMed

    Biswas, Chinmay; Dey, Piyali; Gotyal, B S; Satpathy, Subrata

    2015-04-01

    The fungal entomopathogen Beauveria bassiana is a promising biocontrol agent for many pests. Some B. bassiana strains have been found effective against jute pests. To monitor the survival of field released B. bassiana a rapid and efficient detection technique is essential. Conventional methods such as plating method or direct culture method which are based on cultivation on selective media followed by microscopy are time consuming and not so sensitive. PCR based methods are rapid, sensitive and reliable. A single primer PCR may fail to amplify some of the strains. However, multiplex PCR increases the possibility of detection as it uses multiple primers. Therefore, in the present investigation a multiplex PCR protocol was developed by multiplexing three primers SCA 14, SCA 15 and SCB 9 to detect field released B. bassiana strains from soil as well as foliage of jute field. Using our multiplex PCR protocol all the five B. bassiana strains could be detected from soil and three strains viz., ITCC 6063, ITCC 4563 and ITCC 4796 could be detected even from the crop foliage after 45 days of spray. PMID:25680421

  4. Equivalence of time-multiplexed and frequency-multiplexed signals in digital communications.

    NASA Technical Reports Server (NTRS)

    Timor, U.

    1972-01-01

    In comparing different techniques for multiplexing N binary data signals into a single channel, time-division multiplexing (TDM) is known to have a theoretic efficiency of 100 percent (neglecting sync power) and thus seems to outperform frequency-division multiplexing systems (FDM). By considering more general FDM systems, we will show that both TDM and FDM are equivalent and have an efficiency of 100 percent. The difference between the systems is in the multiplexing and demultiplexing subsystems, but not in the performance or in the generated waveforms.

  5. Recent developments in multiplexing techniques for immunohistochemistry

    PubMed Central

    Dixon, Angela R; Bathany, Cédric; Tsuei, Michael; White, Joshua; Barald, Kate F; Takayama, Shuichi

    2016-01-01

    Methods to detect immuno-labelled molecules at increasingly higher resolution, even when present at low levels, are revolutionizing immunohistochemistry (IHC). These technologies can be valuable for management and examination of rare patient tissue specimens, and for improved accuracy of early disease detection. The purpose of this mini-review is to highlight recent multiplexing methods that are candidates for more prevalent use in clinical research and potential translation to the clinic. Multiplex IHC methods, which permit identification of at least 3 and up to 30 discrete antigens, have been divided into whole section staining and spatially-patterned staining categories. Associated signal enhancement technologies that can enhance performance and throughput of multiplex IHC assays are also discussed. Each multiplex IHC technique, detailed herein, is associated with several advantages as well as tradeoffs that must be taken into consideration for proper evaluation and use of the methods. PMID:26289603

  6. A design for a 32-channel multiplexer

    NASA Astrophysics Data System (ADS)

    Martinson, P. F.

    1981-01-01

    A time scan analog system multiplexer used for recording data during flight trials of unmanned aircraft navigation sensors is described. The 32 inputs are buffered, then multiplexed in groups of 8 onto 4 lines. These four are multiplexed onto one line and the signal passes through the output buffer to the recorder. Aircraft attitude, heading and height are recorded. Signals from a camera and a kinetheodolite tracking system are synchronized. Operating conditions were simulated using a helicopter. Noise and drift are due to the recorder. The multiplexer copes well with signals of several kHz bandwidth (raw data) and signals of a few Hz bandwidth (processed data, test signals). It can be used with any multitrack tape recorder having an FM recording format on at least two tracks. It can be converted for use in a digital telemetry system.

  7. Interlaboratory transfer of a PCR multiplex method for simultaneous detection of four genetically modified maize lines: Bt11, MON810, T25, and GA21.

    PubMed

    Hernández, Marta; Rodríguez-Lázaro, David; Zhang, David; Esteve, Teresa; Pla, Maria; Prat, Salomé

    2005-05-01

    The number of cultured hectares and commercialized genetically modified organisms (GMOs) has increased exponentially in the past 9 years. Governments in many countries have established a policy of labeling all food and feed containing or produced by GMOs. Consequently, versatile, laboratory-transferable GMO detection methods are in increasing demand. Here, we describe a qualitative PCR-based multiplex method for simultaneous detection and identification of four genetically modified maize lines: Bt11, MON810, T25, and GA21. The described system is based on the use of five primers directed to specific sequences in these insertion events. Primers were used in a single optimized multiplex PCR reaction, and sequences of the amplified fragments are reported. The assay allows amplification of the MON810 event from the 35S promoter to the hsp intron yielding a 468 bp amplicon. Amplification of the Bt11 and T25 events from the 35S promoter to the PAT gene yielded two different amplicons of 280 and 177 bp, respectively, whereas amplification of the 5' flanking region of the GA21 gave rise to an amplicon of 72 bp. These fragments are clearly distinguishable in agarose gels and have been reproduced successfully in a different laboratory. Hence, the proposed method comprises a rapid, simple, reliable, and sensitive (down to 0.05%) PCR-based assay, suitable for detection of these four GM maize lines in a single reaction. PMID:15853368

  8. Correlated edge overlaps in multiplex networks.

    PubMed

    Baxter, Gareth J; Bianconi, Ginestra; da Costa, Rui A; Dorogovtsev, Sergey N; Mendes, José F F

    2016-07-01

    We develop the theory of sparse multiplex networks with partially overlapping links based on their local treelikeness. This theory enables us to find the giant mutually connected component in a two-layer multiplex network with arbitrary correlations between connections of different types. We find that correlations between the overlapping and nonoverlapping links markedly change the phase diagram of the system, leading to multiple hybrid phase transitions. For assortative correlations we observe recurrent hybrid phase transitions. PMID:27575144

  9. Switchable holograms and approaches to storage multiplexing

    NASA Astrophysics Data System (ADS)

    Domash, Lawrence H.; Chen, Yong-Ming; Snowbell, Michael; Gozewski, Conrad M.

    1994-09-01

    Holographic data storage requires reference beam encoding for multiplexing and demultiplexing. Electrically switchable holographic composites (ESHC) based on a Polaroid photopolymer films are being investigated as a basis for several types of reference beam encoding devices. A laboratory demonstration reported here recorded four holograms in a photoresponsive storage medium. Both random phase encoding and angle multiplexing approaches were tested. ESHC encoding devices have much higher diffraction efficiency than spatial light modulators used in many other encoding schemes.

  10. Correlated edge overlaps in multiplex networks

    NASA Astrophysics Data System (ADS)

    Baxter, Gareth J.; Bianconi, Ginestra; da Costa, Rui A.; Dorogovtsev, Sergey N.; Mendes, José F. F.

    2016-07-01

    We develop the theory of sparse multiplex networks with partially overlapping links based on their local treelikeness. This theory enables us to find the giant mutually connected component in a two-layer multiplex network with arbitrary correlations between connections of different types. We find that correlations between the overlapping and nonoverlapping links markedly change the phase diagram of the system, leading to multiple hybrid phase transitions. For assortative correlations we observe recurrent hybrid phase transitions.

  11. Vasectomy reversal.

    PubMed

    Belker, A M

    1987-02-01

    A vasovasostomy may be performed on an outpatient basis with local anesthesia, but also may be performed on an outpatient basis with epidural or general anesthesia. Local anesthesia is preferred by most of my patients, the majority of whom choose this technique. With proper preoperative and intraoperative sedation, patients sleep lightly through most of the procedure. Because of the length of time often required for bilateral microsurgical vasoepididymostomy, epidural or general anesthesia and overnight hospitalization are usually necessary. Factors influencing the preoperative choice for vasovasostomy or vasoepididymostomy in patients undergoing vasectomy reversal are considered. The preoperative planned choice of vasovasostomy or vasoepididymostomy for patients having vasectomy reversal described herein does not have the support of all urologists who regularly perform these procedures. My present approach has evolved as the data reported in Tables 1 and 2 have become available, but it may change as new information is evaluated. However, it offers a logical method for planning choices of anesthesia and inpatient or outpatient status for patients undergoing vasectomy reversal procedures. PMID:3811050

  12. Multiplexing of fiber optic Bragg grating sensors

    NASA Astrophysics Data System (ADS)

    Chan, Kok Cheung Peter

    2000-11-01

    The main objective of this project was to develop a novel technique for multiplexing fiber Bragg grating sensors for strain measurements. Multiplexing is a very important issue for fiber Bragg grating sensors, as it allows them to be used for distributed sensing where their greatest impact is anticipated. Three types of multiplexed fiber Bragg grating sensor system prototypes were developed in this work. Most effort was devoted to a frequency-modulated continuous wave technique for multiplexing fiber Bragg grating sensors. A detailed mathematical analysis of the frequency-modulated continuous wave multiplexing technique was performed. It was identified that the technique can be used to multiplex up to 32 fiber Bragg grating sensors of the same nominal Bragg wavelength with a theoretical crosstalk performance of below -48 dB. This level of crosstalk corresponds to a wavelength detected error of well below 1 pm if fiber Bragg gratings having a bandwidth of around 0.2 nm are used. A few hundreds of sensors could be multiplexed by combining the frequency-modulated continuous wave technique with the well known wavelength-division-multiplexing technique. The practical factors which limit the performance, including the effect of biasing from the optimal working condition and the effect of non-ideal frequency sweeping intensity modulation, were investigated. The system performance, in terms of power budget and inter-sensor crosstalk for a serial and parallel architecture was also determined. A series of experiments were carried out to verify the principle of operation and to study the effects arising from the various practical performance limiting factors and from different network architectures. A three sensor system was experimentally demonstrated with -30 dB crosstalk level and with 2 μɛ resolution in terms of root-mean-square strain value. The system performance was found to be limited by the residual amplitude modulation due to the non-ideal frequency response of

  13. A novel, multiplex, real-time PCR–based approach for the detection of the commonly occurring pathogenic fungi and bacteria

    PubMed Central

    2013-01-01

    Background Polymerase chain reaction (PCR)-based techniques are widely used to identify fungal and bacterial infections. There have been numerous reports of different, new, real-time PCR-based pathogen identification methods although the clinical practicability of such techniques is not yet fully clarified. The present study focuses on a novel, multiplex, real-time PCR-based pathogen identification system developed for rapid differentiation of the commonly occurring bacterial and fungal causative pathogens of bloodstream infections. Results A multiplex, real-time PCR approach is introduced for the detection and differentiation of fungi, Gram-positive (G+) and Gram-negative (G-) bacteria. The Gram classification is performed with the specific fluorescence resonance energy transfer (FRET) probes recommended for LightCycler capillary real-time PCR. The novelty of our system is the use of a non-specific SYBR Green dye instead of labelled anchor probes or primers, to excite the acceptor dyes on the FRET probes. In conjunction with this, the use of an intercalating dye allows the detection of fungal amplicons. With the novel pathogen detection system, fungi, G + and G- bacteria in the same reaction tube can be differentiated within an hour after the DNA preparation via the melting temperatures of the amplicons and probes in the same tube. Conclusions This modified FRET technique is specific and more rapid than the gold-standard culture-based methods. The fact that fungi, G + and G- bacteria were successfully identified in the same tube within an hour after the DNA preparation permits rapid and early evidence-based management of bloodstream infections in clinical practice. PMID:24364823

  14. Multiplexed modulation of behavioral choice

    PubMed Central

    Palmer, Chris R.; Barnett, Megan N.; Copado, Saul; Gardezy, Fred; Kristan, William B.

    2014-01-01

    Stimuli in the environment, as well as internal states, influence behavioral choice. Of course, animals are often exposed to multiple external and internal factors simultaneously, which makes the ultimate determinants of behavior quite complex. We observed the behavioral responses of European leeches, Hirudo verbana, as we varied one external factor (surrounding water depth) with either another external factor (location of tactile stimulation along the body) or an internal factor (body distention following feeding). Stimulus location proved to be the primary indicator of behavioral response. In general, anterior stimulation produced shortening behavior, midbody stimulation produced local bending, and posterior stimulation usually produced either swimming or crawling but sometimes a hybrid of the two. By producing a systematically measured map of behavioral responses to body stimulation, we found wide areas of overlap between behaviors. When we varied the surrounding water depth, this map changed significantly, and a new feature – rotation of the body along its long axis prior to swimming – appeared. We found additional interactions between water depth and time since last feeding. A large blood meal initially made the animals crawl more and swim less, an effect that was attenuated as water depth increased. The behavioral map returned to its pre-feeding form after approximately 3 weeks as the leeches digested their blood meal. In summary, we found multiplexed impacts on behavioral choice, with the map of responses to tactile stimulation modified by water depth, which itself modulated the impact that feeding had on the decision to swim or crawl. PMID:24902753

  15. Intracavity absorption multiplexed sensor network based on dense wavelength division multiplexing filter.

    PubMed

    Zhang, Haiwei; Lu, Ying; Duan, Liangcheng; Zhao, Zhiqiang; Shi, Wei; Yao, Jianquan

    2014-10-01

    We report the system design and experimental verification of an intracavity absorption multiplexed sensor network with hollow core photonic crystal fiber (HCPCF) sensors and dense wavelength division multiplexing (DWDM) filters. Compared with fiber Bragg grating (FBG), it is easier for the DWDM to accomplish a stable output. We realize the concentration detection of three gas cells filled with acetylene. The sensitivity is up to 100 ppmV at 1536.71 nm. Voltage gradient is firstly used to optimize the intracavity sensor network enhancing the detection efficiency up to 6.5 times. To the best of our knowledge, DWDM is firstly used as a wavelength division multiplexing device to realize intracavity absorption multiplexed sensor network. It make it possible to realize high capacity intracavity sensor network via multiplexed technique. PMID:25322029

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

    PubMed

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

    2016-03-01

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

  17. PCR-based isolation and identification of full-length low-molecular-weight glutenin subunit genes in bread wheat (Triticum aestivum L.).

    PubMed

    Zhang, Xiaofei; Liu, Dongcheng; Jiang, Wei; Guo, Xiaoli; Yang, Wenlong; Sun, Jiazhu; Ling, Hongqing; Zhang, Aimin

    2011-12-01

    Low-molecular-weight glutenin subunits (LMW-GSs) are encoded by a multi-gene family and are essential for determining the quality of wheat flour products, such as bread and noodles. However, the exact role or contribution of individual LMW-GS genes to wheat quality remains unclear. This is, at least in part, due to the difficulty in characterizing complete sequences of all LMW-GS gene family members in bread wheat. To identify full-length LMW-GS genes, a polymerase chain reaction (PCR)-based method was established, consisting of newly designed conserved primers and the previously developed LMW-GS gene molecular marker system. Using the PCR-based method, 17 LMW-GS genes were identified and characterized in Xiaoyan 54, of which 12 contained full-length sequences. Sequence alignments showed that 13 LMW-GS genes were identical to those found in Xiaoyan 54 using the genomic DNA library screening, and the other four full-length LMW-GS genes were first isolated from Xiaoyan 54. In Chinese Spring, 16 unique LMW-GS genes were isolated, and 13 of them contained full-length coding sequences. Additionally, 16 and 17 LMW-GS genes in Dongnong 101 and Lvhan 328 (chosen from the micro-core collections of Chinese germplasm), respectively, were also identified. Sequence alignments revealed that at least 15 LMW-GS genes were common in the four wheat varieties, and allelic variants of each gene shared high sequence identities (>95%) but exhibited length polymorphism in repetitive regions. This study provides a PCR-based method for efficiently identifying LMW-GS genes in bread wheat, which will improve the characterization of complex members of the LMW-GS gene family and facilitate the understanding of their contributions to wheat quality. PMID:21830110

  18. The physiologic state of Escherichia coli O157:H7 does not affect its detection in two commercial real-time PCR-based tests

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Multiplex real-time PCR detection of Escherichia coli O157:H7 is an efficient molecular tool with high sensitivity and specificity for meat safety and quality assurance in the beef industry. The Biocontrol GDS and the DuPont Qualicon BAX®-RT rapid detection systems are two commercial tests based on...

  19. Method and apparatus for multiplexing switch signals

    NASA Technical Reports Server (NTRS)

    Hannaford, Blake (Inventor)

    1989-01-01

    Apparatus for multiplexing switch state signals comprises a plurality of switches and parallel weighted resistors connected in series between circuit ground and a node at a utilization device. The resistors are weighted as a function of a power of the same base, such as the power of the base 2, for coding the multiplexed switch state signals. A constant current source connected between the node and circuit ground drives current over a single cable conductor through the resistor. Each switch may be independently closed to change the switch state voltage signals multiplexed to the node. An analog-to-digital converter connected between the node and circuit ground demultiplexes the switch state signals received at the node and provides a switch state signal at each analog-to-digital output corresponding to the state of the switches at the moment. A potentiometer may replace a resistor and bypass switch combination in a position where the potentiometer has a maximum value of the lowest power of the base in order to multiplex a true analog voltage signal while switch state signals are unambiguously coded and multiplexed. The potentiometer in the least significant position permits the analog value to be in the range from 0 to a maximum corresponding to the least significant position of the switch state encoding. The invention may be used in redundancy systems by duplicating the invention with corresponding switches in each duplication ganged to open and close simultaneously upon operation of a pushbutton switch.

  20. Giant components in directed multiplex networks.

    PubMed

    Azimi-Tafreshi, N; Dorogovtsev, S N; Mendes, J F F

    2014-11-01

    We describe the complex global structure of giant components in directed multiplex networks that generalizes the well-known bow-tie structure, generic for ordinary directed networks. By definition, a directed multiplex network contains vertices of one type and directed edges of m different types. In directed multiplex networks, we distinguish a set of different giant components based on the existence of directed paths of different types between their vertices such that for each type of edges, the paths run entirely through only edges of that type. If, in particular, m=2, we define a strongly viable component as a set of vertices in which for each type of edges each two vertices are interconnected by at least two directed paths in both directions, running through the edges of only this type. We show that in this case, a directed multiplex network contains in total nine different giant components including the strongly viable component. In general, the total number of giant components is 3^{m}. For uncorrelated directed multiplex networks, we obtain exactly the size and the emergence point of the strongly viable component and estimate the sizes of other giant components. PMID:25493836

  1. Optical encryption system using quadrature multiplexing

    NASA Astrophysics Data System (ADS)

    Islam, Mohammed Nazrul; Alam, Mohammad S.

    2006-08-01

    Optical security systems have attracted much research interest recently for information security and fraud deterrent applications. A number of encryption techniques have been proposed in the literature, which includes double random-phase encryption, polarization encoding, encryption and verification using a multiplexed minimum average correlation energy phase-encrypted filter. Most of these reports employ a pseudo-random code for each information to be encrypted, where it requires individual storage capacity or transmission channel for further processing of each information. The objective of this paper is to develop an optical encryption system employing quadrature multiplexing to enhance the storage/transmission capacity of the system. Two information signals are encrypted using the same code but employing two orthogonal functions and then they are multiplexed together in the same domain. As the orthogonal functions have zero cross-correlation between them, so the encrypted information are expected to be unaffected by each other. Each encryption and multiplexing process can accommodate two information signals for a single code and a single storage cell or transmission channel. The same process can be performed in multiple steps to increase the multiplexing capability of the system. For decryption purpose, the composite encoded signal is correlated using the appropriate code and the appropriate function. The proposed technique has been found to work excellent in computer simulation with binary as well as gray level images. It has also been verified that the encrypted images remain secure, because no unwanted reproduction is possible without having the appropriate code and function.

  2. Chemically modified primers for improved multiplex PCR

    PubMed Central

    Shum, Jonathan; Paul, Natasha

    2009-01-01

    Multiplexed PCR, the amplification of multiple targets in a single reaction, presents a new set of challenges that further complicate more traditional PCR set-ups. These complications include a greater probability for non-specific amplicon formation and for imbalanced amplification of different targets, each of which can compromise quantification and detection of multiple targets. Despite these difficulties, multiplex PCR is frequently used in such applications as pathogen detection, RNA quantification, mutation analysis and now, next generation DNA sequencing. Herein, we investigate the utility of primers with one or two thermolabile 4-oxo-1-pentyl phosphotriester modifications in improving multiplex PCR performance. Initial endpoint and real-time analyses reveal a decrease in off-target amplification and subsequent increase in amplicon yield. Furthermore, the use of modified primers in multiplex set-ups revealed a greater limit of detection and more uniform amplification of each target as compared to unmodified primers. Overall, the thermolabile modified primers present a novel and exciting avenue in improving multiplex PCR performance. PMID:19258004

  3. Giant components in directed multiplex networks

    NASA Astrophysics Data System (ADS)

    Azimi-Tafreshi, N.; Dorogovtsev, S. N.; Mendes, J. F. F.

    2014-11-01

    We describe the complex global structure of giant components in directed multiplex networks that generalizes the well-known bow-tie structure, generic for ordinary directed networks. By definition, a directed multiplex network contains vertices of one type and directed edges of m different types. In directed multiplex networks, we distinguish a set of different giant components based on the existence of directed paths of different types between their vertices such that for each type of edges, the paths run entirely through only edges of that type. If, in particular, m =2 , we define a strongly viable component as a set of vertices in which for each type of edges each two vertices are interconnected by at least two directed paths in both directions, running through the edges of only this type. We show that in this case, a directed multiplex network contains in total nine different giant components including the strongly viable component. In general, the total number of giant components is 3m. For uncorrelated directed multiplex networks, we obtain exactly the size and the emergence point of the strongly viable component and estimate the sizes of other giant components.

  4. Rapid Simultaneous Detection of Enterovirus and Parechovirus RNAs in Clinical Samples by One-Step Real-Time Reverse Transcription-PCR Assay ▿

    PubMed Central

    Bennett, Susan; Harvala, Heli; Witteveldt, Jeroen; McWilliam Leitch, E. Carol; McLeish, Nigel; Templeton, Kate; Gunson, Rory; Carman, William F.; Simmonds, Peter

    2011-01-01

    Enteroviruses (EVs) are recognized as the major etiological agent in meningitis in children and young adults. The use of molecular techniques, such as PCR, has substantially improved the sensitivity of enterovirus detection compared to that of virus culture methods. PCR-based methods also can detect a much wider range of EV variants, including those within species A, as well as human parechoviruses (HPeVs) that often grow poorly in vitro and which previously have been underdiagnosed by traditional methods. To exploit these developments, we developed a real-time one-step reverse transcription-PCR (RT-PCR) for the rapid and sensitive detection of EV and HPeV in clinical specimens. Two commercially available RT-PCR kits were used (method I, Platinum one-step kit; method II, Express qPCR one-step kit) with primers and probes targeting the EV and HPeV 5′-untranslated regions (5′UTR). Amplification dynamics (threshold cycle [CT]values and efficiencies) of absolutely quantified full-length RNA transcripts representative of EV species A to D and HPeV were similar, demonstrating the effectiveness of both assays across the range of currently described human EV and HPeV variants. Probit analysis of multiple endpoint replicates demonstrated comparable sensitivities of the assays for EV and HPeV (method I, approximately 10 copies per reaction for both targets; method II, 20 copies per reaction). CT values were highly reproducible on repeat testing of positive controls within assays and between assay runs. Considering the sample turnaround time of less than 3 h, the multiplexed one-step RT-PCR method provides rapid diagnostic testing for EV and HPeV in cases of suspected central nervous system infections in a clinically relevant time frame. PMID:21593263

  5. Multiplexed image storage by electromagnetically induced transparency in a solid

    NASA Astrophysics Data System (ADS)

    Heinze, G.; Rentzsch, N.; Halfmann, T.

    2012-11-01

    We report on frequency- and angle-multiplexed image storage by electromagnetically induced transparency (EIT) in a Pr3+:Y2SiO5 crystal. Frequency multiplexing by EIT relies on simultaneous storage of light pulses in atomic coherences, driven in different frequency ensembles of the inhomogeneously broadened solid medium. Angular multiplexing by EIT relies on phase matching of the driving laser beams, which permits simultaneous storage of light pulses propagating under different angles into the crystal. We apply the multiplexing techniques to increase the storage capacity of the EIT-driven optical memory, in particular to implement multiplexed storage of larger two-dimensional amounts of data (images). We demonstrate selective storage and readout of images by frequency-multiplexed EIT and angular-multiplexed EIT, as well as the potential to combine both multiplexing approaches towards further enhanced storage capacities.

  6. An X-band SQUID Multiplexer

    NASA Astrophysics Data System (ADS)

    Hahn, I.; Bumble, B.; LeDuc, H. G.; Weilert, M.; Day, P.

    2006-09-01

    We are developing a microwave readout multiplexer for arrays of superconducting quantum interference devices (SQUIDs). A series of microwave resonators with frequencies ˜10 GHz are each loaded by a dc SQUID to a degree that depends on the flux state of the SQUID. By using resonators with high quality factors and slightly different resonance frequencies, many of these resonator-coupled SQUIDs may be read out with a single excitation line and cryogenic amplifier. Our multiplexer is similar to the one demonstrated by Irwin and Lehnert except for the use of higher-frequency, fully-lithographed transmission line resonators. We discuss a technique for modulating the SQUID array in series that alleviates the need to individually flux-bias the SQUIDs. The multiplexer has applications to the readout of detector arrays for astronomy as well as medical magnetic imaging.

  7. Superconducting Digital Multiplexers for Sensor Arrays

    NASA Technical Reports Server (NTRS)

    Kadin, Alan M.; Brock, Darren K.; Gupta, Deepnarayan

    2004-01-01

    Arrays of cryogenic microbolometers and other cryogenic detectors are being developed for infrared imaging. If the signal from each sensor is amplified, multiplexed, and digitized using superconducting electronics, then this data can be efficiently read out to ambient temperature with a minimum of noise and thermal load. HYPRES is developing an integrated system based on SQUID amplifiers, a high-resolution analog-to-digital converter (ADC) based on RSFQ (rapid single flux quantum) logic, and a clocked RSFQ multiplexer. The ADC and SQUIDs have already been demonstrated for other projects, so this paper will focus on new results of a digital multiplexer. Several test circuits have been fabricated using Nb Josephson technology and are about to be tested at T = 4.2 K, with a more complete prototype in preparation.

  8. Metric projection for dynamic multiplex networks.

    PubMed

    Jurman, Giuseppe

    2016-08-01

    Evolving multiplex networks are a powerful model for representing the dynamics along time of different phenomena, such as social networks, power grids, biological pathways. However, exploring the structure of the multiplex network time series is still an open problem. Here we propose a two-step strategy to tackle this problem based on the concept of distance (metric) between networks. Given a multiplex graph, first a network of networks is built for each time step, and then a real valued time series is obtained by the sequence of (simple) networks by evaluating the distance from the first element of the series. The effectiveness of this approach in detecting the occurring changes along the original time series is shown on a synthetic example first, and then on the Gulf dataset of political events. PMID:27626089

  9. Automated Methods for Multiplexed Pathogen Detection

    SciTech Connect

    Straub, Tim M.; Dockendorff, Brian P.; Quinonez-Diaz, Maria D.; Valdez, Catherine O.; Shutthanandan, Janani I.; Tarasevich, Barbara J.; Grate, Jay W.; Bruckner-Lea, Cindy J.

    2005-09-01

    Detection of pathogenic microorganisms in environmental samples is a difficult process. Concentration of the organisms of interest also co-concentrates inhibitors of many end-point detection methods, notably, nucleic acid methods. In addition, sensitive, highly multiplexed pathogen detection continues to be problematic. The primary function of the BEADS (Biodetection Enabling Analyte Delivery System) platform is the automated concentration and purification of target analytes from interfering substances, often present in these samples, via a renewable surface column. In one version of BEADS, automated immunomagnetic separation (IMS) is used to separate cells from their samples. Captured cells are transferred to a flow-through thermal cycler where PCR, using labeled primers, is performed. PCR products are then detected by hybridization to a DNA suspension array. In another version of BEADS, cell lysis is performed, and community RNA is purified and directly labeled. Multiplexed detection is accomplished by direct hybridization of the RNA to a planar microarray. The integrated IMS/PCR version of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river water samples. Multiplexed PCR assays for the simultaneous detection of E. coli O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for the detection of as few as 100 cells for each organism. Results for the RNA version of BEADS are also showing promising results. Automation yields highly purified RNA, suitable for multiplexed detection on microarrays, with microarray detection specificity equivalent to PCR. Both versions of the BEADS platform show great promise for automated pathogen detection from environmental samples. Highly multiplexed pathogen detection using PCR continues to be problematic, but may be required for trace detection in large volume samples. The RNA approach solves the issues of highly multiplexed PCR and provides ''live vs. dead'' capabilities. However

  10. Chromosome-Directed PCR-Based Detection and Quantification of Bacillus cereus Group Members with Focus on B. thuringiensis Serovar israelensis Active against Nematoceran Larvae

    PubMed Central

    Hendriksen, Niels B.; Melin, Petter; Lundström, Jan O.; Sundh, Ingvar

    2015-01-01

    Bacillus thuringiensis serovar israelensis is a wide-spread soil bacterium affiliated with the B. cereus group (Bcg) and is widely used in biocontrol products applied against mosquito and black fly larvae. For monitoring and quantification of applied B. thuringiensis serovar israelensis and its effect on indigenous B. thuringiensis serovar israelensis and Bcg assemblages, efficient and reliable tools are essential. The abundance and properties of B. thuringiensis serovar israelensis strains in the environment traditionally have been investigated with cultivation-dependent techniques, which are hampered by low sensitivity and the morphological similarity between B. cereus and B. thuringiensis. Currently available PCR-based detection and quantification tools target markers located on plasmids. In this study, a new cultivation-independent PCR-based method for efficient and specific quantification of B. thuringiensis serovar israelensis and Bcg is presented, utilizing two sets of PCR primers targeting the bacterial chromosome. Sequence database searches and empirical tests performed on target and nontarget species, as well as on bulk soil DNA samples, demonstrated that this diagnostic tool is specific for B. thuringiensis serovar israelensis and Bcg. The method will be useful for comparisons of Bcg and B. thuringiensis serovar israelensis abundances in the same samples. Moreover, the effect of B. thuringiensis serovar israelensis-based insecticide application on the total Bcg assemblages, including indigenous populations, can be investigated. This type of information is valuable in risk assessment and policy making for use of B. thuringiensis serovar israelensis in the environment. PMID:25979887

  11. Eigenvector centrality of nodes in multiplex networks.

    PubMed

    Solá, Luis; Romance, Miguel; Criado, Regino; Flores, Julio; García del Amo, Alejandro; Boccaletti, Stefano

    2013-09-01

    We extend the concept of eigenvector centrality to multiplex networks, and introduce several alternative parameters that quantify the importance of nodes in a multi-layered networked system, including the definition of vectorial-type centralities. In addition, we rigorously show that, under reasonable conditions, such centrality measures exist and are unique. Computer experiments and simulations demonstrate that the proposed measures provide substantially different results when applied to the same multiplex structure, and highlight the non-trivial relationships between the different measures of centrality introduced. PMID:24089967

  12. Line graphs for a multiplex network.

    PubMed

    Criado, Regino; Flores, Julio; García Del Amo, Alejandro; Romance, Miguel; Barrena, Eva; Mesa, Juan A

    2016-06-01

    It is well known that line graphs offer a good summary of the graphs properties, which make them easier to analyze and highlight the desired properties. We extend the concept of line graph to multiplex networks in order to analyze multi-plexed and multi-layered networked systems. As these structures are very rich, different approaches to this notion are required to capture a variety of situations. Some relationships between these approaches are established. Finally, by means of some simulations, the potential utility of this concept is illustrated. PMID:27368798

  13. Cooperative spreading processes in multiplex networks

    NASA Astrophysics Data System (ADS)

    Wei, Xiang; Chen, Shihua; Wu, Xiaoqun; Ning, Di; Lu, Jun-an

    2016-06-01

    This study is concerned with the dynamic behaviors of epidemic spreading in multiplex networks. A model composed of two interacting complex networks is proposed to describe cooperative spreading processes, wherein the virus spreading in one layer can penetrate into the other to promote the spreading process. The global epidemic threshold of the model is smaller than the epidemic thresholds of the corresponding isolated networks. Thus, global epidemic onset arises in the interacting networks even though an epidemic onset does not arise in each isolated network. Simulations verify the analysis results and indicate that cooperative spreading processes in multiplex networks enhance the final infection fraction.

  14. Multiplexed imaging of intracellular protein networks.

    PubMed

    Grecco, Hernán E; Imtiaz, Sarah; Zamir, Eli

    2016-08-01

    Cellular functions emerge from the collective action of a large number of different proteins. Understanding how these protein networks operate requires monitoring their components in intact cells. Due to intercellular and intracellular molecular variability, it is important to monitor simultaneously multiple components at high spatiotemporal resolution. However, inherent trade-offs narrow the boundaries of achievable multiplexed imaging. Pushing these boundaries is essential for a better understanding of cellular processes. Here the motivations, challenges and approaches for multiplexed imaging of intracellular protein networks are discussed. © 2016 International Society for Advancement of Cytometry. PMID:27183498

  15. Nanowire sensors for multiplexed detection of biomolecules

    PubMed Central

    He, Bo; Morrow, Thomas J; Keating, Christine D

    2009-01-01

    Nanowire-based detection strategies provide promising new routes to bioanalysis that could one day revolutionize the healthcare industry. This review covers recent developments in nanowire sensors for multiplexed detection of biomolecules such as nucleic acids and proteins. We focus on encoded nanowire suspension arrays and semiconductor nanowire-based field-effect transistors. Nanowire assembly and integration with microchip technology is emphasized as a key step toward the ultimate goal of multiplexed detection at the point of care using portable, low power, electronic biosensor chips. PMID:18804551

  16. Multimode fiber optic wavelength division multiplexing

    NASA Technical Reports Server (NTRS)

    Spencer, J. L.

    1982-01-01

    Optical wavelength division multiplexing (WDM) systems, with signals transmitted on different wavelengths through a single optical fiber, can have increased bandwidth and fault isolation properties over single wavelength optical systems. Two WDM system designs that might be used with multimode fibers are considered and a general description of the components which could be used to implement the system are given. The components described are sources, multiplexers, demultiplexers, and detectors. Emphasis is given to the demultiplexer technique which is the major developmental component in the WDM system.

  17. Line graphs for a multiplex network

    NASA Astrophysics Data System (ADS)

    Criado, Regino; Flores, Julio; García del Amo, Alejandro; Romance, Miguel; Barrena, Eva; Mesa, Juan A.

    2016-06-01

    It is well known that line graphs offer a good summary of the graphs properties, which make them easier to analyze and highlight the desired properties. We extend the concept of line graph to multiplex networks in order to analyze multi-plexed and multi-layered networked systems. As these structures are very rich, different approaches to this notion are required to capture a variety of situations. Some relationships between these approaches are established. Finally, by means of some simulations, the potential utility of this concept is illustrated.

  18. Polarization-multiplexed encoding at nanometer scales.

    PubMed

    Macias-Romero, C; Munro, P R T; Török, P

    2014-10-20

    Optical data storage was developed using binary encoding primarily due to signal to noise ratio considerations. We report on a multiplexing method that allows a seven fold storage increase, per storage layer, per side, and propose one that can yield theoretically a 20+ fold increase. Multiplexing is achieved by encoding information in polarization via appropriately oriented nanostructures that emit strongly polarized light when excited by unpolarized light. The storage increase is possible due to the significantly reduced crosstalk that results form using unpolarized light. PMID:25401656

  19. Wavelength-division multiplexing of nano-optomechanical doubly clamped beam systems.

    PubMed

    Sauer, Vincent T K; Diao, Zhu; Freeman, Mark R; Hiebert, Wayne K

    2015-05-01

    Wavelength-division multiplexing is demonstrated for a set of two doubly clamped beams. Using a single input/output waveguide in a nanophotonic detection system, the two mechanical beams are independently addressable using different wavelength channels as determined by their respective racetrack resonator detection cavities. The two cavities slightly overlap, which also enables the mechanical frequency of both beams to be detected simultaneously with a single wavelength. Finally, to physically map which wavelength channel corresponds to which specific device, a heating laser is targeted individually on each beam to create a reversible mechanical frequency shift. This multiplexing method would allow for the simpler detection of large arrays of nanomechanical devices in a sensor system. PMID:25927755

  20. Real-time multiplex PCR assays for reliable detection of Clostridium perfringens toxin genes in animal isolates.

    PubMed

    Albini, S; Brodard, I; Jaussi, A; Wollschlaeger, N; Frey, J; Miserez, R; Abril, C

    2008-02-01

    Typing of Clostridium perfringens strains by PCR-based determination of toxin genes proved to be a reliable method for diagnosis of enterotoxaemia in various animal species. We report the establishment and validation of three real-time fluorogenic (TaqMan) multiplex PCRs for the detection of C. perfringens alpha-, beta-, beta2-, epsilon-, entero- and iota-toxin genes. The composition of the PCRs was chosen with regard to robustness of the assays and in order to increase sensitivity compared to the conventional simplex PCRs. The combination of probe dyes selected for the real-time assays (FAM/TAMRA, Cy-5/BHQ-2 and VIC/TAMRA) as well as the designation of the chromosome-borne alpha-toxin as internal positive control allowed the creation of highly specific and sensitive, as well as time and cost effective PCRs. One hundred and three strains of C. perfringens isolated in Switzerland derived from clinical or suspected cases of enterotoxaemia in 10 different animal species were tested. The toxin genotypes were in agreement in both the conventional PCRs and the newly designed multiplex PCRs. Furthermore, the real-time PCR carried out as simplex allows to quantitate the copy numbers of plasmid-borne toxin genes in relation to the chromosomally located alpha-toxin gene. PMID:17855025

  1. Development of a Multiplex PCR Assay for Rapid Molecular Serotyping of Haemophilus parasuis

    PubMed Central

    Peters, Sarah E.; Wang, Jinhong; Hernandez-Garcia, Juan; Weinert, Lucy A.; Luan, Shi-Lu; Chaudhuri, Roy R.; Angen, Øystein; Aragon, Virginia; Williamson, Susanna M.; Langford, Paul R.; Rycroft, Andrew N.; Wren, Brendan W.; Maskell, Duncan J.; Tucker, Alexander W.

    2015-01-01

    Haemophilus parasuis causes Glässer's disease and pneumonia in pigs. Indirect hemagglutination (IHA) is typically used to serotype this bacterium, distinguishing 15 serovars with some nontypeable isolates. The capsule loci of the 15 reference strains have been annotated, and significant genetic variation was identified between serovars, with the exception of serovars 5 and 12. A capsule locus and in silico serovar were identified for all but two nontypeable isolates in our collection of >200 isolates. Here, we describe the development of a multiplex PCR, based on variation within the capsule loci of the 15 serovars of H. parasuis, for rapid molecular serotyping. The multiplex PCR (mPCR) distinguished between all previously described serovars except 5 and 12, which were detected by the same pair of primers. The detection limit of the mPCR was 4.29 × 105 ng/μl bacterial genomic DNA, and high specificity was indicated by the absence of reactivity against closely related commensal Pasteurellaceae and other bacterial pathogens of pigs. A subset of 150 isolates from a previously sequenced H. parasuis collection was used to validate the mPCR with 100% accuracy compared to the in silico results. In addition, the two in silico-nontypeable isolates were typeable using the mPCR. A further 84 isolates were analyzed by mPCR and compared to the IHA serotyping results with 90% concordance (excluding those that were nontypeable by IHA). The mPCR was faster, more sensitive, and more specific than IHA, enabling the differentiation of 14 of the 15 serovars of H. parasuis. PMID:26424843

  2. A Multiplexed Diagnostic Platform for Point-of-Care Pathogen Detection

    SciTech Connect

    Regan, J F; Letant, S E; Adams, K L; Mahnke, R C; Nguyen, N T; Dzenitis, J M; Hindson, B J; Hadley, D R; Makarewicz, T J; Henderer, B D; Breneman, J W; Tammero, L F; Ortiz, J I; Derlet, R W; Cohen, S; Colston, W W; McBride, M T; Birch, J M

    2008-02-04

    We developed an automated point-of-care diagnostic instrument that is capable of analyzing nasal swab samples for the presence of respiratory diseases. This robust instrument, called FluIDx, performs autonomous multiplexed RT-PCR reactions that are analyzed by microsphere xMAP technology. We evaluated the performance of FluIDx, in comparison rapid tests specific for influenza and respiratory syncytial virus, in a clinical study performed at the UC Davis Medical Center. The clinical study included samples positive for RSV (n = 71), influenza A (n = 16), influenza B (n = 4), adenovirus (n = 5), parainfluenza virus (n = 2), and 44 negative samples, according to a composite reference method. FluIDx and the rapid tests detected 85.9% and 62.0% of the RSV positive samples, respectively. Similar sensitivities were recorded for the influenza B samples; whereas the influenza A samples were poorly detected, likely due to the utilization of an influenza A signature that did not accurately match currently circulating influenza A strains. Data for all pathogens were compiled and indicate that FluIDx is more sensitive than the rapid tests, detecting 74.2% (95% C.I. of 64.7-81.9%) of the positive samples in comparison to 53.6% (95% C.I. of 43.7-63.2%) for the rapid tests. The higher sensitivity of FluIDx was partially offset by a lower specificity, 77.3% versus 100.0%. Overall, these data suggest automated flow-through PCR-based instruments that perform multiplexed assays can successfully screen clinical samples for infectious diseases.

  3. PCR-Based Serotyping of Streptococcus pneumoniae from Culture-Negative Specimens: Novel Primers for Detection of Serotypes within Serogroup 18.

    PubMed

    Tanmoy, Arif M; Saha, Senjuti; Darmstadt, Gary L; Whitney, Cynthia G; Saha, Samir K

    2016-08-01

    Six multiplex-compatible PCR primers were designed to distinguish Streptococcus pneumoniae serotypes within serogroup 18 from culturable/nonculturable pneumococcal specimens, with no cross-reactivity with other serotypes and respiratory organisms. These primers will aid in the generation of better data on vaccine/nonvaccine serotypes in invasive and carriage pneumococcal surveillance and contribute to future vaccine formulation and impact studies. PMID:27252464

  4. PCR-Based Serotyping of Streptococcus pneumoniae from Culture-Negative Specimens: Novel Primers for Detection of Serotypes within Serogroup 18

    PubMed Central

    Tanmoy, Arif M.; Saha, Senjuti; Darmstadt, Gary L.; Whitney, Cynthia G.

    2016-01-01

    Six multiplex-compatible PCR primers were designed to distinguish Streptococcus pneumoniae serotypes within serogroup 18 from culturable/nonculturable pneumococcal specimens, with no cross-reactivity with other serotypes and respiratory organisms. These primers will aid in the generation of better data on vaccine/nonvaccine serotypes in invasive and carriage pneumococcal surveillance and contribute to future vaccine formulation and impact studies. PMID:27252464

  5. Microwave multiplex readout for superconducting sensors

    NASA Astrophysics Data System (ADS)

    Ferri, E.; Becker, D.; Bennett, D.; Faverzani, M.; Fowler, J.; Gard, J.; Giachero, A.; Hays-Wehle, J.; Hilton, G.; Maino, M.; Mates, J.; Puiu, A.; Nucciotti, A.; Reintsema, C.; Schmidt, D.; Swetz, D.; Ullom, J.; Vale, L.

    2016-07-01

    The absolute neutrino mass scale is still an outstanding challenge in both particle physics and cosmology. The calorimetric measurement of the energy released in a nuclear beta decay is a powerful tool to determine the effective electron-neutrino mass. In the last years, the progress on low temperature detector technologies has allowed to design large scale experiments aiming at pushing down the sensitivity on the neutrino mass below 1 eV. Even with outstanding performances in both energy (~ eV on keV) and time resolution (~ 1 μs) on the single channel, a large number of detectors working in parallel is required to reach a sub-eV sensitivity. Microwave frequency domain readout is the best available technique to readout large array of low temperature detectors, such as Transition Edge Sensors (TESs) or Microwave Kinetic Inductance Detectors (MKIDs). In this way a multiplex factor of the order of thousands can be reached, limited only by the bandwidth of the available commercial fast digitizers. This microwave multiplexing system will be used to readout the HOLMES detectors, an array of 1000 microcalorimeters based on TES sensors in which the 163Ho will be implanted. HOLMES is a new experiment for measuring the electron neutrino mass by means of the electron capture (EC) decay of 163Ho. We present here the microwave frequency multiplex which will be used in the HOLMES experiment and the microwave frequency multiplex used to readout the MKID detectors developed in Milan as well.

  6. Highly multiplexed DNA sequencing by capillary electrophoresis

    SciTech Connect

    Yeung, E.S.; Ueno, K.; Chang, H.T.

    1994-12-31

    It is obvious that irrespective of whichever basic technology is eventually selected to sequence the entire human genome there are substantial gains to be made if a high degree of multiplexing of parallel runs can be implemented. Such multiplexing should not involve expensive instrumentation and should not require additional personnel, or else the main objective of cost reduction will not be satisfied even though the total time for sequencing is reduced. In the last two years, several research groups have shown that capillary electrophoresis (CE) is an attractive alternative for DNA sequencing. Part of the improvement in sequencing speed in CE is counteracted by the inherent ability of slab gels for accommodating multiple lanes in a single run. Recently, the authors have developed several excitation schemes for highly multiplexed capillary electrophoresis. Detection at the pM level was demonstrated. The authors report here the use of a novel excitation geometry to simultaneously monitor 100 capillary tubes during electrophoresis. This represents a truly parallel multiplexing scheme for high-speed DNA sequencing.

  7. Moving through a multiplex holographic scene

    NASA Astrophysics Data System (ADS)

    Mrongovius, Martina

    2013-02-01

    This paper explores how movement can be used as a compositional element in installations of multiplex holograms. My holographic images are created from montages of hand-held video and photo-sequences. These spatially dynamic compositions are visually complex but anchored to landmarks and hints of the capturing process - such as the appearance of the photographer's shadow - to establish a sense of connection to the holographic scene. Moving around in front of the hologram, the viewer animates the holographic scene. A perception of motion then results from the viewer's bodily awareness of physical motion and the visual reading of dynamics within the scene or movement of perspective through a virtual suggestion of space. By linking and transforming the physical motion of the viewer with the visual animation, the viewer's bodily awareness - including proprioception, balance and orientation - play into the holographic composition. How multiplex holography can be a tool for exploring coupled, cross-referenced and transformed perceptions of movement is demonstrated with a number of holographic image installations. Through this process I expanded my creative composition practice to consider how dynamic and spatial scenes can be conveyed through the fragmented view of a multiplex hologram. This body of work was developed through an installation art practice and was the basis of my recently completed doctoral thesis: 'The Emergent Holographic Scene — compositions of movement and affect using multiplex holographic images'.

  8. Immunity of multiplex networks via acquaintance vaccination

    NASA Astrophysics Data System (ADS)

    Wang, Zhen; Zhao, Da-Wei; Wang, Lin; Sun, Gui-Quan; Jin, Zhen

    2015-11-01

    How to find the effective approach of immunizing a population is one open question in the research of complex systems. Up to now, there have been a great number of works focusing on the efficiency of various immunization strategies. However, the majority of these existing achievements are limited to isolated networks, how immunization affects disease spreading in multiplex networks seems to need further exploration. In this letter, we explore the impact of the acquaintance immunization in multiplex networks, where two kinds of immunization strategies, multiplex node-based acquaintance immunization and layer node-based acquaintance immunization, are proposed. With the generating function method, our theoretical framework is able to accurately calculate the critical immunization threshold which is one of the most important indexes to predict the epidemic regime. Moreover, we further uncover that, with the increment of degree correlation between network layers, the immunization threshold declines for multiplex node-based acquaintance immunization, but slowly increases for layer node-based acquaintance immunization.

  9. Multiplexing schemes for quantum repeater networks

    NASA Astrophysics Data System (ADS)

    Aparicio, Luciano; Van Meter, Rodney

    2011-08-01

    When built, quantum repeaters will allow the distribution of entangled quantum states across large distances, playing a vital part in many proposed quantum technologies. Enabling multiple users to connect through the same network will be key to their real-world deployment. Previous work on repeater technologies has focussed only on simple entanglment production, without considering the issues of resource scarcity and competition that necessarily arise in a network setting. In this paper we simulated a thirteen-node network with up to five flows sharing different parts of the network, measuring the total throughput and fairness for each case. Our results suggest that the Internet-like approach of statistical multiplexing use of a congested link gives the highest aggregate throughput. Time division multiplexing and buffer space multiplexing were slightly less effective, but all three schemes allow the sum of multiple flows to substantially exceed that of any one flow, improving over circuit switching by taking advantage of resources that are forced to remain idle in circuit switching. All three schemes proved to have excellent fairness. The high performance, fairness and simplicity of implementation support a recommendation of statistical multiplexing for shared quantum repeater networks.

  10. Fiber optics wavelength division multiplexing(components)

    NASA Technical Reports Server (NTRS)

    Hendricks, Herbert D.

    1985-01-01

    The long term objectives are to develop optical multiplexers/demultiplexers, different wavelength and modulation stable semiconductor lasers and high data rate transceivers, as well as to test and evaluate fiber optic networks applicable to the Space Station. Progress in each of the above areas is briefly discussed.

  11. Reverse Phase Protein Arrays for Compound Profiling.

    PubMed

    Moerke, Nathan; Fallahi-Sichani, Mohammad

    2016-01-01

    Reverse phase protein arrays (RPPAs), also called reverse phase lysate arrays (RPLAs), involve immobilizing cell or tissue lysates, in small spots, onto solid supports which are then probed with primary antibodies specific for proteins or post-translational modifications of interest. RPPA assays are well suited for large-scale, high-throughput measurement of protein and PTM levels in cells and tissues. RPPAs are affordable and highly multiplexable, as a large number of arrays can readily be produced in parallel and then probed separately with distinct primary antibodies. This article describes a procedure for treating cells and preparing cell lysates, as well as a procedure for generating RPPAs using these lysates. A method for probing, imaging, and analyzing RPPAs is also described. These procedures are readily adaptable to a wide range of studies of cell signaling in response to drugs and other perturbations. © 2016 by John Wiley & Sons, Inc. PMID:27622568

  12. Antibody-Based Protein Multiplex Platforms: Technical and Operational Challenges

    PubMed Central

    Ellington, Allison A.; Kullo, Iftikhar J.; Bailey, Kent R.; Klee, George G.

    2010-01-01

    BACKGROUND The measurement of multiple protein biomarkers may refine risk stratification in clinical settings. This concept has stimulated development of multiplexed immunoassay platforms that provide multiple, parallel protein measurements on the same specimen. CONTENT We provide an overview of antibody-based multiplexed immunoassay platforms and discuss technical and operational challenges. Multiplexed immunoassays use traditional immunoassay principles in which high-affinity capture ligands are immobilized in parallel arrays in either planar format or on microspheres in suspension. Development of multiplexed immunoassays requires rigorous validation of assay configuration and analytical performance to minimize assay imprecision and inaccuracy. Challenges associated with multiplex configuration include selection and immobilization of capture ligands, calibration, interference between antibodies and proteins and assay diluents, and compatibility of assay limits of quantification. We discuss potential solutions to these challenges. Criteria for assessing analytical multiplex assay performance include the range of linearity, analytical specificity, recovery, and comparison to a quality reference method. Quality control materials are not well developed for multiplexed protein immunoassays, and algorithms for interpreting multiplex quality control data are needed. SUMMARY Technical and operational challenges have hindered implementation of multiplexed assays in clinical settings. Formal procedures that guide multiplex assay configuration, analytical validation, and quality control are needed before broad application of multiplexed arrays can occur in the in vitro diagnostic market. PMID:19959625

  13. Detection of Plasmid-Mediated AmpC β-Lactamase Genes in Clinical Isolates by Using Multiplex PCR

    PubMed Central

    Pérez-Pérez, F. Javier; Hanson, Nancy D.

    2002-01-01

    Therapeutic options for infections caused by gram-negative organisms expressing plasmid-mediated AmpC β-lactamases are limited because these organisms are usually resistant to all the β-lactam antibiotics, except for cefepime, cefpirome, and the carbapenems. These organisms are a major concern in nosocomial infections and should therefore be monitored in surveillance studies. Six families of plasmid-mediated AmpC β-lactamases have been identified, but no phenotypic test can differentiate among them, a fact which creates problems for surveillance and epidemiology studies. This report describes the development of a multiplex PCR for the purpose of identifying family-specific AmpC β-lactamase genes within gram-negative pathogens. The PCR uses six sets of ampC-specific primers resulting in amplicons that range from 190 bp to 520 bp and that are easily distinguished by gel electrophoresis. ampC multiplex PCR differentiated the six plasmid-mediated ampC-specific families in organisms such as Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, and Salmonella enterica serovar Typhimurium. Family-specific primers did not amplify genes from the other families of ampC genes. Furthermore, this PCR-based assay differentiated multiple genes within one reaction. In addition, WAVE technology, a high-pressure liquid chromatography-based separation system, was used as a way of decreasing analysis time and increasing the sensitivity of multiple-gene assays. In conclusion, a multiplex PCR technique was developed for identifying family-specific ampC genes responsible for AmpC β-lactamase expression in organisms with or without a chromosomal AmpC β-lactamase gene. PMID:12037080

  14. TES Detector Noise Limited Readout Using SQUID Multiplexers

    NASA Technical Reports Server (NTRS)

    Staguhn, J. G.; Benford, D. J.; Chervenak, J. A.; Khan, S. A.; Moseley, S. H.; Shafer, R. A.; Deiker, S.; Grossman, E. N.; Hilton, G. C.; Irwin, K. D.

    2004-01-01

    The availability of superconducting Transition Edge Sensors (TES) with large numbers of individual detector pixels requires multiplexers for efficient readout. The use of multiplexers reduces the number of wires needed between the cryogenic electronics and the room temperature electronics and cuts the number of required cryogenic amplifiers. We are using an 8 channel SQUID multiplexer to read out one-dimensional TES arrays which are used for submillimeter astronomical observations. We present results from test measurements which show that the low noise level of the SQUID multiplexers allows accurate measurements of the TES Johnson noise, and that in operation, the readout noise is dominated by the detector noise. Multiplexers for large number of channels require a large bandwidth for the multiplexed readout signal. We discuss the resulting implications for the noise performance of these multiplexers which will be used for the readout of two dimensional TES arrays in next generation instruments.

  15. Capacity limits of spatially multiplexed free-space communication

    NASA Astrophysics Data System (ADS)

    Zhao, Ningbo; Li, Xiaoying; Li, Guifang; Kahn, Joseph M.

    2015-12-01

    Increasing the information capacity per unit bandwidth has been a perennial goal of scientists and engineers. Multiplexing of independent degrees of freedom, such as wavelength, polarization and more recently space, has been a preferred method to increase capacity in both radiofrequency and optical communication. Orbital angular momentum, a physical property of electromagnetic waves discovered recently, has been proposed as a new degree of freedom for multiplexing to achieve capacity beyond conventional multiplexing techniques, and has generated widespread and significant interest in the scientific community. However, the capacity of orbital angular momentum multiplexing has not been established or compared to other multiplexing techniques. Here, we show that orbital angular momentum multiplexing is not an optimal technique for realizing the capacity limits of a free-space communication channel and is outperformed by both conventional line-of-sight multi-input multi-output transmission and spatial-mode multiplexing.

  16. A Population Genetic Database of Cat Breeds Developed in Coordination with a Domestic Cat STR Multiplex*

    PubMed Central

    Menotti-Raymond, Marilyn; David, Victor A.; Weir, Bruce S.; O’Brien, Stephen J.

    2015-01-01

    A simple tandem repeat (STR) PCR-based typing system developed for the genetic individualization of domestic cat samples has been used to generate a population genetic database of domestic cat breeds. A panel of 10 tetranucleotide STR loci and a gender-identifying sequence tagged site (STS) were co-amplified in genomic DNA of 1043 individuals representing 38 cat breeds. The STR panel exhibits relatively high heterozygosity in cat breeds, with an average 10-locus heterozygosity of 0.71, which represents an average of 38 breed-specific heterozygosities for the 10-member panel. When the entire set of breed individuals was analyzed as a single population, a heterozygosity of 0.87 was observed. Heterozygosities obtained for the 10 loci range from 0.72 to 0.96. The power for genetic individualization of domestic cat samples of the multiplex is high, with a probability of match (pm) of 6.2E-14, using a conservative θ = 0.05. PMID:22268511

  17. Usefulness of Seminested Multiplex PCR in Surveillance of Imported Malaria in Spain

    PubMed Central

    Rubio, J. M.; Benito, A.; Berzosa, P. J.; Roche, J.; Puente, S.; Subirats, M.; López-Vélez, R.; García, L.; Alvar, J.

    1999-01-01

    The use of a new PCR-based method for the diagnosis of malaria in the Spanish Malaria Reference Laboratory has promoted an increase in confirmed cases of malaria. From August 1997 to July 1998, a total of 192 whole-blood samples and 71 serum samples from 168 patients were received from the hospitals of the Spanish National Health System. Most of the patients came from west-central African countries (85%). This molecular method showed more sensitivity and specificity than microscopy, detecting 12.4% more positive samples than microscopy and 13% of mixed infections undetectable by Giemsa stain. Plasmodium falciparum was the main species detected, with 68% of the total positive malaria cases, followed by Plasmodium malariae (29%), Plasmodium vivax (14%), and Plasmodium ovale (7%), including mixed infections in all cases. This report consists of the first wide, centralized survey of malaria surveillance in Spain. The reference laboratory conducted the analysis of all imported cases in order to detect trends in acquisition. The use of a seminested multiplex PCR permitted confirmation of the origins of the infections and the Plasmodium species involved and confirmation of the effectiveness of drug treatments. This PCR also allowed the detection of the presence in Spain of primaquine-tolerant P. vivax strains from west-central Africa, as well as the detection of a P. falciparum infection induced by transfusion. PMID:10488189

  18. A Multiplexed Amplicon Approach for Detecting Gene Fusions by Next-Generation Sequencing.

    PubMed

    Beadling, Carol; Wald, Abigail I; Warrick, Andrea; Neff, Tanaya L; Zhong, Shan; Nikiforov, Yuri E; Corless, Christopher L; Nikiforova, Marina N

    2016-03-01

    Chromosomal rearrangements that result in oncogenic gene fusions are clinically important drivers of many cancer types. Rapid and sensitive methods are therefore needed to detect a broad range of gene fusions in clinical specimens that are often of limited quantity and quality. We describe a next-generation sequencing approach that uses a multiplex PCR-based amplicon panel to interrogate fusion transcripts that involve 19 driver genes and 94 partners implicated in solid tumors. The panel also includes control assays that evaluate the 3'/5' expression ratios of 12 oncogenic kinases, which might be used to infer gene fusion events when the partner is unknown or not included on the panel. There was good concordance between the solid tumor fusion gene panel and other methods, including fluorescence in situ hybridization, real-time PCR, Sanger sequencing, and other next-generation sequencing panels, because 40 specimens known to harbor gene fusions were correctly identified. No specific fusion reads were observed in 59 fusion-negative specimens. The 3'/5' expression ratio was informative for fusions that involved ALK, RET, and NTRK1 but not for BRAF or ROS1 fusions. However, among 37 ALK or RET fusion-negative specimens, four exhibited elevated 3'/5' expression ratios, indicating that fusions predicted solely by 3'/5' read ratios require confirmatory testing. PMID:26747586

  19. A degenerate PCR-based strategy as a means of identifying homologues of aminoglycoside and β-lactam resistance genes in the gut microbiota

    PubMed Central

    2014-01-01

    Background The potential for the human gut microbiota to serve as a reservoir for antibiotic resistance genes has been the subject of recent discussion. However, this has yet to be investigated using a rapid PCR-based approach. In light of this, here we aim to determine if degenerate PCR primers can detect aminoglycoside and β-lactam resistance genes in the gut microbiota of healthy adults, without the need for an initial culture-based screen for resistant isolates. In doing so, we would determine if the gut microbiota of healthy adults, lacking recent antibiotic exposure, is a reservoir for resistance genes. Results The strategy employed resulted in the identification of numerous aminoglycoside (acetylation, adenylation and phosphorylation) and β-lactam (including blaOXA, blaTEM, blaSHV and blaCTX-M) resistance gene homologues. On the basis of homology, it would appear that these genes originated from different bacterial taxa, with members of the Enterobacteriaceae being a particularly rich source. The results demonstrate that, even in the absence of recent antibiotic exposure, the human gut microbiota is a considerable reservoir for antibiotic resistance genes. Conclusions This study has demonstrated that the gut can be a significant source of aminoglycoside and β-lactam resistance genes, even in the absence of recent antibiotic exposure. The results also demonstrate that PCR-based approaches can be successfully applied to detect antibiotic resistance genes in the human gut microbiota, without the need to isolate resistant strains. This approach could also be used to rapidly screen other complex environments for target genes. PMID:24499167

  20. PCR-Based Simple Subgrouping Is Validated for Classification of Gliomas and Defines Negative Prognostic Copy Number Aberrations in IDH Mutant Gliomas

    PubMed Central

    Nakae, Shunsuke; Sasaki, Hikaru; Hayashi, Saeko; Hattori, Natsuki; Kumon, Masanobu; Nishiyama, Yuya; Adachi, Kazuhide; Nagahisa, Shinya; Hayashi, Takuro; Inamasu, Joji; Abe, Masato; Hasegawa, Mitsuhiro; Hirose, Yuichi

    2015-01-01

    Genetic subgrouping of gliomas has been emphasized recently, particularly after the finding of isocitrate dehydrogenase 1 (IDH1) mutations. In a previous study, we investigated whole-chromosome copy number aberrations (CNAs) of gliomas and have described genetic subgrouping based on CNAs and IDH1 mutations. Subsequently, we classified gliomas using simple polymerase chain reaction (PCR)-based methods to improve the availability of genetic subgrouping. We selected IDH1/2 and TP53 as markers and analyzed 237 adult supratentorial gliomas using Sanger sequencing. Using these markers, we classified gliomas into three subgroups that were strongly associated with patient prognoses. These included IDH mutant gliomas without TP53 mutations, IDH mutant gliomas with TP53 mutations, and IDH wild-type gliomas. IDH mutant gliomas without TP53 mutations, which mostly corresponded to gliomas carrying 1p19q co-deletions, showed lower recurrence rates than the other 2 groups. In the other high-recurrence groups, the median progression-free survival (PFS) and overall survival (OS) of patients with IDH mutant gliomas with TP53 mutations were significantly longer than those of patients with IDH wild-type gliomas. Notably, most IDH mutant gliomas with TP53 mutations had at least one of the CNAs +7q, +8q, −9p, and −11p. Moreover, IDH mutant gliomas with at least one of these CNAs had a significantly worse prognosis than did other IDH mutant gliomas. PCR-based mutation analyses of IDH and TP53 were sufficient for simple genetic diagnosis of glioma that were strongly associated with prognosis of patients and enabled us to detect negative CNAs in IDH mutant gliomas. PMID:26558387

  1. PCR-based identification of eight Lactobacillus species and 18 hr-HPV genotypes in fixed cervical samples of South African women at risk of HIV and BV.

    PubMed

    Dols, Joke A M; Reid, Gregor; Kort, Remco; Schuren, Frank H J; Tempelman, Hugo; Bontekoe, Tj Romke; Korporaal, Hans; Van der Veer, E M; Smit, Pieter W; Boon, Mathilde E

    2012-06-01

    Vaginal lactobacilli assessed by PCR-based microarray and PCR-based genotyping of HPV in South African women at risk for HIV and BV. Vaginal lactobacilli can be defined by microarray techniques in fixed cervical samples of South African women. Cervical brush samples suspended in the coagulant fixative BoonFix of one hundred women attending a health centre for HIV testing in South Africa were available for this study. In the Ndlovu Medical Centre in Elandsdoorn, South Africa, identification of 18 hr-HPV genotypes was done using the INNO-LiPA method. An inventory of lactobacilli organisms was performed using microarray technology. On the basis of the Lactobacillus and Lactobacillus biofilm scoring, the cases were identified as Leiden bacterial vaginosis (BV) negative (BV-; n = 41), Leiden BV intermediate (BV±; n = 25), and Leiden BV positive (BV+; n = 34). Fifty-one women were HIV positive and 49 HIV negative. Out of the 51 HIV positive women, 35 were HPV infected. These 51 HIV positive women were frequently infected with HPV16 and HPV18. In addition, HPV35, HPV52, HPV33, and HPV66 were often detected in these samples. Lactobacillus salivarius and Lactobacillus iners were the most prevalent lactobacilli as established by the microarray technique. In women with HPV infection, the prevalence of Lactobacillus crispatus was significantly reduced. In both HIV and HPV infection, a similar (but not identical) shift in the composition of the lactobacillus flora was observed. We conclude that there is a shift in the composition of vaginal lactobacilli in HIV-infected women. Because of the prominence of HPV35, HPV52, HPV33, and HPV66, vaccination for exclusively HPV16 and HPV18 might be insufficient in South African HIV+ women. PMID:22021225

  2. Virtual-image generation in 360-degree viewable image-plane disk-type multiplex holography.

    PubMed

    Cheng, Yih-Shyang; Chen, Zheng-Feng; Chen, Chih-Hung

    2013-04-22

    By shifting the rotational axis of the recording film and recording the individual image-plane holograms in reversed sequence with the real-image holographic system [Opt. Express 18, 14012 (2010)], the disk-type multiplex hologram can be made to generate virtual image for walk-around viewing if the recording reference source point is maintained on the symmetry axis of hologram disk. Theoretical formulation and numerical simulation show the characteristics of the reconstructed image. Experimental results are also shown for qualitative comparison. PMID:23609740

  3. Universal Multiplex PCR: a novel method of simultaneous amplification of multiple DNA fragments

    PubMed Central

    2012-01-01

    Background Multiplex PCR has been successfully applied in many areas since it was first reported in 1988; however, it suffers from poor universality. Results A novel method called Universal Multiplex PCR (UM-PCR) was created, which simultaneously amplifies multiple target fragments from genomic DNA. The method has two steps. First, the universal adapter-F and universal adapter-R are connected to the forward primers and the reverse primers, respectively. Hairpin structures and cross dimers of five pairs of adapter-primers are detected. Second, UM-PCR amplification is implemented using a novel PCR procedure termed “Two Rounds Mode” (three and 28–32 cycles). The first round (the first three cycles) is named the “One by One Annealing Round”. The second round (28–32 cycles) combines annealing with extension. In the first two cycles of the first round, primers only amplify the specific templates; there are no templates for the universal adapters. The templates of universal adapters begin to be synthesized from the second cycle of the first round, and universal adapters and primers commence full amplification from the third cycle of the first round. Conclusions UM-PCR greatly improves the universality of multiplex PCR. UM-PCR could rapidly detect the genetic purity of maize seeds. In addition, it could be applied in other areas, such as analysis of polymorphisms, quantitative assays and identifications of species. PMID:22894545

  4. Integrated mode converter for mode division multiplexing

    NASA Astrophysics Data System (ADS)

    Perez-Galacho, Diego; Alonso-Ramos, Carlos Alberto; Marris-Morini, Delphine; Vakarin, Vladyslav; Le Roux, Xavier; Ortega-Moñux, Alejandro; Wangüemert-Perez, Juan Gonzalo; Vivien, Laurent

    2016-05-01

    The ever growing demands of bandwidth in optical communication systems are making traditional Wavelength Division Multiplexing (WDM) based systems to reach its limit. In order to cope with future bandwidth demand is necessary to use new levels of orthogonality, such as the waveguide mode or the polarization state. Mode Division Multiplexing (MDM) has recently attracted attention as a possible solution to increase aggregate bandwidth. In this work we discuss the proposition a of mode converter that can cover the whole C-Band of optical communications. The Mode Converter is based on two Multimode Interference (MMI) couplers and a phase shifter. Insertion loss (IL) below 0.2 dB and Extinction ratio (ER) higher than 20 dB in a broad bandwidth range of 1.5 μm to 1.6 μm have been estimated. The total length of the device is less than 30 μm.

  5. Tunable lifetime multiplexing using luminescent nanocrystals

    NASA Astrophysics Data System (ADS)

    Lu, Yiqing; Zhao, Jiangbo; Zhang, Run; Liu, Yujia; Liu, Deming; Goldys, Ewa M.; Yang, Xusan; Xi, Peng; Sunna, Anwar; Lu, Jie; Shi, Yu; Leif, Robert C.; Huo, Yujing; Shen, Jian; Piper, James A.; Robinson, J. Paul; Jin, Dayong

    2014-01-01

    Optical multiplexing plays an important role in applications such as optical data storage, document security, molecular probes and bead assays for personalized medicine. Conventional fluorescent colour coding is limited by spectral overlap and background interference, restricting the number of distinguishable identities. Here, we show that tunable luminescent lifetimes τ in the microsecond region can be exploited to code individual upconversion nanocrystals. In a single colour band, one can generate more than ten nanocrystal populations with distinct lifetimes ranging from 25.6 µs to 662.4 µs and decode their well-separated lifetime identities, which are independent of both colour and intensity. Such `τ-dots' potentially suit multichannel bioimaging, high-throughput cytometry quantification, high-density data storage, as well as security codes to combat counterfeiting. This demonstration extends the optical multiplexing capability by adding the temporal dimension of luminescent signals, opening new opportunities in the life sciences, medicine and data security.

  6. Spin and wavelength multiplexed nonlinear metasurface holography

    PubMed Central

    Ye, Weimin; Zeuner, Franziska; Li, Xin; Reineke, Bernhard; He, Shan; Qiu, Cheng-Wei; Liu, Juan; Wang, Yongtian; Zhang, Shuang; Zentgraf, Thomas

    2016-01-01

    Metasurfaces, as the ultrathin version of metamaterials, have caught growing attention due to their superior capability in controlling the phase, amplitude and polarization states of light. Among various types of metasurfaces, geometric metasurface that encodes a geometric or Pancharatnam–Berry phase into the orientation angle of the constituent meta-atoms has shown great potential in controlling light in both linear and nonlinear optical regimes. The robust and dispersionless nature of the geometric phase simplifies the wave manipulation tremendously. Benefitting from the continuous phase control, metasurface holography has exhibited advantages over conventional depth controlled holography with discretized phase levels. Here we report on spin and wavelength multiplexed nonlinear metasurface holography, which allows construction of multiple target holographic images carried independently by the fundamental and harmonic generation waves of different spins. The nonlinear holograms provide independent, nondispersive and crosstalk-free post-selective channels for holographic multiplexing and multidimensional optical data storages, anti-counterfeiting, and optical encryption. PMID:27306147

  7. Multiplexed Energy Coupler for Rotating Equipment

    NASA Technical Reports Server (NTRS)

    Zhao, Xiaoliang

    2011-01-01

    A multiplexing antenna assembly can efficiently couple AC signal/energy into, or out of, rotating equipment. The unit only passes AC energy while blocking DC energy. Concentric tubes that are sliced into multiple pieces are assembled together so that, when a piece from an outer tube aligns well with an inner tube piece, efficient energy coupling is achieved through a capacitive scheme. With N outer pieces and M inner pieces, an effective N x M combination can be achieved in a multiplexed manner. The energy coupler is non-contact, which is useful if isolation from rotating and stationary parts is required. Additionally, the innovation can operate in high temperatures. Applications include rotating structure sensing, non-contact energy transmission, etc.

  8. Optimal estimator for tomographic fluorescence lifetime multiplexing

    PubMed Central

    Hou, Steven S.; Bacskai, Brian J.; Kumar, Anand T. N.

    2016-01-01

    We use the model resolution matrix to analytically derive an optimal Bayesian estimator for multiparameter inverse problems that simultaneously minimizes inter-parameter cross talk and the total reconstruction error. Application of this estimator to time-domain diffuse fluorescence imaging shows that the optimal estimator for lifetime multiplexing is identical to a previously developed asymptotic time-domain (ATD) approach, except for the inclusion of a diagonal regularization term containing decay amplitude uncertainties. We show that, while the optimal estimator and ATD provide zero cross talk, the optimal estimator provides lower reconstruction error, while ATD results in superior relative quantitation. The framework presented here is generally applicable to other multiplexing problems where the simultaneous and accurate relative quantitation of multiple parameters is of interest. PMID:27192234

  9. Spin and wavelength multiplexed nonlinear metasurface holography

    NASA Astrophysics Data System (ADS)

    Ye, Weimin; Zeuner, Franziska; Li, Xin; Reineke, Bernhard; He, Shan; Qiu, Cheng-Wei; Liu, Juan; Wang, Yongtian; Zhang, Shuang; Zentgraf, Thomas

    2016-06-01

    Metasurfaces, as the ultrathin version of metamaterials, have caught growing attention due to their superior capability in controlling the phase, amplitude and polarization states of light. Among various types of metasurfaces, geometric metasurface that encodes a geometric or Pancharatnam-Berry phase into the orientation angle of the constituent meta-atoms has shown great potential in controlling light in both linear and nonlinear optical regimes. The robust and dispersionless nature of the geometric phase simplifies the wave manipulation tremendously. Benefitting from the continuous phase control, metasurface holography has exhibited advantages over conventional depth controlled holography with discretized phase levels. Here we report on spin and wavelength multiplexed nonlinear metasurface holography, which allows construction of multiple target holographic images carried independently by the fundamental and harmonic generation waves of different spins. The nonlinear holograms provide independent, nondispersive and crosstalk-free post-selective channels for holographic multiplexing and multidimensional optical data storages, anti-counterfeiting, and optical encryption.

  10. Spin and wavelength multiplexed nonlinear metasurface holography.

    PubMed

    Ye, Weimin; Zeuner, Franziska; Li, Xin; Reineke, Bernhard; He, Shan; Qiu, Cheng-Wei; Liu, Juan; Wang, Yongtian; Zhang, Shuang; Zentgraf, Thomas

    2016-01-01

    Metasurfaces, as the ultrathin version of metamaterials, have caught growing attention due to their superior capability in controlling the phase, amplitude and polarization states of light. Among various types of metasurfaces, geometric metasurface that encodes a geometric or Pancharatnam-Berry phase into the orientation angle of the constituent meta-atoms has shown great potential in controlling light in both linear and nonlinear optical regimes. The robust and dispersionless nature of the geometric phase simplifies the wave manipulation tremendously. Benefitting from the continuous phase control, metasurface holography has exhibited advantages over conventional depth controlled holography with discretized phase levels. Here we report on spin and wavelength multiplexed nonlinear metasurface holography, which allows construction of multiple target holographic images carried independently by the fundamental and harmonic generation waves of different spins. The nonlinear holograms provide independent, nondispersive and crosstalk-free post-selective channels for holographic multiplexing and multidimensional optical data storages, anti-counterfeiting, and optical encryption. PMID:27306147

  11. Multiplexing Short Primers for Viral Family PCR

    SciTech Connect

    Gardner, S N; Hiddessen, A L; Hara, C A; Williams, P L; Wagner, M; Colston, B W

    2008-06-26

    We describe a Multiplex Primer Prediction (MPP) algorithm to build multiplex compatible primer sets for large, diverse, and unalignable sets of target sequences. The MPP algorithm is scalable to larger target sets than other available software, and it does not require a multiple sequence alignment. We applied it to questions in viral detection, and demonstrated that there are no universally conserved priming sequences among viruses and that it could require an unfeasibly large number of primers ({approx}3700 18-mers or {approx}2000 10-mers) to generate amplicons from all sequenced viruses. We then designed primer sets separately for each viral family, and for several diverse species such as foot-and-mouth disease virus, hemagglutinin and neuraminidase segments of influenza A virus, Norwalk virus, and HIV-1.

  12. Spectrally multiplexed chromatic confocal multipoint sensing.

    PubMed

    Hillenbrand, Matthias; Lorenz, Lucia; Kleindienst, Roman; Grewe, Adrian; Sinzinger, Stefan

    2013-11-15

    We present a concept for chromatic confocal distance sensing that employs two levels of spectral multiplexing for the parallelized evaluation of multiple lateral measurement points; at the first level, the chromatic confocal principle is used to encode distance information within the spectral distribution of the sensor signal. For lateral multiplexing, the total spectral bandwidth of the sensor is split into bands. Each band is assigned to a different lateral measurement point by a segmented diffractive element. Based on this concept, we experimentally demonstrate a chromatic confocal three-point sensor that is suitable for harsh production environments, since it works with a single-point spectrometer and does not require scanning functionality. The experimental system has a working distance of more than 50 mm, a measurement range of 9 mm, and an axial resolution of 50 μm. PMID:24322108

  13. Optimal distributions for multiplex logistic networks.

    PubMed

    Solá Conde, Luis E; Used, Javier; Romance, Miguel

    2016-06-01

    This paper presents some mathematical models for distribution of goods in logistic networks based on spectral analysis of complex networks. Given a steady distribution of a finished product, some numerical algorithms are presented for computing the weights in a multiplex logistic network that reach the equilibrium dynamics with high convergence rate. As an application, the logistic networks of Germany and Spain are analyzed in terms of their convergence rates. PMID:27368801

  14. Physicians' Attitudes About Multiplex Tumor Genomic Testing

    PubMed Central

    Gray, Stacy W.; Hicks-Courant, Katherine; Cronin, Angel; Rollins, Barrett J.; Weeks, Jane C.

    2014-01-01

    Purpose Although predictive multiplex somatic genomic tests hold the potential to transform care by identifying targetable alterations in multiple cancer genes, little is known about how physicians will use such tests in practice. Participants and Methods Before the initiation of enterprise-wide multiplex testing at a major cancer center, we surveyed all clinically active adult cancer physicians to assess their current use of somatic testing, their attitudes about multiplex testing, and their genomic confidence. Results A total of 160 physicians participated (response rate, 61%): 57% were medical oncologists; 29%, surgeons; 14% radiation oncologists; 37%, women; and 83%, research principal investigators. Twenty-two percent of physicians reported low confidence in their genomic knowledge. Eighteen percent of physicians anticipated testing patients infrequently (≤ 10%), whereas 25% anticipate testing most patients (≥ 90%). Higher genomic confidence was associated with wanting to test a majority of patients (adjusted odds ratio [OR], 6.09; 95% CI, 2.1 to 17.5) and anticipating using actionable (adjusted OR, 2.46; 95% CI, 1.2 to 5.2) or potentially actionable (adjusted OR, 2.89; 95% CI, 1.1 to 7.9) test results to inform treatment recommendations. Forty-two percent of physicians endorsed disclosure of uncertain genomic findings to patients. Conclusion Physicians at a tertiary-care National Cancer Institute–designated comprehensive cancer center varied considerably in how they planned to incorporate predictive multiplex somatic genomic tests into practice and in their attitudes about the disclosure of genomic information of uncertain significance. Given that many physicians reported low genomic confidence, evidence-based guidelines and enhanced physician genomic education efforts may be needed to ensure that genomically guided cancer care is adequately delivered. PMID:24663044

  15. Space division multiplexing in access networks

    NASA Astrophysics Data System (ADS)

    Effenberger, Frank J.

    2015-01-01

    Space division multiplexing (SDM) has received a lot of attention in the past years, as it is seen as the final frontier of fiber optic capacity improvement for long haul transmission. Its use in access networks is even more interesting, due to the different design optimization goals in access versus transport. This paper explores some of the applications of SDM in access that have the potential for early adoption.

  16. Multiplexed fluorescence detector system for capillary electrophoresis

    DOEpatents

    Yeung, Edward S.; Taylor, John A.

    1994-06-28

    A fluorescence detection system for capillary electrophoresis is provided wherein the detection system can simultaneously excite fluorescence and substantially simultaneously monitor separations in multiple capillaries. This multiplexing approach involves laser irradiation of a sample in a plurality of capillaries through optical fibers that are coupled individually with the capillaries. The array is imaged orthogonally through a microscope onto a charge-coupled device camera for signal analysis.

  17. Multiplexed fluorescence detector system for capillary electrophoresis

    DOEpatents

    Yeung, Edward S.; Taylor, John A.

    1996-03-12

    A fluorescence detection system for capillary electrophoresis is provided wherein the detection system can simultaneously excite fluorescence and substantially simultaneously monitor separations in multiple capillaries. This multiplexing approach involves laser irradiation of a sample in a plurality of capillaries through optical fibers that are coupled individually with the capillaries. The array is imaged orthogonally through a microscope onto a charge-coupled device camera for signal analysis.

  18. Multiplexed fluorescence detector system for capillary electrophoresis

    DOEpatents

    Yeung, E.S.; Taylor, J.A.

    1994-06-28

    A fluorescence detection system for capillary electrophoresis is provided wherein the detection system can simultaneously excite fluorescence and substantially simultaneously monitor separations in multiple capillaries. This multiplexing approach involves laser irradiation of a sample in a plurality of capillaries through optical fibers that are coupled individually with the capillaries. The array is imaged orthogonally through a microscope onto a charge-coupled device camera for signal analysis. 14 figures.

  19. Multiplexed fluorescence detector system for capillary electrophoresis

    DOEpatents

    Yeung, E.S.; Taylor, J.A.

    1996-03-12

    A fluorescence detection system for capillary electrophoresis is provided wherein the detection system can simultaneously excite fluorescence and substantially simultaneously monitor separations in multiple capillaries. This multiplexing approach involves laser irradiation of a sample in a plurality of capillaries through optical fibers that are coupled individually with the capillaries. The array is imaged orthogonally through a microscope onto a charge-coupled device camera for signal analysis. 14 figs.

  20. Selecting optimal oligonucleotide primers for multiplex PCR.

    PubMed

    Nicodème, P; Steyaert, J M

    1997-01-01

    We investigate the problem of designing efficient multiplex PCR for medical applications. We show that the problem is NP-complete by transformation to the Multiple Choice Matching problem and give an efficient approximation algorithm. We developed this algorithm in a computer program that predicts which genomic regions may be simultaneously amplified by PCR. Practical use of the software shows that the method can treat 250 non-polymorphic loci with less than 5 simultaneous experiments. PMID:9322038

  1. Broadband Hybrid Holographic Multiplexing with Geometric Metasurfaces.

    PubMed

    Huang, Lingling; Mühlenbernd, Holger; Li, Xiaowei; Song, Xu; Bai, Benfeng; Wang, Yongtian; Zentgraf, Thomas

    2015-11-01

    An effective way for broadband holographic multiplexing based on geometric metasurfaces is demonstrated by the integration of several recording channels into a single device. Each image can be individually addressed with a unique set of parameters, such as circular polarization, position, and angle. Such a technique paves the way for a wide range of applications related to optical patterning, encryption, and information processing. PMID:26398589

  2. Microgels for multiplex and direct fluorescence detection

    NASA Astrophysics Data System (ADS)

    Causa, Filippo; Aliberti, Anna; Cusano, Angela M.; Battista, Edmondo; Netti, Paolo A.

    2015-05-01

    Blood borne oligonucleotides fragments contain useful clinical information whose detection and monitoring represent the new frontier in liquid biopsy as they can transform the current diagnosis procedure. For instance, recent studies have identified a new class of circulating biomarkers such as s miRNAs, and demonstrated that changes in their concentration are closely associated with the development of cancer and other pathologies. However, direct detection of miRNAs in body fluids is particularly challenging and demands high sensitivity -concentration range between atto to femtomolarspecificity, and multiplexing Here we report on engineered multifunctional microgels and innovative probe design for a direct and multiplex detection of relevant clinical miRNAs in fluorescence by single particle assay. Polyethyleneglycol-based microgels have a coreshell architecture with two spectrally encoded fluorescent dyes for multiplex analyses and are endowed with fluorescent probes for miRNA detection. Encoding and detection fluorescence signals are distinguishable by not overlapping emission spectra. Tuneable fluorescence probe conjugation and corresponding emission confinement on single microgel allows for enhanced target detection. Such suspension array has indeed high selectivity and sensitivity with a detection limit of 10-15 M and a dynamic range from 10-9 to 10-15 M. We believe that sensitivity in the fM concentration range, signal background minimization, multiplexed capability and direct measurement of such microgels will translate into diagnostic benefits opening up new roots toward liquid biopsy in the context of point-of-care testing through an easy and fast detection of sensitive diagnostic biomarkers directly in serum.

  3. Wireless Multiplexed Surface Acoustic Wave Sensors Project

    NASA Technical Reports Server (NTRS)

    Youngquist, Robert C.

    2014-01-01

    Wireless Surface Acoustic Wave (SAW) Sensor is a new technology for obtaining multiple, real-time measurements under extreme environmental conditions. This project plans to develop a wireless multiplexed sensor system that uses SAW sensors, with no batteries or semiconductors, that are passive and rugged, can operate down to cryogenic temperatures and up to hundreds of degrees C, and can be used to sense a wide variety of parameters over reasonable distances (meters).

  4. Optimal distributions for multiplex logistic networks

    NASA Astrophysics Data System (ADS)

    Solá Conde, Luis E.; Used, Javier; Romance, Miguel

    2016-06-01

    This paper presents some mathematical models for distribution of goods in logistic networks based on spectral analysis of complex networks. Given a steady distribution of a finished product, some numerical algorithms are presented for computing the weights in a multiplex logistic network that reach the equilibrium dynamics with high convergence rate. As an application, the logistic networks of Germany and Spain are analyzed in terms of their convergence rates.

  5. Emergence of Multiplex Communities in Collaboration Networks

    PubMed Central

    Nicosia, Vincenzo; Bianconi, Ginestra; Latora, Vito

    2016-01-01

    Community structures in collaboration networks reflect the natural tendency of individuals to organize their work in groups in order to better achieve common goals. In most of the cases, individuals exploit their connections to introduce themselves to new areas of interests, giving rise to multifaceted collaborations which span different fields. In this paper, we analyse collaborations in science and among movie actors as multiplex networks, where the layers represent respectively research topics and movie genres, and we show that communities indeed coexist and overlap at the different layers of such systems. We then propose a model to grow multiplex networks based on two mechanisms of intra and inter-layer triadic closure which mimic the real processes by which collaborations evolve. We show that our model is able to explain the multiplex community structure observed empirically, and we infer the strength of the two underlying social mechanisms from real-world systems. Being also able to correctly reproduce the values of intra-layer and inter-layer assortativity correlations, the model contributes to a better understanding of the principles driving the evolution of social networks. PMID:26815700

  6. Emergence of Multiplex Communities in Collaboration Networks.

    PubMed

    Battiston, Federico; Iacovacci, Jacopo; Nicosia, Vincenzo; Bianconi, Ginestra; Latora, Vito

    2016-01-01

    Community structures in collaboration networks reflect the natural tendency of individuals to organize their work in groups in order to better achieve common goals. In most of the cases, individuals exploit their connections to introduce themselves to new areas of interests, giving rise to multifaceted collaborations which span different fields. In this paper, we analyse collaborations in science and among movie actors as multiplex networks, where the layers represent respectively research topics and movie genres, and we show that communities indeed coexist and overlap at the different layers of such systems. We then propose a model to grow multiplex networks based on two mechanisms of intra and inter-layer triadic closure which mimic the real processes by which collaborations evolve. We show that our model is able to explain the multiplex community structure observed empirically, and we infer the strength of the two underlying social mechanisms from real-world systems. Being also able to correctly reproduce the values of intra-layer and inter-layer assortativity correlations, the model contributes to a better understanding of the principles driving the evolution of social networks. PMID:26815700

  7. Multiplex congruence network of natural numbers

    NASA Astrophysics Data System (ADS)

    Yan, Xiao-Yong; Wang, Wen-Xu; Chen, Guan-Rong; Shi, Ding-Hua

    2016-03-01

    Congruence theory has many applications in physical, social, biological and technological systems. Congruence arithmetic has been a fundamental tool for data security and computer algebra. However, much less attention was devoted to the topological features of congruence relations among natural numbers. Here, we explore the congruence relations in the setting of a multiplex network and unveil some unique and outstanding properties of the multiplex congruence network. Analytical results show that every layer therein is a sparse and heterogeneous subnetwork with a scale-free topology. Counterintuitively, every layer has an extremely strong controllability in spite of its scale-free structure that is usually difficult to control. Another amazing feature is that the controllability is robust against targeted attacks to critical nodes but vulnerable to random failures, which also differs from ordinary scale-free networks. The multi-chain structure with a small number of chain roots arising from each layer accounts for the strong controllability and the abnormal feature. The multiplex congruence network offers a graphical solution to the simultaneous congruences problem, which may have implication in cryptography based on simultaneous congruences. Our work also gains insight into the design of networks integrating advantages of both heterogeneous and homogeneous networks without inheriting their limitations.

  8. Multiplexed Holographic Data Storage in Bacteriorhodopsin

    NASA Technical Reports Server (NTRS)

    Mehrl, David J.; Krile, Thomas F.

    1997-01-01

    High density optical data storage, driven by the information revolution, remains at the forefront of current research areas. Much of the current research has focused on photorefractive materials (SBN and LiNbO3) and polymers, despite various problems with expense, durability, response time and retention periods. Photon echo techniques, though promising, are questionable due to the need for cryogenic conditions. Bacteriorhodopsin (BR) films are an attractive alternative recording medium. Great strides have been made in refining BR, and materials with storage lifetimes as long as 100 days have recently become available. The ability to deposit this robust polycrystalline material as high quality optical films suggests the use of BR as a recording medium for commercial optical disks. Our own recent research has demonstrated the suitability of BR films for real time spatial filtering and holography. We propose to fully investigate the feasibility of performing holographic mass data storage in BR. Important aspects of the problem to be investigated include various data multiplexing techniques (e.g. angle- amplitude- and phase-encoded multiplexing, and in particular shift-multiplexing), multilayer recording techniques, SLM selection and data readout using crossed polarizers for noise rejection. Systems evaluations of storage parameters, including access times, memory refresh constraints, erasure, signal-to-noise ratios and bit error rates, will be included in our investigations.

  9. Emergence of Chimera in Multiplex Network

    NASA Astrophysics Data System (ADS)

    Ghosh, Saptarshi; Jalan, Sarika

    2016-06-01

    Chimera is a relatively new emerging phenomenon where coexistence of synchronous and asynchronous states is observed in symmetrically coupled dynamical units. We report the observation of the chimera state in multiplex networks where individual layer is represented by 1-d lattice with nonlocal interactions. While, multiplexing does not change the type of the chimera state and retains the multi-chimera state displayed by the isolated networks, it changes the regions of the incoherence. We investigate the emergence of coherent-incoherent bifurcation upon varying the control parameters, namely, the coupling strength and the network size. Additionally, we investigate the effect of initial condition on the dynamics of the chimera state. Using a measure based on the differences between the neighboring nodes which distinguishes smooth and nonsmooth spatial profiles, we find the critical coupling strength for the transition to the chimera state. Observing chimera in a multiplex network with one-to-one inter layer coupling is important to gain insight to many real world complex systems which inherently posses multilayer architecture.

  10. Multiplex congruence network of natural numbers.

    PubMed

    Yan, Xiao-Yong; Wang, Wen-Xu; Chen, Guan-Rong; Shi, Ding-Hua

    2016-01-01

    Congruence theory has many applications in physical, social, biological and technological systems. Congruence arithmetic has been a fundamental tool for data security and computer algebra. However, much less attention was devoted to the topological features of congruence relations among natural numbers. Here, we explore the congruence relations in the setting of a multiplex network and unveil some unique and outstanding properties of the multiplex congruence network. Analytical results show that every layer therein is a sparse and heterogeneous subnetwork with a scale-free topology. Counterintuitively, every layer has an extremely strong controllability in spite of its scale-free structure that is usually difficult to control. Another amazing feature is that the controllability is robust against targeted attacks to critical nodes but vulnerable to random failures, which also differs from ordinary scale-free networks. The multi-chain structure with a small number of chain roots arising from each layer accounts for the strong controllability and the abnormal feature. The multiplex congruence network offers a graphical solution to the simultaneous congruences problem, which may have implication in cryptography based on simultaneous congruences. Our work also gains insight into the design of networks integrating advantages of both heterogeneous and homogeneous networks without inheriting their limitations. PMID:27029650

  11. Multiplex congruence network of natural numbers

    PubMed Central

    Yan, Xiao-Yong; Wang, Wen-Xu; Chen, Guan-Rong; Shi, Ding-Hua

    2016-01-01

    Congruence theory has many applications in physical, social, biological and technological systems. Congruence arithmetic has been a fundamental tool for data security and computer algebra. However, much less attention was devoted to the topological features of congruence relations among natural numbers. Here, we explore the congruence relations in the setting of a multiplex network and unveil some unique and outstanding properties of the multiplex congruence network. Analytical results show that every layer therein is a sparse and heterogeneous subnetwork with a scale-free topology. Counterintuitively, every layer has an extremely strong controllability in spite of its scale-free structure that is usually difficult to control. Another amazing feature is that the controllability is robust against targeted attacks to critical nodes but vulnerable to random failures, which also differs from ordinary scale-free networks. The multi-chain structure with a small number of chain roots arising from each layer accounts for the strong controllability and the abnormal feature. The multiplex congruence network offers a graphical solution to the simultaneous congruences problem, which may have implication in cryptography based on simultaneous congruences. Our work also gains insight into the design of networks integrating advantages of both heterogeneous and homogeneous networks without inheriting their limitations. PMID:27029650

  12. Dentification of four vectors of human Plasmodium spp. by multiplex PCR: Anopheles rangeli, An. strodei, An. triannulatus, and An. trinkae (Diptera: Culicidae: Nyssorhynchus).

    PubMed

    Fritz, G N; Engman, S; Rodriguez, R; Wilkerson, R C

    2004-11-01

    One of the major obstacles for studies of the biology, ecology, and behavior of Neotropical vectors of human Plasmodium has been the lack of reliable and efficient means of identifying many species. Although the subgenus Nyssorhynchus includes most species responsible for human transmission in South America, there are no polymerase chain reaction (PCR)-based techniques for identifying members of this taxon. We describe the first multiplex PCR for identifying four species in the subgenus Nyssorhynchus that are vectors of human Plasmodium spp. Four species specific primers, together with a universal primer that anneals to the 5.8S rDNA region, produce amplicons of the internal transcribed spacer two with base pair sizes of 131,308,371, and 441 for An. triannulatus, An. trinkae, An. strodei, and An. rangeli, respectively. PMID:15605651

  13. Identification of blood meal source and infection with Trypanosoma cruzi of Chagas disease vectors using a multiplex cytochrome b polymerase chain reaction assay.

    PubMed

    Mota, Javier; Chacon, Juan C; Gutiérrez-Cabrera, Ana E; Sánchez-Cordero, Víctor; Wirtz, Robert A; Ordoñez, Rosalinda; Panzera, Francisco; Ramsey, Janine M

    2007-01-01

    Long-term control of triatomine bugs in Chagas endemic regions will depend on a full understanding of vector-parasite-host interactions. Herein we describe a cytochrome b multiplex polymerase chain reaction (PCR)-based strategy for blood meal source identification in bug foregut contents. This technique discriminates human from animal blood, and has been tested in five Triatoma species from México. Host identification has been validated for human, four rodent species, two bat species, dog, rabbit, sheep, and opossum. In addition, Trypanosoma cruzi can be identified simultaneously using S34/S67-specific kinetoplast DNA primers. Both host and parasite identification were possible as long as 10 weeks after bug feeding, and in samples stored up to 6 years. The blood meal identification procedure described here represents a powerful tool for large-scale studies identifying the biological, ecological, and environmental variables associated with Chagas disease transmission. PMID:18021027

  14. Time division multiplexed orbital angular momentum access system

    NASA Astrophysics Data System (ADS)

    Shi, Jianyang; Fang, Yuan; Chi, Nan

    2016-03-01

    We propose and experimentally demonstrate time division multiplexed orbital angular momentum (OAM) access system to increase transmission capacity and spectral efficiency. In this system, data carried on different time tributaries share the same OAM mode. Multiple time division multiplexed OAM modes are multiplexed to realize two-dimensional (time dimension and OAM dimension) multiplexing. Therefore, the capacity and spectral efficiency of the access system will increase. The orthogonality between optical time division multiplexing (OTDM) and OAM techniques is also verified in our experiment. In a proof-of-concept experiment, 2×5-Gbps return-to-zero signal over OAM mode +4 is transmitted and investigated. The bit error ratio performance after transmission in this system can be smaller than 1×10-9. Results show that the proposed time division multiplexed OAM access system is suitable for future broadband access network.

  15. Multiplex PCR for rapid diagnosis of tuberculous meningitis.

    PubMed

    Kusum, Sharma; Aman, Sharma; Pallab, Ray; Kumar, Sharma Shiv; Manish, Modi; Sudesh, Prabhakar; Subhash, Varma; Meera, Sharma

    2011-10-01

    Rapid and specific diagnosis of tubercular meningitis is of paramount importance to decrease morbidity and mortality. The aim of the study was to evaluate multiplex PCR using protein b, MPB 64, and IS6110 primers directed against M. tuberculosis complex for the diagnosis of tuberculous meningitis (TBM). Multiplex PCR was performed on 18 TBM confirmed cases (culture was positive), 92 clinically suspected TBM cases and 100 non-TBM (control group) patients. Multiplex PCR had a sensitivity of 94.4% for confirmed cases and specificity of 100% for confirmed TBM cases. In 92 clinically diagnosed but unconfirmed TBM cases, multiplex PCR was positive in 84.78% cases. The overall sensitivity of microscopy, culture and multiplex cases were 1.81, 16.73, and 86.63% and specificity was 100, 100, and 100% respectively. Multiplex PCR using protein b, MPB 64, and IS6110 primers has a high sensitivity and specificity in diagnosis of tubercular meningitis. PMID:21455603

  16. Demonstration of hybrid orbital angular momentum multiplexing and time-division multiplexing passive optical network.

    PubMed

    Wang, Andong; Zhu, Long; Liu, Jun; Du, Cheng; Mo, Qi; Wang, Jian

    2015-11-16

    Mode-division multiplexing passive optical network (MDM-PON) is a promising scheme for next-generation access networks to further increase fiber transmission capacity. In this paper, we demonstrate the proof-of-concept experiment of hybrid mode-division multiplexing (MDM) and time-division multiplexing (TDM) PON architecture by exploiting orbital angular momentum (OAM) modes. Bidirectional transmissions with 2.5-Gbaud 4-level pulse amplitude modulation (PAM-4) downstream and 2-Gbaud on-off keying (OOK) upstream are demonstrated in the experiment. The observed optical signal-to-noise ratio (OSNR) penalties for downstream and upstream transmissions at a bit-error rate (BER) of 2 × 10(-3) are less than 2.0 dB and 3.0 dB, respectively. PMID:26698429

  17. Mycobacterial Interspersed Repetitive-Unit–Variable-Number Tandem-Repeat (MIRU-VNTR) Genotyping of Mycobacterium intracellulare for Strain Comparison with Establishment of a PCR-Based Database

    PubMed Central

    Iakhiaeva, Elena; McNulty, Steven; Brown Elliott, Barbara A.; Falkinham, Joseph O.; Williams, Myra D.; Vasireddy, Ravikiran; Wilson, Rebecca W.; Turenne, Christine

    2013-01-01

    Strain comparison is important to population genetics and to evaluate relapses in patients with Mycobacterium avium complex (MAC) lung disease, but the “gold standard” of pulsed-field gel electrophoresis (PFGE) is time-consuming and complex. We used variable-number tandem repeats (VNTR) for fingerprinting of respiratory isolates of M. intracellulare from patients with underlying bronchiectasis, to establish a nonsequence-based database for population analysis. Different genotypes identified by PFGE underwent species identification using a 16S rRNA gene multiplex PCR. Genotypes of M. intracellulare were confirmed by internal transcribed spacer 1 (ITS1) sequencing and characterized using seven VNTR primers. The pattern of VNTR amplicon sizes and repeat number defined each specific VNTR type. Forty-two VNTR types were identified among 84 genotypes. PFGE revealed most isolates with the same VNTR type to be clonal or exhibit similar grouping of bands. Repetitive sequence-based PCR (rep-PCR) showed minimal pattern diversity between VNTR types compared to PFGE. Fingerprinting of relapse isolates from 31 treated patients using VNTR combined with 16S multiplex PCR unambiguously and reliably distinguished different genotypes from the same patient, with results comparable to those of PFGE. VNTR for strain comparison is easier and faster than PFGE, is as accurate as PFGE, and does not require sequencing. Starting with a collection of 167 M. intracellulare isolates, VNTR distinguished M. intracellulare into 42 clonal groups. Comparison of isolates from different geographic areas, habitats, and clinical settings is now possible. PMID:23175249

  18. Development of a high-throughput real time PCR based on a hot-start alternative for Pfu mediated by quantum dots

    NASA Astrophysics Data System (ADS)

    Sang, Fuming; Yang, Yang; Yuan, Lin; Ren, Jicun; Zhang, Zhizhou

    2015-09-01

    Hot start (HS) PCR is an excellent alternative for high-throughput real time PCR due to its ability to prevent nonspecific amplification at low temperature. Development of a cost-effective and simple HS PCR technique to guarantee high-throughput PCR specificity and consistency still remains a great challenge. In this study, we systematically investigated the HS characteristics of QDs triggered in real time PCR with EvaGreen and SYBR Green I dyes by the analysis of amplification curves, standard curves and melting curves. Two different kinds of DNA polymerases, Pfu and Taq, were employed. Here we showed that high specificity and efficiency of real time PCR were obtained in a plasmid DNA and an error-prone two-round PCR assay using QD-based HS PCR, even after an hour preincubation at 50 °C before real time PCR. Moreover, the results obtained by QD-based HS PCR were comparable to a commercial Taq antibody DNA polymerase. However, no obvious HS effect of QDs was found in real time PCR using Taq DNA polymerase. The findings of this study demonstrated that a cost-effective high-throughput real time PCR based on QD triggered HS PCR could be established with high consistency, sensitivity and accuracy.Hot start (HS) PCR is an excellent alternative for high-throughput real time PCR due to its ability to prevent nonspecific amplification at low temperature. Development of a cost-effective and simple HS PCR technique to guarantee high-throughput PCR specificity and consistency still remains a great challenge. In this study, we systematically investigated the HS characteristics of QDs triggered in real time PCR with EvaGreen and SYBR Green I dyes by the analysis of amplification curves, standard curves and melting curves. Two different kinds of DNA polymerases, Pfu and Taq, were employed. Here we showed that high specificity and efficiency of real time PCR were obtained in a plasmid DNA and an error-prone two-round PCR assay using QD-based HS PCR, even after an hour

  19. Fixed nuclei as alternative template of BIOMED-2 multiplex polymerase chain reaction for immunoglobulin gene clonality testing in B-cell malignancies

    PubMed Central

    Tang, Yuan; Chen, Jie; Wang, Jianchao; Zheng, Ke; Liao, Dianying; Liao, Xiaomei; Liu, Weiping; Wang, Lin

    2015-01-01

    Evaluation of immunoglobulin (Ig) gene rearrangements with BIOMED-2 multiplex PCR has become a standard detection of clonality in mature B cell malignancies. Conventionally, this method is relatively labor-intensive and time-consuming, as it requires DNA isolation from bone marrow aspirates (BM) or peripheral blood (PB) in patients with BM or PB involvement. On the other hand, fluorescence in situ hybridization (FISH) is routinely used as genetic screening in B cell malignancies, but the surplus fixed nuclei initially prepared for FISH usually turn useless afterwards. We sought to use these surplus nuclei after FISH as a template to perform PCR-based Ig gene clonality testing. Templates of 12 patients with mature B cell malignancies, which consisted of both DNA isolated with commercial DNA isolation kit from fresh BM or PB (DNA group) and the fixed nuclei initially prepared for FISH (nuclei group) from the same individuals, were subjected to PCR with BIOMED-2 primer sets for immunoglobulin heavy chain and kappa light chain under recommended conditions. Our result, for the first time, showed a high consistency between the two groups in detecting B cell clonality, which indicates that nuclei for FISH can function as a reliable template comparable to fresh tissue-isolated DNA in PCR based Ig clonality testing. This offers a simple, rapid and more economical alternative to standard Ig testing based on regular DNA. PMID:27069754

  20. Communicability reveals a transition to coordinated behavior in multiplex networks

    NASA Astrophysics Data System (ADS)

    Estrada, Ernesto; Gómez-Gardeñes, Jesús

    2014-04-01

    We analyze the flow of information in multiplex networks by means of the communicability function. First, we generalize this measure from its definition from simple graphs to multiplex networks. Then, we study its relevance for the analysis of real-world systems by studying a social multiplex where information flows using formal-informal channels and an air transportation system where the layers represent different air companies. Accordingly, the communicability, which is essential for the good performance of these complex systems, emerges at a systemic operation point in the multiplex where the performance of the layers operates in a coordinated way very differently from the state represented by a collection of unconnected networks.

  1. Diffraction model of peristrophic multiplexing with spherical reference wave.

    PubMed

    Yoshida, Shuhei; Takahata, Yosuke; Horiuchi, Shuma; Yamamoto, Manabu

    2015-02-01

    Multiplexing recording is a primary contributor to determining the recording density in holographic data storage. Therefore, many different kinds of recording methods have been proposed. Among them, the method that utilizes spherical waves as reference waves is characterized by the ability to enable multiplexing recording only by moving (shifting or rotating) the recording medium. In our research, we propose a theoretical diffraction model of peristrophic multiplexing with a spherical reference wave and evaluate the diffraction efficiency; this multiplexing recording method has incorporated spherical reference waves in rotation of the media. Additionally, we verify the effectiveness of the model by comparing it with experimental results. PMID:26366593

  2. Shift-peristrophic multiplexing for holographic data storage

    NASA Astrophysics Data System (ADS)

    Kurata, Hiroyuki; Mori, Jun; Tsukamoto, Yu; Yamamoto, Keiko; Yoshida, Shuhei; Yamamoto, Manabu

    2014-09-01

    Holographic data storage (HDS) is a promising technology that has huge capacity. A multiplexing method plays a significant role in increasing the data capacity. Various multiplexing methods have been researched so far. In this paper, we proposed shift-peristrophic multiplexing using spherical reference wave and experimentally verified that this method is efficiently increase the data capacity. A series of holograms was recorded with shift multiplexing and rotating recording material with the axis of rotation being perpendicular to the material's surface. This method can realize more than 1 Tbits/inch2 data density recording. Furthermore if we maximize the performance of a recording medium, several TB per disk capacity would be available.

  3. Collaborative trial validation studies of real-time PCR-based GMO screening methods for detection of the bar gene and the ctp2-cp4epsps construct.

    PubMed

    Grohmann, Lutz; Brünen-Nieweler, Claudia; Nemeth, Anne; Waiblinger, Hans-Ulrich

    2009-10-14

    Polymerase Chain Reaction (PCR)-based screening methods targeting genetic elements commonly used in genetically modified (GM) plants are important tools for the detection of GM materials in food, feed, and seed samples. To expand and harmonize the screening capability of enforcement laboratories, the German Federal Office of Consumer Protection and Food Safety conducted collaborative trials for interlaboratory validation of real-time PCR methods for detection of the phosphinothricin acetyltransferase (bar) gene from Streptomyces hygroscopicus and a construct containing the 5-enolpyruvylshikimate-3-phosphate synthase gene from Agrobacterium tumefaciens sp. strain CP4 (ctp2-cp4epsps), respectively. To assess the limit of detection, precision, and accuracy of the methods, laboratories had to analyze two sets of 18 coded genomic DNA samples of events LLRice62 and MS8 with the bar method and NK603 and GT73 with the ctp2-cp4epsps method at analyte levels of 0, 0.02, and 0.1% GM content, respectively. In addition, standard DNAs were provided to the laboratories to generate calibration curves for copy number quantification of the bar and ctp2-cp4epsps target sequences present in the test samples. The study design and the results obtained are discussed with respect to the difficult issue of developing general guidelines and concepts for the collaborative trial validation of qualitative PCR screening methods. PMID:19807158

  4. Prevalence of mip virulence gene and PCR-base sequence typing of Legionella pneumophila from cooling water systems of two cities in Iran.

    PubMed

    Ahmadrajabi, Roya; Shakibaie, Mohammad Reza; Iranmanesh, Zahra; Mollaei, Hamid Reza; Sobhanipoor, Mohammad Hossein

    2016-07-01

    Legionella pneumophila is the primary respiratory pathogen and mostly transmitted to human through water cooling systems and cause mild to severe pneumonia with high mortality rate especially in elderly both in hospitals and community. However, current Legionella risk assessments may be compromised by uncertainties in Legionella detection methods. Here, we investigated the presence of L. pneumophila mip gene in water samples collected from different hospitals cooling towers, nursing homes and building/hotels water coolants from two geographical locations of Iran (Kerman and Bam cities) during summer season of 2015 by both nested and real-time PCR methods. Analysis of the 128 water samples for presence of the mip gene by nested-PCR revealed, 18 (23%) positive cases in Kerman and 7(14%) in Bam. However, when samples were tested by real-time PCR, we identified 4 more new cases of L. pneumophila in the hospitals as well as nursing homes water systems that were missed by nested-PCR. The highest rate of contamination was detected in water obtained from hospitals cooling towers in both the cities (p≤0.05). Dendrogram analysis and clonal relationship by PCR-base sequence typing (SBT) of the L. pneumophila genomic DNAs in Kerman water samples showed close clonal similarities among the isolates, in contrast, isolates identified from Bam city demonstrated two fingerprint patterns. The clones from hospital water samples were more related to the L. pneumophila serogroup- 1. PMID:27028760

  5. A PCR-Based Diagnostic System for Differentiating Two Weevil Species (Coleoptera: Curculionidae) of Economic Importance to the Chilean Citrus Industry.

    PubMed

    Aguirre, C; Olivares, N; Luppichini, P; Hinrichsen, P

    2015-02-01

    A PCR-based method was developed to identify Naupactus cervinus (Boheman) and Naupactus xanthographus (Germar), two curculionids affecting the citrus industry in Chile. The quarantine status of these two species depends on the country to which fruits are exported. This identification method was developed because it is not possible to discriminate between these two species at the egg stage. The method is based on the species-specific amplification of sequences of internal transcribed spacers, for which we cloned and sequenced these genome fragments from each species. We designed an identification system based on two duplex-PCR reactions. Each one contains the species-specific primer set and a second generic primer set that amplify a short 18S region common to coleopterans, to avoid false negatives. The marker system is able to differentiate each Naupactus species at any life stage, and with a diagnostic sensitivity to 0.045 ng of genomic DNA. This PCR kit was validated by samples collected from different citrus production areas throughout Chile and showed 100% accuracy in differentiating the two Naupactus species. PMID:26470110

  6. PCR-based prevalence of a fatal reovirus of the blue crab, Callinectes sapidus (Rathbun) along the northern Atlantic coast of the USA.

    PubMed

    Flowers, E M; Simmonds, K; Messick, G A; Sullivan, L; Schott, E J

    2016-06-01

    There is a need for more information on the relationship between diseases and fluctuations of wild populations of marine animals. In the case of Callinectes sapidus reovirus 1 (CsRV1, also known as RLV), there is a lack of baseline information on range, prevalence and outbreaks, from which to develop an understanding of population-level impacts. An RT-qPCR assay was developed that is capable of detecting 10 copies of the CsRV1 genome. In collaboration with state, federal and academic partners, blue crabs were collected from sites throughout the north-eastern United States to assess the northern range of this pathogen. In addition, archived crab samples from the Chesapeake Bay were assessed for CsRV1 by RT-qPCR and histology. PCR-based assessments indicate that CsRV1 was present at all but one site. Prevalence of CsRV1 as assessed by RT-qPCR was highly variable between locations, and CsRV1 prevalence varied between years at a given location. Mean CsRV1 prevalence as assessed by RT-qPCR was >15% each year, and peak prevalence was 79%. The wide geographic range and highly variable prevalence of CsRV1 indicate that more study is needed to understand CsRV1 dynamics and the role the virus plays in blue crab natural mortality. PMID:26249243

  7. Discovery and characterization of RecA protein of thermophilic bacterium Thermus thermophilus MAT72 phage Tt72 that increases specificity of a PCR-based DNA amplification.

    PubMed

    Stefanska, Aleksandra; Kaczorowska, Anna-Karina; Plotka, Magdalena; Fridjonsson, Olafur H; Hreggvidsson, Gudmundur O; Hjorleifsdottir, Sigridur; Kristjansson, Jakob K; Dabrowski, Slawomir; Kaczorowski, Tadeusz

    2014-07-20

    The recA gene of newly discovered Thermus thermophilus MAT72 phage Tt72 (Myoviridae) was cloned and overexpressed in Escherichia coli. The 1020-bp gene codes for a 339-amino-acid polypeptide with an Mr of 38,155 which shows 38.7% positional identity to the E. coli RecA protein. When expressed in E. coli, the Tt72 recA gene did not confer the ability to complement the ultraviolet light (254nm) sensitivity of an E. coli recA mutant. Tt72 RecA protein has been purified with good yield to catalytic and electrophoretic homogeneity using a three-step chromatography procedure. Biochemical characterization indicated that the protein can pair and promote ATP-dependent strand exchange reaction resulting in formation of a heteroduplex DNA at 60°C under conditions otherwise optimal for E. coli RecA. When the Tt72 RecA protein was included in a standard PCR-based DNA amplification reaction, the specificity of the PCR assays was significantly improved by eliminating non-specific products. PMID:24786823

  8. A simple method of DNA isolation from jute (Corchorus olitorius) seed suitable for PCR-based detection of the pathogen Macrophomina phaseolina (Tassi) Goid.

    PubMed

    Biswas, C; Dey, P; Satpathy, S; Sarkar, S K; Bera, A; Mahapatra, B S

    2013-02-01

    A simple method was developed for isolating DNA from jute seed, which contains high amounts of mucilage and secondary metabolites, and a PCR protocol was standardized for detecting the seedborne pathogen Macrophomina phaseolina. The cetyl trimethyl ammonium bromide method was modified with increased salt concentration and a simple sodium acetate treatment to extract genomic as well as fungal DNA directly from infected jute seed. The Miniprep was evaluated along with five other methods of DNA isolation in terms of yield and quality of DNA and number of PCR positive samples. The Miniprep consistently recovered high amounts of DNA with good spectral qualities at A260/A280. The DNA isolated from jute seed was found suitable for PCR amplification. Macrophomina phaseolina could be detected by PCR from artificially inoculated as well as naturally infected jute seeds. The limit of PCR-based detection of M. phaseolina in jute seed was determined to be 0·62 × 10(-7) CFU g(-1) seed. PMID:23121598

  9. High thickness acrylamide photopolymer for peristrophic multiplexing

    NASA Astrophysics Data System (ADS)

    Ortuño, M.; Fernández, E.; Márquez, A.; Gallego, S.; Neipp, C.; Pascual, I.

    2006-05-01

    The acrylamide photolymers are considered interesting materials for holographic media. They have high diffraction efficiency (ratio of the intensities of the diffracted and the incident beams), an intermediate energetic sensitivity among other materials and post-processing steps are not necessary, therefore the media is not altered. The layers of these materials, about 1 mm thick, are a suitable media for recording many diffraction gratings in the same volume of photopolymer using peristrophic multiplexing technique, with great practical importance in the field of holographic memories type WORM (write once read many). In this work we study the recording of diffraction gratings by peristrophic multiplexing with axis of rotation perpendicular to the recording media. The photopolymer is composed of acrylamide as the polymerizable monomer, triethanolamine as radical generator, yellowish eosin as sensitizer and a binder of polyvinyl alcohol. We analyze the holographic behaviour of the material during recording and reconstruction of diffraction gratings using a continuous Nd:YAG laser (532 nm) at an intensity of 5 mW/cm2 as recording laser. The response of the material is monitored after recording with an He-Ne laser. We study the recording process of unslanted diffraction gratings of 1125 lines/mm. The diffraction efficiency of each hologram is seen to decrease as the number of holograms recorded increases, due to consumption of the available dynamic range, in a constant exposure scheduling. It can be seen that the photopolymer works well with high energy levels, without excessive dispersion of light by noise gratings. In order to homogenize the diffraction efficiency of each hologram we use the method proposed by Pu. This method is designed to share all or part of the avaliable dynamic range of the recording material among the holograms to be multiplexed. Using exposure schedules derived from this method we have used 3 scheduling recordings from the algorithm used

  10. Multiplexed Western Blotting Using Microchip Electrophoresis.

    PubMed

    Jin, Shi; Furtaw, Michael D; Chen, Huaxian; Lamb, Don T; Ferguson, Stephen A; Arvin, Natalie E; Dawod, Mohamed; Kennedy, Robert T

    2016-07-01

    Western blotting is a commonly used protein assay that combines the selectivity of electrophoretic separation and immunoassay. The technique is limited by long time, manual operation with mediocre reproducibility, and large sample consumption, typically 10-20 μg per assay. Western blots are also usually used to measure only one protein per assay with an additional housekeeping protein for normalization. Measurement of multiple proteins is possible; however, it requires stripping membranes of antibody and then reprobing with a second antibody. Miniaturized alternatives to Western blot based on microfluidic or capillary electrophoresis have been developed that enable higher-throughput, automation, and greater mass sensitivity. In one approach, proteins are separated by electrophoresis on a microchip that is dragged along a polyvinylidene fluoride membrane so that as proteins exit the chip they are captured on the membrane for immunoassay. In this work, we improve this method to allow multiplexed protein detection. Multiple injections made from the same sample can be deposited in separate tracks so that each is probed with a different antibody. To further enhance multiplexing capability, the electrophoresis channel dimensions were optimized for resolution while keeping separation and blotting times to less than 8 min. Using a 15 μm deep × 50 μm wide × 8.6 cm long channel, it is possible to achieve baseline resolution of proteins that differ by 5% in molecular weight, e.g., ERK1 (44 kDa) from ERK2 (42 kDa). This resolution allows similar proteins detected by cross-reactive antibodies in a single track. We demonstrate detection of 11 proteins from 9 injections from a single Jurkat cell lysate sample consisting of 400 ng of total protein using this procedure. Thus, multiplexed Western blots are possible without cumbersome stripping and reprobing steps. PMID:27270033

  11. Effects of Holding Time, Storage, and the Preservation of Samples on Sample Integrity for the Detection of Fecal Indicator Bacteria by Quantitative Polymerase Chain Reaction (qPCR)-based assays.

    EPA Science Inventory

    The purpose of this project was to answer questions related to storage of samples to be analyzed by the quantitative polymerase chain reaction (qPCR)-based assays for fecal indicator bacteria. The project was divided into two parts. The first part was to determine if filters th...

  12. Performance of Panfungal- and Specific-PCR-Based Procedures for Etiological Diagnosis of Invasive Fungal Diseases on Tissue Biopsy Specimens with Proven Infection: a 7-Year Retrospective Analysis from a Reference Laboratory

    PubMed Central

    Bernal-Martinez, L.; Castelli, M. V.; Rodriguez-Tudela, J. L.; Cuenca-Estrella, M.

    2014-01-01

    A retrospective analysis of real-time PCR (RT-PCR) results for 151 biopsy samples obtained from 132 patients with proven invasive fungal diseases was performed. PCR-based techniques proved to be fast and sensitive and enabled definitive diagnosis in all cases studied, with detection of a total of 28 fungal species. PMID:24574295

  13. Reverse phase protein microarrays advance to use in clinical trials.

    PubMed

    Mueller, Claudius; Liotta, Lance A; Espina, Virginia

    2010-12-01

    Individualizing cancer therapy for molecular targeted inhibitors requires a new class of molecular profiling technology that can map the functional state of the cancer cell signal pathways containing the drug targets. Reverse phase protein microarrays (RPMA) are a technology platform designed for quantitative, multiplexed analysis of specific phosphorylated, cleaved, or total (phosphorylated and non-phosphorylated) forms of cellular proteins from a limited amount of sample. This class of microarray can be used to interrogate tissue samples, cells, serum, or body fluids. RPMA were previously a research tool; now this technology has graduated to use in research clinical trials with clinical grade sensitivity and precision. In this review we describe the application of RPMA for multiplexed signal pathway analysis in therapeutic monitoring, biomarker discovery, and evaluation of pharmaceutical targets, and conclude with a summary of the technical aspects of RPMA construction and analysis. PMID:20974554

  14. Reversible Thermoset Adhesives

    NASA Technical Reports Server (NTRS)

    Mac Murray, Benjamin C. (Inventor); Tong, Tat H. (Inventor); Hreha, Richard D. (Inventor)

    2016-01-01

    Embodiments of a reversible thermoset adhesive formed by incorporating thermally-reversible cross-linking units and a method for making the reversible thermoset adhesive are provided. One approach to formulating reversible thermoset adhesives includes incorporating dienes, such as furans, and dienophiles, such as maleimides, into a polymer network as reversible covalent cross-links using Diels Alder cross-link formation between the diene and dienophile. The chemical components may be selected based on their compatibility with adhesive chemistry as well as their ability to undergo controlled, reversible cross-linking chemistry.

  15. Hardware Counter Multiplexing V1.2

    Energy Science and Technology Software Center (ESTSC)

    2000-10-13

    The Hardware Counter Multiplexer works with the built-in counter registers on computer processors. These counters record varius low-level events as software runs, but they can cannot record all possible events at the same time. This software helps work around that limitation by counting a series of different events in sequence over a period of time. This in turn allows programmers to measure interesting combinations of events, rather than single events. The software is designed tomore » work with multithreaded or single-threaded programs.« less

  16. Multiplexed microsatellite markers for seven Metarhizium species.

    PubMed

    Mayerhofer, Johanna; Lutz, Andy; Widmer, Franco; Rehner, Stephen A; Leuchtmann, Adrian; Enkerli, Jürg

    2015-11-01

    Cross-species transferability of 41 previously published simple sequence repeat (SSR) markers was assessed for 11 species of the entomopathogenic fungus Metarhizium. A collection of 65 Metarhizium strains including all 54 used in a recent phylogenetic revision of the genus were characterized. Between 15 and 34 polymorphic SSR markers produced scorable PCR amplicons in seven species, including M. anisopliae, M. brunneum, M. guizhouense, M. lepidiotae, M. majus, M. pingshaense, and M. robertsii. To provide genotyping tools for concurrent analysis of these seven species fifteen markers grouped in five multiplex pools were selected based on high allelic diversity and easy scorability of SSR chromatograms. PMID:26407949

  17. Two wavelength division multiplexing WAN trials

    SciTech Connect

    Lennon, W.J.; Thombley, R.L.

    1995-01-20

    Lawrence Livermore National Laboratory, as a super-user, supercomputer, and super-application site, is anticipating the future bandwidth and protocol requirements necessary to connect to other such sites as well as to connect to remote-sited control centers and experiments. In this paper the authors discuss their vision of the future of Wide Area Networking, describe the plans for a wavelength division multiplexed link connecting Livermore with the University of California at Berkeley and describe plans for a transparent, {approx} 10 Gb/s ring around San Francisco Bay.

  18. Replicator dynamics with diffusion on multiplex networks

    NASA Astrophysics Data System (ADS)

    Requejo, R. J.; Díaz-Guilera, A.

    2016-08-01

    In this study we present an extension of the dynamics of diffusion in multiplex graphs, which makes the equations compatible with the replicator equation with mutations. We derive an exact formula for the diffusion term, which shows that, while diffusion is linear for numbers of agents, it is necessary to account for nonlinear terms when working with fractions of individuals. We also derive the transition probabilities that give rise to such macroscopic behavior, completing the bottom-up description. Finally, it is shown that the usual assumption of constant population sizes induces a hidden selective pressure due to the diffusive dynamics, which favors the increase of fast diffusing strategies.

  19. Preliminary study of visual effect of multiplex hologram

    NASA Astrophysics Data System (ADS)

    Fu, Huaiping; Xiong, Bingheng; Yang, Hong; Zhang, Xueguo

    2004-06-01

    The process of any movement of real object can be recorded and displayed by a multiplex holographic stereogram. An embossing multiplex holographic stereogram and a multiplex rainbow holographic stereogram have been made by us, the multiplex rainbow holographic stereogram reconstructs the dynamic 2D line drawing of speech organs, the embossing multiplex holographic stereogram reconstructs the process of an old man drinking water. In this paper, we studied the visual result of an embossing multiplex holographic stereogram made with 80 films of 2-D pictures. Forty-eight persons of aged from 13 to 67 were asked to see the hologram and then to answer some questions about the feeling of viewing. The results indicate that this kind of holograms could be accepted by human visual sense organ without any problem. This paper also discusses visual effect of the multiplex holography stereograms base on visual perceptual psychology. It is open out that the planar multiplex holograms can be recorded and present the movement of real animal and object. Not only have the human visual perceptual constancy for shape, just as that size, color, etc... but also have visual perceptual constancy for binocular parallax.

  20. 47 CFR 73.319 - FM multiplex subcarrier technical standards.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 4 2014-10-01 2014-10-01 false FM multiplex subcarrier technical standards. 73.319 Section 73.319 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) BROADCAST RADIO SERVICES RADIO BROADCAST SERVICES FM Broadcast Stations § 73.319 FM multiplex subcarrier technical standards. (a) The technical specifications in...

  1. 47 CFR 73.319 - FM multiplex subcarrier technical standards.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 4 2012-10-01 2012-10-01 false FM multiplex subcarrier technical standards. 73.319 Section 73.319 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) BROADCAST RADIO SERVICES RADIO BROADCAST SERVICES FM Broadcast Stations § 73.319 FM multiplex subcarrier technical standards. (a) The technical specifications in...

  2. Flexible wavelength de-multiplexer for elastic optical networking.

    PubMed

    Zhou, Rui; Gutierrez Pascual, M Deseada; Anandarajah, Prince M; Shao, Tong; Smyth, Frank; Barry, Liam P

    2016-05-15

    We report an injection locked flexible wavelength de-multiplexer (de-mux) that shows 24-h frequency stability of 1 kHz for optical comb-based elastic optical networking applications. We demonstrate 50 GHz, 87.5 GHz equal spacing and 6.25G-25G-50 GHz, 75G-50G-100 GHz unequal spacing for the de-multiplexer outputs. We also implement an unequally spaced (75G-50G-100 GHz), mixed symbol rate (12.5 GBaud and 40 GBaud) and modulation format (polarization division multiplexed quadrature phase shift keying and on-off keying) wavelength division multiplexed transmission system using the de-multiplexer outputs. The results show 0.6 dB receiver sensitivity penalty, at 7% hard decision forward error correction coding limit, of the 100 km transmitted de-mux outputs when compared to comb source seeding laser back-to-back. PMID:27176972

  3. Cascading processes on multiplex networks: Impact of weak layers

    NASA Astrophysics Data System (ADS)

    Lee, Kyu-Min; Goh, Kwang-Il

    Many real-world complex systems such as biological and socio-technological systems consist of manifold layers in multiplex networks. The multiple network layers give rise to the nonlinear effect for the emergent dynamics of systems. Especially, the weak layers plays the significant role in nonlinearity of multiplex networks, which can be neglected in single-layer network framework overlaying all layers. Here we present a simple model of cascades on multiplex networks of heterogeneous layers. The model is simulated on the multiplex network of international trades. We found that the multiplex model produces more catastrophic cascading failures which were the result of collective behaviors from coupling layers rather than the simple summation effect. Therefore risks can be systematically underestimated in simply overlaid network system because the impact of weak layers is overlooked. Our simple theoretical model would have some implications to investigate and design optimal real-world complex systems.

  4. Wavelength division multiplexing WDM, CWDM and DWDM applications

    NASA Astrophysics Data System (ADS)

    Vasile, Irina Bristena; Vasile, Alexandru; Luciana, Stan; Tache, Mihaela

    2007-05-01

    The fiber optics has become the most preferred media for this very large data traffic. TDM (Time Division Multiplexing) has been the most practical method to divide the significant capacity of a single fiber optics into several communication channels. This technology is still limited by the large complexity of high-flow modulation and multiplexing equipment. Presently, a complementary approach proves its potential: Wavelength-Division Multiplexing (WDM). The evolution of WDM allows now a very small spacing between channels wavelength, in nm, generating DWDM (Dense Wavelength Division Multiplexing). The networks with individual fibers including more than 100 independent optic channels, as well as those with bidirectional flow are already available on the market. CWDM (Coarse Wavelength Division Multiplexing) represents an economical application of a mature technology which may provide options where the capacity of fibers is limited.

  5. Assessment of real-time PCR based methods for quantification of pollen-mediated gene flow from GM to conventional maize in a field study.

    PubMed

    Pla, Maria; La Paz, José-Luis; Peñas, Gisela; García, Nora; Palaudelmàs, Montserrat; Esteve, Teresa; Messeguer, Joaquima; Melé, Enric

    2006-04-01

    Maize is one of the main crops worldwide and an increasing number of genetically modified (GM) maize varieties are cultivated and commercialized in many countries in parallel to conventional crops. Given the labeling rules established e.g. in the European Union and the necessary coexistence between GM and non-GM crops, it is important to determine the extent of pollen dissemination from transgenic maize to other cultivars under field conditions. The most widely used methods for quantitative detection of GMO are based on real-time PCR, which implies the results are expressed in genome percentages (in contrast to seed or grain percentages). Our objective was to assess the accuracy of real-time PCR based assays to accurately quantify the contents of transgenic grains in non-GM fields in comparison with the real cross-fertilization rate as determined by phenotypical analysis. We performed this study in a region where both GM and conventional maize are normally cultivated and used the predominant transgenic maize Mon810 in combination with a conventional maize variety which displays the characteristic of white grains (therefore allowing cross-pollination quantification as percentage of yellow grains). Our results indicated an excellent correlation between real-time PCR results and number of cross-fertilized grains at Mon810 levels of 0.1-10%. In contrast, Mon810 percentage estimated by weight of grains produced less accurate results. Finally, we present and discuss the pattern of pollen-mediated gene flow from GM to conventional maize in an example case under field conditions. PMID:16604462

  6. A Duplex PCR-Based Assay for Measuring the Amount of Bacterial Contamination in a Nucleic Acid Extract from a Culture of Free-Living Protists

    PubMed Central

    Marron, Alan O.; Akam, Michael; Walker, Giselle

    2013-01-01

    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 to develop q

  7. Antibiotic resistance and molecular typing among cockle (Anadara granosa) strains of Vibrio parahaemolyticus by polymerase chain reaction (PCR)-based analysis.

    PubMed

    Sahilah, A M; Laila, R A S; Sallehuddin, H Mohd; Osman, H; Aminah, A; Ahmad Azuhairi, A

    2014-02-01

    Genomic DNA of Vibrio parahaemolyticus were characterized by antibiotic resistance, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis. These isolates originated from 3 distantly locations of Selangor, Negeri Sembilan and Melaka (East coastal areas), Malaysia. A total of 44 (n = 44) of tentatively V. parahaemolyticus were also examined for the presence of toxR, tdh and trh gene. Of 44 isolates, 37 were positive towards toxR gene; while, none were positive to tdh and trh gene. Antibiotic resistance analysis showed the V. parahaemolyticus isolates were highly resistant to bacitracin (92%, 34/37) and penicillin (89%, 33/37) followed by resistance towards ampicillin (68%, 25/37), cefuroxime (38%, 14/37), amikacin (6%, 2/37) and ceftazidime (14%, 5/37). None of the V. parahaemolyticus isolates were resistant towards chloramphenicol, ciprofloxacin, ceftriaxone, enrofloxacin, norfloxacin, streptomycin and vancomycin. Antibiogram patterns exhibited, 9 patterns and phenotypically less heterogenous when compared to PCR-based techniques using ERIC- and RAPD-PCR. The results of the ERIC- and RAPD-PCR were analyzed using GelCompare software. ERIC-PCR with primers ERIC1R and ERIC2 discriminated the V. parahaemolyticus isolates into 6 clusters and 21 single isolates at a similarity level of 80%. While, RAPD-PCR with primer Gen8 discriminated the V. parahaemolyticus isolates into 11 clusters and 10 single isolates and Gen9 into 8 clusters and 16 single isolates at the same similarity level examined. Results in the presence study demonstrated combination of phenotypically and genotypically methods show a wide heterogeneity among cockle isolates of V. parahaemolyticus. PMID:24068534

  8. Multi-center evaluation of the novel fully-automated PCR-based Idylla™ BRAF Mutation Test on formalin-fixed paraffin-embedded tissue of malignant melanoma.

    PubMed

    Melchior, Linea; Grauslund, Morten; Bellosillo, Beatriz; Montagut, Clara; Torres, Erica; Moragón, Ester; Micalessi, Isabel; Frans, Johan; Noten, Veerle; Bourgain, Claire; Vriesema, Renske; van der Geize, Robert; Cokelaere, Kristof; Vercooren, Nancy; Crul, Katrien; Rüdiger, Thomas; Buchmüller, Diana; Reijans, Martin; Jans, Caroline

    2015-12-01

    The advent of BRAF-targeted therapies led to increased survival in patients with metastatic melanomas harboring a BRAF V600 mutation (implicated in 46-48% of malignant melanomas). The Idylla(™) System (Idylla(™)), i.e., the real-time-PCR-based Idylla(™) BRAF Mutation Test performed on the fully-automated Idylla(™) platform, enables detection of the most frequent BRAF V600 mutations (V600E/E2/D, V600K/R/M) in tumor material within approximately 90 min and with 1% detection limit. Idylla(™) performance was determined in a multi-center study by analyzing BRAF mutational status of 148 archival formalin-fixed paraffin-embedded (FFPE) tumor samples from malignant melanoma patients, and comparing Idylla(™) results with assessments made by commercial or in-house routine diagnostic methods. Of the 148 samples analyzed, Idylla(™) initially recorded 7 insufficient DNA input calls and 15 results discordant with routine method results. Further analysis learned that the quality of 8 samples was insufficient for Idylla(™) testing, 1 sample had an invalid routine test result, and Idylla(™) results were confirmed in 10 samples. Hence, Idylla(™) identified all mutations present, including 7 not identified by routine methods. Idylla(™) enables fully automated BRAF V600 testing directly on FFPE tumor tissue with increased sensitivity, ease-of-use, and much shorter turnaround time compared to existing diagnostic tests, making it a tool for rapid, simple and highly reliable analysis of therapeutically relevant BRAF mutations, in particular for diagnostic units without molecular expertise and infrastructure. PMID:26407762

  9. Rapid and Accurate Determination of Lipopolysaccharide O-Antigen Types in Klebsiella pneumoniae with a Novel PCR-Based O-Genotyping Method

    PubMed Central

    Shih, Yun-Jui; Cheong, Cheng-Man; Yi, Wen-Ching

    2015-01-01

    Klebsiella pneumoniae, a Gram-negative bacillus that causes life-threatening infections in both hospitalized patients and ambulatory persons, can be classified into nine lipopolysaccharide (LPS) O-antigen serotypes. The O-antigen type has important clinical and epidemiological significance. However, K. pneumoniae O serotyping is cumbersome, and the reagents are not commercially available. To overcome the limitations of conventional serotyping methods, we aimed to create a rapid and accurate PCR method for K. pneumoniae O genotyping. We sequenced the genetic determinants of LPS O antigen from serotypes O1, O2a, O2ac, O3, O4, O5, O8, O9, and O12. We established a two-step genotyping scheme, based on the two genomic regions associated with O-antigen biosynthesis. The first set of PCR primers, which detects alleles at the wzm-wzt loci of the wb gene cluster, distinguishes between O1/O2, O3, O4, O5, O8, O9, and O12. The second set of PCR primers, which detects alleles at the wbbY region, further differentiates between O1, O2a, and O2ac. We verified the specificity of O genotyping against the O-serotype reference strains. We then tested the sensitivity and specificity of O genotyping in K. pneumoniae, using the 56 K-serotype reference strains with known O serotypes determined by an inhibition enzyme-linked immunosorbent assay (iELISA). There is a very good correlation between the O genotypes and classical O serotypes. Three discrepancies were observed and resolved by nucleotide sequencing—all in favor of O genotyping. The PCR-based O genotyping, which can be easily performed in clinical and research microbiology laboratories, is a rapid and accurate method for determining the LPS O-antigen types of K. pneumoniae isolates. PMID:26719438

  10. Genetic Variability of Yersinia pestis Isolates as Predicted by PCR-Based IS100 Genotyping and Analysis of Structural Genes Encoding Glycerol-3-Phosphate Dehydrogenase (glpD)

    PubMed Central

    Motin, Vladimir L.; Georgescu, Anca M.; Elliott, Jeffrey M.; Hu, Ping; Worsham, Patricia L.; Ott, Linda L.; Slezak, Tomas R.; Sokhansanj, Bahrad A.; Regala, Warren M.; Brubaker, Robert R.; Garcia, Emilio

    2002-01-01

    A PCR-based genotyping system that detects divergence of IS100 locations within the Yersinia pestis genome was used to characterize a large collection of isolates of different biovars and geographical origins. Using sequences derived from the glycerol-negative biovar orientalis strain CO92, a set of 27 locus-specific primers was designed to amplify fragments between the end of IS100 and its neighboring gene. Geographically diverse members of the orientalis biovar formed a homogeneous group with identical genotype with the exception of strains isolated in Indochina. In contrast, strains belonging to the glycerol-positive biovar antiqua showed a variety of fingerprinting profiles. Moreover, strains of the biovar medievalis (also glycerol positive) clustered together with the antiqua isolates originated from Southeast Asia, suggesting their close phylogenetic relationships. Interestingly, a Manchurian biovar antiqua strain Nicholisk 51 displayed a genotyping pattern typical of biovar orientalis isolates. Analysis of the glycerol pathway in Y. pestis suggested that a 93-bp deletion within the glpD gene encoding aerobic glycerol-3-phosphate dehydrogenase might account for the glycerol-negative phenotype of the orientalis biovar. The glpD gene of strain Nicholisk 51 did not possess this deletion, although it contained two nucleotide substitutions characteristic of the glpD version found exclusively in biovar orientalis strains. To account for this close relationship between biovar orientalis strains and the antiqua Nicholisk 51 isolate, we postulate that the latter represents a variant of this biovar with restored ability to ferment glycerol. The fact that such a genetic lesion might be repaired as part of the natural evolutionary process suggests the existence of genetic exchange between different Yersinia strains in nature. The relevance of this observation on the emergence of epidemic Y. pestis strains is discussed. PMID:11807062

  11. Bacterial diversity and real-time PCR based assessment of linA and linB gene distribution at hexachlorocyclohexane contaminated sites.

    PubMed

    Lal, Devi; Jindal, Swati; Kumari, Hansi; Jit, Simran; Nigam, Aeshna; Sharma, Pooja; Kumari, Kirti; Lal, Rup

    2015-03-01

    The disposal of hexachlorocyclohexane (HCH) muck has created large number of HCH dumpsites all over the world from where the harmful HCH isomers are leaking into the environment. Bacteria have evolved at such contaminated sites that have the ability to degrade HCH. Degradation of various HCH isomers in bacterial strains is mediated primarily by two genes: linA and linB which encode dehydrochlorinase and haloalkane dehalogenase respectively. In this study we explored one such highly contaminated HCH dumpsite located in Lucknow, Uttar Pradesh, India. To assess the biostimulation potential of the contaminated site, microbial diversity study and real-time PCR based quantification of lin genes was carried out. The soil samples from dumpsite and surrounding areas were found to be highly contaminated with HCH residue levels as high as 1.8 × 10(5)  mg kg(-1). The residues were detected in areas upto 13 km from the dumpsite. Sphingomonads, Chromohalobacter, and Marinobacter were the dominant genera present at the dump-site. Role of Sphingomonads in HCH degradation has been well documented. The highest copy numbers of linA and linB genes as determined using real-time PCR were 6.2 × 10(4) and 5.3 × 10(5), respectively, were found in sample from the dump site. The presence of Sphingomonads, linA, and linB genes from HCH contaminated soil indicates the presence of indigenous bacterial communities capable of HCH degradation. PMID:24002962

  12. Multiplexed Colorimetric Solid-Phase Extraction

    NASA Technical Reports Server (NTRS)

    Gazda, Daniel B.; Fritz, James S.; Porter, Marc D.

    2009-01-01

    Multiplexed colorimetric solid-phase extraction (MC-SPE) is an extension of colorimetric solid-phase extraction (C-SPE) an analytical platform that combines colorimetric reagents, solid phase extraction, and diffuse reflectance spectroscopy to quantify trace analytes in water. In CSPE, analytes are extracted and complexed on the surface of an extraction membrane impregnated with a colorimetric reagent. The analytes are then quantified directly on the membrane surface using a handheld diffuse reflectance spectrophotometer. Importantly, the use of solid-phase extraction membranes as the matrix for impregnation of the colorimetric reagents creates a concentration factor that enables the detection of low concentrations of analytes in small sample volumes. In extending C-SPE to a multiplexed format, a filter holder that incorporates discrete analysis channels and a jig that facilitates the concurrent operation of multiple sample syringes have been designed, enabling the simultaneous determination of multiple analytes. Separate, single analyte membranes, placed in a readout cartridge create unique, analyte-specific addresses at the exit of each channel. Following sample exposure, the diffuse reflectance spectrum of each address is collected serially and the Kubelka-Munk function is used to quantify each water quality parameter via calibration curves. In a demonstration, MC-SPE was used to measure the pH of a sample and quantitate Ag(I) and Ni(II).

  13. Multiplexed quantification for data-independent acquisition.

    PubMed

    Minogue, Catherine E; Hebert, Alexander S; Rensvold, Jarred W; Westphall, Michael S; Pagliarini, David J; Coon, Joshua J

    2015-03-01

    Data-independent acquisition (DIA) strategies provide a sensitive and reproducible alternative to data-dependent acquisition (DDA) methods for large-scale quantitative proteomic analyses. Unfortunately, DIA methods suffer from incompatibility with common multiplexed quantification methods, specifically stable isotope labeling approaches such as isobaric tags and stable isotope labeling of amino acids in cell culture (SILAC). Here we expand the use of neutron-encoded (NeuCode) SILAC to DIA applications (NeuCoDIA), producing a strategy that enables multiplexing within DIA scans without further convoluting the already complex MS(2) spectra. We demonstrate duplex NeuCoDIA analysis of both mixed-ratio (1:1 and 10:1) yeast and mouse embryo myogenesis proteomes. Analysis of the mixed-ratio yeast samples revealed the strong accuracy and precision of our NeuCoDIA method, both of which were comparable to our established MS(1)-based quantification approach. NeuCoDIA also uncovered the dynamic protein changes that occur during myogenic differentiation, demonstrating the feasibility of this methodology for biological applications. We consequently establish DIA quantification of NeuCode SILAC as a useful and practical alternative to DDA-based approaches. PMID:25621425

  14. Multiplexed protein profiling by sequential affinity capture.

    PubMed

    Ayoglu, Burcu; Birgersson, Elin; Mezger, Anja; Nilsson, Mats; Uhlén, Mathias; Nilsson, Peter; Schwenk, Jochen M

    2016-04-01

    Antibody microarrays enable parallelized and miniaturized analysis of clinical samples, and have proven to provide novel insights for the analysis of different proteomes. However, there are concerns that the performance of such direct labeling and single antibody assays are prone to off-target binding due to the sample context. To improve selectivity and sensitivity while maintaining the possibility to conduct multiplexed protein profiling, we developed a multiplexed and semi-automated sequential capture assay. This novel bead-based procedure encompasses a first antigen capture, labeling of captured protein targets on magnetic particles, combinatorial target elution and a read-out by a secondary capture bead array. We demonstrate in a proof-of-concept setting that target detection via two sequential affinity interactions reduced off-target contribution, while lowered background and noise levels, improved correlation to clinical values compared to single binder assays. We also compared sensitivity levels with single binder and classical sandwich assays, explored the possibility for DNA-based signal amplification, and demonstrate the applicability of the dual capture bead-based antibody microarray for biomarker analysis. Hence, the described concept enhances the possibilities for antibody array assays to be utilized for protein profiling in body fluids and beyond. PMID:26935855

  15. Multiplexing holograms in an acrylamide photopolymer

    NASA Astrophysics Data System (ADS)

    Fernández, Elena; Ortuño, Manuel; Márquez, Andrés; Gallego, Sergi; Pascual, Inmaculada

    2006-04-01

    A peristrophic multiplexing method is used to store various diffraction gratings at the same spot in the material. This material is formed of acrylamide photopolymers which are considered interesting materials for recording holographic memories. They have high diffraction efficiency (ratio between diffracted and incident beams), high energetic sensitivity and optical quality, and developing processes are not necessary. In this work, the photopolymer is composed of acrylamide (AA) as the polymerizable monomer, triethanolamine (TEA) as radical generator, N,N'methylene-bis-acrylamide (BMA) as crosslinker, yellowish eosin (YE) as sensitizer and a binder of polyvinyl alcohol (PVA). The layers of material obtained are approximately 1 mm thick. Using holographic recording schedules, the exposure energy each hologram should receive in order to achieve uniform diffraction efficiency is optimized. The purpose of these recording schedules is to enable full advantage to be taken of the whole dynamic range of the material and to share it between the individual holograms. The Scheduled Exposure Method (SEM) and the Incremental Exposure Method (IEM) are the two multiplexing schedules used to determine the recording times. Having determined these times, the results obtained with both methods are compared to ascertain which method enables the greatest number of holograms to be recorded with the highest, most uniform diffraction efficiencies.

  16. Mesoscopic quantum multiplex for channeling bunches

    NASA Astrophysics Data System (ADS)

    Shen, Jing

    1998-09-01

    (1) Bogacz-Cline channeling is an interesting idea that can transform a bunch of low particle intensity to a collider of high luminosity but it was maintained as impossible to carry out because of three technical problems. (2) The first of which is discussed in this paper, and it is how to get billions particles from each bunch to enter into and channel through a single crystal channel. (3) Two basic difficulties of entrance are discussed in this paper. The first is due to the Heisenberg's uncertainty, and the second is the dimension reduction of a beam bunch in crystal from 3D to 1D. (4) To overcome these difficulties, a hybrid device named Mesoscopic Quantum Multiplex (MQM) is designed to achieve entrance and channeling. It is a quantum generalization of classical multiplex in detector readout electronics for the classical-quantum interface. It is made by nano-crystalline technology. (5) The MQM can channel the Richter-Kimura-Takada flat e± beams of NLC-JLC, and low emittance p or heavy ion beams as well as the Bogacz-Cline μ± beams, and the Nagamine-Chu cool μ± beams.

  17. Pneumatic Valve Operated by Multiplex Pneumatic Transmission

    NASA Astrophysics Data System (ADS)

    Nishioka, Yasutaka; Suzumori, Koichi; Kanda, Takefumi; Wakimoto, Shuichi

    A pneumatic system has several advantages, which are cheapness, lightweight, and reliability to human and environment. These advantages are adapted to some research areas, such as industrial lines, medical and nursing cares, and rehabilitation tools. However, the pneumatic system needs several devices; compressor, air tube, and control valve. This research aim to downsize pneumatic system. In this paper, a new method of multiplex pneumatic transmission for multi-pneumatic servo system is proposed. The valve for this system consists of two vibrators supported by springs, which was designed with simple and cheap structure. The working principle of the valve is vibrators resonance from multiplex pneumatic transmission and it is possible to work as ON/OFF valves without electric wire. Dynamic simulation was used to confirm the working principle of the resonance driving system. A prototype device confirming the principle was designed and developed based on the simulation. The experiments show that this new control system works very well to control two separated valves through single pneumatic tube.

  18. Multiplexed microsatellite recovery using massively parallel sequencing

    USGS Publications Warehouse

    Jennings, T.N.; Knaus, B.J.; Mullins, T.D.; Haig, S.M.; Cronn, R.C.

    2011-01-01

    Conservation and management of natural populations requires accurate and inexpensive genotyping methods. Traditional microsatellite, or simple sequence repeat (SSR), marker analysis remains a popular genotyping method because of the comparatively low cost of marker development, ease of analysis and high power of genotype discrimination. With the availability of massively parallel sequencing (MPS), it is now possible to sequence microsatellite-enriched genomic libraries in multiplex pools. To test this approach, we prepared seven microsatellite-enriched, barcoded genomic libraries from diverse taxa (two conifer trees, five birds) and sequenced these on one lane of the Illumina Genome Analyzer using paired-end 80-bp reads. In this experiment, we screened 6.1 million sequences and identified 356958 unique microreads that contained di- or trinucleotide microsatellites. Examination of four species shows that our conversion rate from raw sequences to polymorphic markers compares favourably to Sanger- and 454-based methods. The advantage of multiplexed MPS is that the staggering capacity of modern microread sequencing is spread across many libraries; this reduces sample preparation and sequencing costs to less than $400 (USD) per species. This price is sufficiently low that microsatellite libraries could be prepared and sequenced for all 1373 organisms listed as 'threatened' and 'endangered' in the United States for under $0.5M (USD).

  19. Controllability of asynchronous Boolean multiplex control networks

    NASA Astrophysics Data System (ADS)

    Luo, Chao; Wang, Xingyuan; Liu, Hong

    2014-09-01

    In this article, the controllability of asynchronous Boolean multiplex control networks (ABMCNs) is studied. First, the model of Boolean multiplex control networks under Harvey' asynchronous update is presented. By means of semi-tensor product approach, the logical dynamics is converted into linear representation, and a generalized formula of control-depending network transition matrices is achieved. Second, a necessary and sufficient condition is proposed to verify that only control-depending fixed points of ABMCNs can be controlled with probability one. Third, using two types of controls, the controllability of system is studied and formulae are given to show: (a) when an initial state is given, the reachable set at time s under a group of specified controls; (b) the reachable set at time s under arbitrary controls; (c) the specific probability values from a given initial state to destination states. Based on the above formulae, an algorithm to calculate overall reachable states from a specified initial state is presented. Moreover, we also discuss an approach to find the particular control sequence which steers the system between two states with maximum probability. Examples are shown to illustrate the feasibility of the proposed scheme.

  20. Multiplexed Quantification for Data-Independent Acquisition

    PubMed Central

    Minogue, Catherine E.; Hebert, Alexander S.; Rensvold, Jarred W.; Westphall, Michael S.; Pagliarini, David J.; Coon, Joshua J.

    2015-01-01

    Data-independent acquisition (DIA) strategies provide a sensitive and reproducible alternative to data-dependent acquisition (DDA) methods for large-scale quantitative proteomic analyses. Unfortunately, DIA methods suffer from incompatibility with common multiplexed quantification methods, specifically stable isotope labeling approaches such as isobaric tags and stable isotope labeling of amino acids in cell culture (SILAC). Here we expand the use of neutron-encoded (NeuCode) SILAC to DIA applications (NeuCoDIA), producing a strategy that enables multiplexing within DIA scans without further convoluting the already complex MS2 spectra. We demonstrate duplex NeuCoDIA analysis of both mixed-ratio (1:1 and 10:1) yeast and mouse embryo myogenesis proteomes. Analysis of the mixed-ratio yeast samples revealed the strong accuracy and precision of our NeuCoDIA method, both of which were comparable to our established MS1-based quantification approach. NeuCoDIA also uncovered the dynamic protein changes that occur during myogenic differentiation, demonstrating the feasibility of this methodology for biological applications. We consequently establish DIA quantification of NeuCode SILAC as a useful and practical alternative to DDA-based approaches. PMID:25621425

  1. Dispersion-reduction technique using subcarrier multiplexing

    SciTech Connect

    Sargis, P.D.; Haigh, R.E.; McCammon, K.G.

    1995-10-18

    We have developed a novel dispersion-reduction technique using subcarrier multiplexing (SCM) which permits the transmission of multiple 2.5 Gbit/s data channels over hundreds of kilometers of conventional fiber-optic cable with negligible dispersion. Using a lithium niobate external modulator having a modulation bandwidth of 20 GHz, we are able to multiplex several high-speed data channels at a single wavelength. At the receiving end, we demultiplex the data and detect each channel using a 2-GHz bandwidth optical detector. All of the hardware in our system consists of off-the-shelf components and can be integrated to reduce the overall cost. We demonstrated our dispersion-reduction technique in a recent field trial by transmitting two 2.5 Gbit/s data channels over 90 km of commercially-installed single-mode fiber, followed by 210 km of spooled fiber. For comparison, we substituted the 300 km of fiber with equivalent optical attenuation. We also ran computer simulations to evaluate link behavior. Technical details and field trial results will be presented.

  2. Optically-synchronized encoder and multiplexer scheme for interleaved photonics analog-to-digital conversion

    NASA Astrophysics Data System (ADS)

    Villa, Carlos; Kumavor, Patrick; Donkor, Eric

    2008-04-01

    Photonics Analog-to-Digital Converters (ADCs) utilize a train of optical pulses to sample an electrical input waveform applied to an electrooptic modulator or a reverse biased photodiode. In the former, the resulting train of amplitude-modulated optical pulses is detected (converter to electrical) and quantized using a conversional electronics ADC- as at present there are no practical, cost-effective optical quantizers available with performance that rival electronic quantizers. In the latter, the electrical samples are directly quantized by the electronics ADC. In both cases however, the sampling rate is limited by the speed with which the electronics ADC can quantize the electrical samples. One way to increase the sampling rate by a factor N is by using the time-interleaved technique which consists of a parallel array of N electrical ADC converters, which have the same sampling rate but different sampling phase. Each operating at a quantization rate of fs/N where fs is the aggregated sampling rate. In a system with no real-time operation, the N channels digital outputs are stored in memory, and then aggregated (multiplexed) to obtain the digital representation of the analog input waveform. Alternatively, for real-time operation systems the reduction of storing time in the multiplexing process is desired to improve the time response of the ADC. The complete elimination of memories come expenses of concurrent timing and synchronization in the aggregation of the digital signal that became critical for a good digital representation of the analog signal waveform. In this paper we propose and demonstrate a novel optically synchronized encoder and multiplexer scheme for interleaved photonics ADCs that utilize the N optical signals used to sample different phases of an analog input signal to synchronize the multiplexing of the resulting N digital output channels in a single digital output port. As a proof of concept, four 320 Megasamples/sec 12-bit of resolution digital

  3. A novel multiplex RT-qPCR method based on dual-labelled probes suitable for typing all known genotypes of viral haemorrhagic septicaemia virus.

    PubMed

    Vázquez, D; López-Vázquez, C; Skall, H F; Mikkelsen, S S; Olesen, N J; Dopazo, C P

    2016-04-01

    Viral haemorrhagic septicaemia (VHS) is a notifiable fish disease, whose causative agent is a rhabdovirus isolated from a wide range of fish species, not only in fresh but also in marine and brackish waters. Phylogenetic studies have identified four major genotypes, with a strong geographical relationship. In this study, we have designed and validated a new procedure - named binary multiplex RT-qPCR (bmRT-qPCR) - for simultaneous detection and typing of all four genotypes of VHSV by real-time RT-PCR based on dual-labelled probes and composed by two multiplex systems designed for European and American/Asiatic isolates, respectively, using a combination of three different fluorophores. The specificity of the procedure was assessed by including a panel of 81 VHSV isolates covering all known genotypes and subtypes of the virus, and tissue material from experimentally infected rainbow trout, resulting in a correct detection and typing of all strains. The analytical sensitivity was evaluated in a comparative assay with titration in cell culture, observing that both methods provided similar limits of detection. The proposed method can be a powerful tool for epidemiological analysis of VHSV by genotyping unknown samples within a few hours. PMID:25952496

  4. Combining Forces - The Use of Landsat TM Satellite Imagery, Soil Parameter Information, and Multiplex PCR to Detect Coccidioides immitis Growth Sites in Kern County, California

    PubMed Central

    Lauer, Antje; Talamantes, Jorge; Castañón Olivares, Laura Rosío; Medina, Luis Jaime; Baal, Joe Daryl Hugo; Casimiro, Kayla; Shroff, Natasha; Emery, Kirt W.

    2014-01-01

    Coccidioidomycosis is a fungal disease acquired through the inhalation of spores of Coccidioides spp., which afflicts primarily humans and other mammals. It is endemic to areas in the southwestern United States, including the San Joaquin Valley portion of Kern County, California, our region of interest (ROI). Recently, incidence of coccidioidomycosis, also known as valley fever, has increased significantly, and several factors including climate change have been suggested as possible drivers for this observation. Up to date details about the ecological niche of C. immitis have escaped full characterization. In our project, we chose a three-step approach to investigate this niche: 1) We examined Landsat-5-Thematic-Mapper multispectral images of our ROI by using training pixels at a 750 m×750 m section of Sharktooth Hill, a site confirmed to be a C. immitis growth site, to implement a Maximum Likelihood Classification scheme to map out the locations that could be suitable to support the growth of the pathogen; 2) We used the websoilsurvey database of the US Department of Agriculture to obtain soil parameter data; and 3) We investigated soil samples from 23 sites around Bakersfield, California using a multiplex Polymerase Chain Reaction (PCR) based method to detect the pathogen. Our results indicated that a combination of satellite imagery, soil type information, and multiplex PCR are powerful tools to predict and identify growth sites of C. immitis. This approach can be used as a basis for systematic sampling and investigation of soils to detect Coccidioides spp. PMID:25380290

  5. Reverse Correlation in Neurophysiology

    ERIC Educational Resources Information Center

    Ringach, Dario; Shapley, Robert

    2004-01-01

    This article presents a review of reverse correlation in neurophysiology. We discuss the basis of reverse correlation in linear transducers and in spiking neurons. The application of reverse correlation to measure the receptive fields of visual neurons using white noise and m-sequences, and classical findings about spatial and color processing in…

  6. Multiplex PCR for the detection of Piscirickettsia salmonis, Vibrio anguillarum, Aeromonas salmonicida and Streptococcus phocae in Chilean marine farms.

    PubMed

    Tapia-Cammas, D; Yañez, A; Arancibia, G; Toranzo, A E; Avendaño-Herrera, R

    2011-12-01

    A multiplex (m-)PCR-based protocol was designed for the simultaneous detection of the main marine bacterial pathogens in Chilean salmon farms: Streptococcus phocae, Aeromonas salmonicida, Vibrio anguillarum and Piscirickettsia salmonis. Each of the 4 oligonucleotide primer pairs exclusively amplified the target gene of the specific bacterial pathogen. The detection limit of the m-PCR using purified total bacterial DNA was 50 pg microl(-1) for V anguillarum, 500 fg microl(-1) for P. salmonis, and 5 pg microl(-1) for S. phocae and A. salmonicida. This corresponded to average limits in the m-PCR sensitivity of 3.69 x 10(5) CFU ml(-1) of V anguillarum, 1.26 x 10(4) CFU m(-1) of S. phocae, and 5.33 x 10(4) CFU ml(-1) of A. salmonicida, while the detection limits for the spiked fish tissues, regardless of the sample (spleen, kidney, liver or muscle) were 2.64 +/- 0.54 x 10(7) CFU g(-1) for V. anguillarum, 9.03 +/- 1.84 x 10(5) CFU g(-1) for S. phocae, 3.8 +/- 0.78 x 10(3) CFU mg(-1) for A. salmonicida and 100 P. salmonis cells. However, high amounts of DNA from 3 bacterial species had a reduction of -1 log-unit on the amplification sensitivity of S. phocae or A. salmonicida when these were present in lower concentration in the multiplex reaction. The assay described in this study is a rapid, sensitive and efficient tool to detect the presence of S. phocae, A. salmonicida, V. anguillarum and P. salmonis simultaneously from pure cultures and tissues from clinically diseased fish. Therefore, it may be a useful alternative to culture-based methods for the diagnosis of infections in fish obtained from Chilean salmon farms. PMID:22303630

  7. Methods for detection and differentiation of existing and new crinivirus species through multiplex and degenerate primer RT-PCR.

    PubMed

    Wintermantel, William M; Hladky, Laura L

    2010-12-01

    A method was developed for rapid identification and differentiation of both known and novel crinivirus species involving both multiplex and degenerate reverse transcription-polymerase chain reaction (RT-PCR). The multiplex method can discriminate among known criniviruses infecting vegetable and small fruit crops, and rapidly identify viruses associated with disease symptoms, as well as identification of mixed crinivirus infections. Four host groups for multiplex detection of criniviruses were selected based on the types of crops where specific criniviruses would be expected to occur. Each detection group contained three to four crop-specific primers designed to the same region of the gene encoding the highly conserved RNA-dependent RNA polymerase gene (RdRp) of criniviruses for rapid, single-reaction determination of which crinivirus(es) may be infecting a plant. Degenerate reverse primers used for RT and in PCR were designed to amplify all members of each host group, and were coupled with species-specific forward primers resulting in four separate single-reaction cocktails for detection of most criniviruses sequenced to date, whether present in single or mixed virus infections. Additional viruses can be added to multiplex detection by adjustment of primer concentration for balanced detection of target viruses. In order to identify unknown putative criniviruses or those for which sequence information is not yet available, a genus-wide, universal degenerate primer set was developed. These primers also targeted the crinivirus RdRp gene, and amplify a wide range of crinivirus sequences. Both detection systems can be used with most RNA extraction methods, and with RT-PCR reagents common in most laboratories. PMID:20833203

  8. Mode- and wavelength-division multiplexed transmission using all-fiber mode multiplexer based on mode selective couplers.

    PubMed

    Chang, Sun Hyok; Chung, Hwan Seok; Ryf, Roland; Fontaine, Nicolas K; Han, Changyo; Park, Kyung Jun; Kim, Kwangjoon; Lee, Jyung Chan; Lee, Jong Hyun; Kim, Byoung Yoon; Kim, Young Kie

    2015-03-23

    We propose all-fiber mode multiplexer composed of two consecutive LP₁₁ mode selective couplers that allows for the multiplexing of LP₀₁ mode and two-fold degenerate LP₁₁ modes. We demonstrate WDM transmission of 32 wavelength channels with 100 GHz spacing, each carrying 3 modes of 120 Gb/s polarization division multiplexed quadrature phase shifted keying (PDM-QPSK) signal, over 560 km of few-mode fiber (FMF). Long distance transmission is achieved by 6×6 multiple-input multiple-output digital signal processing and modal differential group delay compensated link of FMF. The all-fiber mode multiplexer has considerable potential to be used in mode- and wavelength-division multiplexed transmission. PMID:25837061

  9. Detection of free-living amoebae by using multiplex quantitative PCR.

    PubMed

    Le Calvez, Thomas; Trouilhé, Marie-Cécile; Humeau, Philippe; Moletta-Denat, Marina; Frère, Jacques; Héchard, Yann

    2012-06-01

    Free-living amoebae (FLA) are protozoa found worldwide in soil and aquatic environments, which are able to colonize man-made water networks. Some FLA have the potential to be pathogenic and others might harbour pathogenic bacteria. Indeed, FLA feed on bacteria, but some bacteria could resist phagocytosis and either survive in FLA or even multiply within FLA. These bacteria are collectively named amoeba resistant bacteria (ARB). The best characterized example is Legionella pneumophila, for which FLA is the main reservoir in the environment. Not only could FLA be a reservoir that protects ARB, some bacteria might become more resistant to treatment and be more virulent. Thus, it is of medical significance to quantify FLA populations in soil, water or the environment. The main limitation for the quantification of FLA is that classical culture is not efficient and reliable for many genera and 'strains'. Thus, several PCR-based quantification methods have been published for various FLA. However, thus far, no method has been published to simultaneously quantify the main FLA genera in the same PCR reaction. In this study, we developed a multiplex qPCR method to detect both Amoebozoan (i.e. Acanthamoeba, Hartmannella and Echinamoeba) and Vahlkampfiidae (i.e. Vahlkampfia and Naegleria) using 18S ribosomal RNA as the target gene. This method was shown to be specific, reliable and sensitive, could be used for the quantification of FLA and is likely to be useful to anticipate risks due to FLA or pathogenic bacteria, such as L. pneumophila. PMID:22449586

  10. Simultaneous typing of seven porcine pathogens by multiplex PCR with a GeXP analyser.

    PubMed

    Hu, Ling; Lin, Xingyu; Nie, Fuping; ZexiaoYang; Yao, Xueping; Li, Guili; Wu, Xulong; Ren, Meishen; Wang, Yin

    2016-06-01

    A novel high-throughput method was developed for simultaneous detection and differentiation of seven porcine pathogens by multiplex PCR based on a GenomeLab Gene Expression Profiler (GeXP) analyser. The pathogens included in this study were pseudorabies virus (PRV), classical swine fever virus (CSFV), African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV), porcine circovirus type 2 (PCV-2) and Japanese encephalitis virus (JEV). Seven pairs of chimeric primers, consisting of a pathogen-specific sequence fused to a universal sequence at the 5' end, were used to initiate the PCR, after which a set of universal primers was used for the subsequent cycles of the PCR. Amplification products were separated by capillary electrophoresis and identified using fluorescence spectrophotometry. The specificity of the GeXP assay was examined with single and mixed pathogen cDNA/DNA templates. The specific DNA product amplification peaks of seven pathogens were observed on the GeXP analyser. Negative controls did not produce DNA products. The sensitivity was evaluated by performing the assay on serial 10-fold dilutions of the plasmids containing the target sequence. Under optimised conditions this assay achieved a sensitivity of 100-1000 copies/μL for a single virus and 1000 copies/μL when all of the seven pre-mixed viral targets were present. Furthermore, the GeXP-PCR assay was 100% specific when 58 clinical samples were tested in comparison with the conventional PCR method. In conclusion, the GeXP assay is a rapid, cost-effective, sensitive, specific and high throughput method for simultaneously detecting seven pathogens that infect swine. PMID:26706731

  11. Quantum Operation Time Reversal

    SciTech Connect

    Crooks, Gavin E.

    2008-03-25

    The dynamics of an open quantum system can be described by a quantum operation: A linear, complete positive map of operators. Here, I exhibit a compact expression for the time reversal of a quantum operation, which is closely analogous to the time reversal of a classical Markov transition matrix. Since open quantum dynamics are stochastic, and not, in general, deterministic, the time reversal is not, in general, an inversion of the dynamics. Rather, the system relaxes toward equilibrium in both the forward and reverse time directions. The probability of a quantum trajectory and the conjugate, time reversed trajectory are related by the heat exchanged with the environment.

  12. A Comparison of Culture- and PCR-Based Methods to Detect Six Major Non-O157 Serogroups of Shiga Toxin-Producing Escherichia coli in Cattle Feces

    PubMed Central

    Noll, Lance W.; Shridhar, Pragathi B.; Dewsbury, Diana M.; Shi, Xiaorong; Cernicchiaro, Natalia; Renter, David G.; Nagaraja, T. G.

    2015-01-01

    Culture-based methods to detect the six major non-O157 (O26, O45, O103, O111, O121 and O145) Shiga toxin-producing E. coli (STEC) are not well established. Our objectives of this study were to develop a culture-based method to detect the six non-O157 serogroups in cattle feces and compare the detection with a PCR method. Fecal samples (n = 576) were collected in a feedlot from 24 pens during a 12-week period and enriched in E. coli broth at 40° C for 6 h. Enriched samples were subjected to immunomagnetic separation, spread-plated onto a selective chromogenic medium, and initially pooled colonies, and subsequently, single colonies were tested by a multiplex PCR targeting six serogroups and four virulence genes, stx1, stx2, eae, and ehxA (culture method). Fecal suspensions, before and after enrichment, were also tested by a multiplex PCR targeting six serogroups and four virulence genes (PCR method). There was no difference in the proportions of fecal samples that tested positive (74.3 vs. 77.4%) for one or more of the six serogroups by either culture or the PCR method. However, each method detected one or more of the six serogroups in samples that were negative by the other method. Both culture method and PCR indicated that O26, O45, and O103 were the dominant serogroups. Higher proportions (P < 0.05) of fecal samples were positive for O26 (44.4 vs. 22.7%) and O121 (22.9 vs. 2.3%) serogroups by PCR than by the culture method. None of the fecal samples contained more than four serogroups. Only a small proportion of the six serogroups (23/640; 3.6%) isolated carried Shiga toxin genes. The culture method and the PCR method detected all six serogroups in samples negative by the other method, highlighting the importance of subjecting fecal samples to both methods for accurate detection of the six non-O157 STEC in cattle feces. PMID:26270482

  13. Novel Subcarrier Multiplexing Technologies for Optical Communication

    NASA Astrophysics Data System (ADS)

    Wang, Xiaolu

    Microwave subcarrier multiplexing (SCM) has recently emerged as a potentially important multiplexing technique for wideband lightwave systems. By using a double beam modulation technique (DBM), in which the information is modulated onto an optical coherent pair instead of a single optical beam, a novel SCM system is proposed. The system, with multi-division and multi-channel capability (encompassing the multiplexing of both multi-channel analog and/or digital signals), potentially has an information bandwidth (IBW) of tens of GHz and is particularly suitable for optical fiber and free-space communications. The principle of the proposed system was first demonstrated by using a standing-wave surface-acoustic -wave optical modulator (SWSAW). The modulator was fabricated on the top of a Ti - LiNbO_3 waveguide. The highest acoustic modulation frequency achieved was 300 MHz, which corresponds to a 600 MHz subcarrier. The laser output, which had been directly modulated by VHF TV signals, passed through the SWSAW modulator and was upconverted to the UHF band. The carrier-to-noise ratio of the upconverted TV signal was measured to be 30 dB. The more advanced way of implementing the proposed SCM is utilizing a frequency-locked-laser (FLL) pair, which has virtually no upper frequency limitation and is readily FM modulated. We have demonstrated, to our knowledge, the first FM modulated FLL pair for optical communication. The subcarrier (locked) frequency of 15 GHz is also believed to be the highest reported today. The multi-channel video signals and high frequency sinusoidal modulations up to 1 GHz, after being FM modulated onto and demodulated from a 15 GHz subcarrier, are displayed directly on standard TV receivers and oscilloscopes. Another novel SCM, with ultra high millimeter -wave frequency subcarriers of up to one hundred GHz, based upon the self-sustained-pulsation (SSP) of the laser diode, was also proposed. A preliminary optical link test with multi

  14. Development of a multiplexed PCR detection method for Barley and Cereal Yellow Dwarf Viruses, Wheat Spindle Streak Virus, Wheat Streak Mosaic Virus and Soil-Borne Wheat Mosaic Virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Barley and Cereal Yellow Dwarf Viruses (B/CYDVs), Wheat Spindle Streak Mosaic (WSSMV), Soil-Borne Wheat Mosaic (SBWMV) Mosaic Virus and Wheat Streak Mosaic Virus (WSMV) constitute the most economically important group of wheat viruses. In this paper, a multiplex reverse transcription polymerase chai...

  15. Development and application of a hexaplex reverse transcription polymerase chain reaction for screening global citrus tristeza virus isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The discovery of the diversity of Citrus tristeza virus (CTV) genotypes has complicated detection and diagnostic measures. To simplify the identification and differentiation of CTV genotypes, an efficient multiplex reverse transcription polymerase chain reaction (M-RT-PCR) technique for the screenin...

  16. Transmission of multiplexed video signals in multimode optical fiber systems

    NASA Technical Reports Server (NTRS)

    White, Preston, III

    1988-01-01

    Kennedy Space Center has the need for economical transmission of two multiplexed video signals along multimode fiberoptic systems. These systems must span unusual distances and must meet RS-250B short-haul standards after reception. Bandwidth is a major problem and studies of the installed fibers, available LEDs and PINFETs led to the choice of 100 MHz as the upper limit for the system bandwidth. Optical multiplexing and digital transmission were deemed inappropriate. Three electrical multiplexing schemes were chosen for further study. Each of the multiplexing schemes included an FM stage to help meet the stringent S/N specification. Both FM and AM frequency division multiplexing methods were investigated theoretically and these results were validated with laboratory tests. The novel application of quadrature amplitude multiplexing was also considered. Frequency division multiplexing of two wideband FM video signal appears the most promising scheme although this application requires high power highly linear LED transmitters. Futher studies are necessary to determine if LEDs of appropriate quality exist and to better quantify performance of QAM in this application.

  17. Multiplexing Strategies for Monolithic Crystal PET Detector Modules

    PubMed Central

    Pierce, L A; Hunter, W C J; Haynor, D R; MacDonald, L R; Kinahan, P E; Miyaoka, R S

    2014-01-01

    To reduce the number of output channels and associated cost in PET detectors, strategies to multiplex the signal channels have been investigated by several researchers. This work aims to find an optimal multiplexing strategy for detector modules consisting of a monolithic LYSO scintillator coupled to a 64-channel PMT. Methods We apply simulated multiplexing strategies to measured data from two continuous miniature crystal element (cMiCE) detector modules. The strategies tested include standard methods such as row-column summation and its variants, as well as new data-driven methods involving the principal components of measured data and variants of those components. The detector positioning resolution and bias is measured for each multiplexing strategy and the results are compared. Results The mean FWHM over the entire detector was 1.23 mm for no multiplexing (64 channels). Using 16 principal component channels yielded a mean FWHM resolution of 1.21 mm, while traditional row/column summation (16 channels) yielded 1.28 mm. Using 8 principal component output channels resulted in a resolution of 1.30 mm. Conclusion Using the principal components of the calibration data to guide the multiplexing scheme appears to be a viable method for reducing the number of output data channels. Further study is needed to determine if the depth-of-interaction resolution can be preserved with this multiplexing scheme. PMID:25146849

  18. Multiplexity versus correlation: the role of local constraints in real multiplexes.

    PubMed

    Gemmetto, V; Garlaschelli, D

    2015-01-01

    Several systems can be represented as multiplex networks, i.e. in terms of a superposition of various graphs, each related to a different mode of connection between nodes. Hence, the definition of proper mathematical quantities aiming at capturing the added level of complexity of those systems is required. Various steps in this direction have been made. In the simplest case, dependencies between layers are measured via correlation-based metrics, a procedure that we show to be equivalent to the use of completely homogeneous benchmarks specifying only global constraints. However, this approach does not take into account the heterogeneity in the degree and strength distributions, which is instead a fundamental feature of real-world multiplexes. In this work, we compare the observed dependencies between layers with the expected values obtained from maximum-entropy reference models that appropriately control for the observed heterogeneity in the degree and strength distributions. This information-theoretic approach results in the introduction of novel and improved multiplexity measures that we test on different datasets, i.e. the International Trade Network and the European Airport Network. Our findings confirm that the use of homogeneous benchmarks can lead to misleading results, and highlight the important role played by the distribution of hubs across layers. PMID:25767040

  19. Multiplexity versus correlation: the role of local constraints in real multiplexes

    NASA Astrophysics Data System (ADS)

    Gemmetto, V.; Garlaschelli, D.

    2015-03-01

    Several systems can be represented as multiplex networks, i.e. in terms of a superposition of various graphs, each related to a different mode of connection between nodes. Hence, the definition of proper mathematical quantities aiming at capturing the added level of complexity of those systems is required. Various steps in this direction have been made. In the simplest case, dependencies between layers are measured via correlation-based metrics, a procedure that we show to be equivalent to the use of completely homogeneous benchmarks specifying only global constraints. However, this approach does not take into account the heterogeneity in the degree and strength distributions, which is instead a fundamental feature of real-world multiplexes. In this work, we compare the observed dependencies between layers with the expected values obtained from maximum-entropy reference models that appropriately control for the observed heterogeneity in the degree and strength distributions. This information-theoretic approach results in the introduction of novel and improved multiplexity measures that we test on different datasets, i.e. the International Trade Network and the European Airport Network. Our findings confirm that the use of homogeneous benchmarks can lead to misleading results, and highlight the important role played by the distribution of hubs across layers.

  20. The evaluation of a PCR-based method for identification of Salmonella enterica serotypes from environmental samples and various food matrices.

    PubMed

    Jean-Gilles Beaubrun, Junia; Cheng, Chorng-Ming; Chen, Kai-Shun; Ewing, Laura; Wang, Hua; Agpaoa, Maria C; Huang, Mei-Chiung J; Dickey, Erin; Du, Jamie M; Williams-Hill, Donna M; Hamilton, Brittany; Micallef, Shirley A; Rosenberg Goldstein, Rachel E; George, Ashish; Joseph, Sam W; Sapkota, Amy R; Jacobson, Andrew P; Tall, Ben D; Kothary, Mahendra H; Dudley, Kim; Hanes, Darcy E

    2012-09-01

    The most commonly used method for serotyping Salmonella spp. is based on the Kaufmann-White scheme, and is composed of serological reactions using antibodies to LPS agglutinins. The multiplex PCR used in this investigation was established by Kim et al. to serotype the 30 most common clinical Salmonella serotypes, as determined by CDC. The PCR assay consists of two five-plex reactions and a single two-plex PCR reaction, based on six genetic loci from Salmonella enterica serotype Typhimurium and four loci from S. enterica serotype Typhi. In this investigation, we further evaluated the method for serotyping Salmonella spp. using a reference collection, environmental samples collected from a Mid-Atlantic region tomato farm study, four food matrices spiked with different Salmonella serotypes and a proficiency test. The PCR assay was first evaluated using DNA isolated from pure cultures of isolates obtained from various clinical and environmental samples, and then DNA isolated from broth cultures of food matrices of "Red round" and Roma tomatoes, Romaine lettuce, green onions and Serrano peppers spiked with serotypes Newport, Typhimurium, Javiana and Saintpaul, respectively. The results showed that the PCR assay correctly serotyped Salmonella spp. from the clinical, environmental, spiked food matrices, and proficiency test samples. These findings are significant because the PCR assay was successful in the identification of Salmonella in the spiked samples in a broth culture containing other non-salmonella organism. This method may be a useful resource for the food safety community. PMID:22608224

  1. Emergence of network features from multiplexity.

    PubMed

    Cardillo, Alessio; Gómez-Gardeñes, Jesús; Zanin, Massimiliano; Romance, Miguel; Papo, David; del Pozo, Francisco; Boccaletti, Stefano

    2013-01-01

    Many biological and man-made networked systems are characterized by the simultaneous presence of different sub-networks organized in separate layers, with links and nodes of qualitatively different types. While during the past few years theoretical studies have examined a variety of structural features of complex networks, the outstanding question is whether such features are characterizing all single layers, or rather emerge as a result of coarse-graining, i.e. when going from the multilayered to the aggregate network representation. Here we address this issue with the help of real data. We analyze the structural properties of an intrinsically multilayered real network, the European Air Transportation Multiplex Network in which each commercial airline defines a network layer. We examine how several structural measures evolve as layers are progressively merged together. In particular, we discuss how the topology of each layer affects the emergence of structural properties in the aggregate network. PMID:23446838

  2. Multiplexed spectroscopy with holographic optical tweezers

    NASA Astrophysics Data System (ADS)

    Cibula, Matthew A.; McIntyre, David H.

    2014-09-01

    We have developed a multiplexed holographic optical tweezers system with an imaging spectrometer to manipulate multiple optically trapped nanosensors and detect multiple fluorescence spectra. The system uses a spatial light modulator (SLM) to control the positions of infrared optical traps in the sample so that multiple nanosensors can be positioned into regions of interest. Spectra of multiple nanosensors are detected simultaneously with the application of an imaging spectrometer. Nanosensors are capable of detecting changes in their environment such as pH, ion concentration, temperature, and voltage by monitoring changes in the nanosensors' emitted fluorescence spectra. We use streptavidin labeled quantum dots bound to the surface of biotin labeled polystyrene microspheres to measure temperature changes by observing a corresponding shift in the wavelength of the spectral peak. The fluorescence is excited at 532 nm with a wide field source.

  3. Prototype data terminal: Multiplexer/demultiplexer

    NASA Technical Reports Server (NTRS)

    Leck, D. E.; Goodwin, J. E.

    1972-01-01

    The design and operation of a quad redundant data terminal and a multiplexer/demultiplexer (MDU) design are described. The most unique feature is the design of the quad redundant data terminal. This is one of the few designs where the unit is fail/op, fail/op, fail/safe. Laboratory tests confirm that the unit will operate satisfactorily with the failure of three out of four channels. Although the design utilizes state-of-the-art technology. The waveform error checks, the voting techniques, and the parity bit checks are believed to be used in unique configurations. Correct word selection routines are also novel, if not unique. The MDU design, while not redundant, utilizes, the latest state-of-the-art advantages of light couplers and integrated circuit amplifiers.

  4. Multiview multiperspective time multiplexed autostereoscopic display

    NASA Astrophysics Data System (ADS)

    Kupiec, Stephen A.; Markov, Vladimir B.; Hopper, Darrel G.; Saini, Gurdial

    2008-02-01

    The implementation of a time multiplexed display capable of eight simultaneously visible viewing zones will be described. The system employs a high speed digital micromirror device (DMD) to allow for the high framerate essential for flicker free display of multiple viewing zones. A combination of custom graphical processor unit (GPU) programming and a correspondingly optimized field programmable gate array (FPGA) DMD driver allows for real time interactive rendering of scenes. The rendering engine is entirely based on off the shelf with the use of a standard DVI-D interface for data transfer to the DMD interface. A rapidly switched LED light engine is employed to overcome the speed limitations of color wheel light sources, as well as providing a highly saturated color gamut. Selection of viewing zones is achieved by the use of a high-speed shutter interfaced directly to the DMD driver for precise synchronization.

  5. Realization of a spin-wave multiplexer.

    PubMed

    Vogt, K; Fradin, F Y; Pearson, J E; Sebastian, T; Bader, S D; Hillebrands, B; Hoffmann, A; Schultheiss, H

    2014-01-01

    Recent developments in the field of spin dynamics--like the interaction of charge and heat currents with magnons, the quasi-particles of spin waves--opens the perspective for novel information processing concepts and potential applications purely based on magnons without the need of charge transport. The challenges related to the realization of advanced concepts are the spin-wave transport in two-dimensional structures and the transfer of existing demonstrators to the micro- or even nanoscale. Here we present the experimental realization of a microstructured spin-wave multiplexer as a fundamental building block of a magnon-based logic. Our concept relies on the generation of local Oersted fields to control the magnetization configuration as well as the spin-wave dispersion relation to steer the spin-wave propagation in a Y-shaped structure. Thus, the present work illustrates unique features of magnonic transport as well as their possible utilization for potential technical applications. PMID:24759754

  6. Multiplex coherent raman spectroscopy detector and method

    NASA Technical Reports Server (NTRS)

    Chen, Peter (Inventor); Joyner, Candace C. (Inventor); Patrick, Sheena T. (Inventor); Guyer, Dean R. (Inventor)

    2004-01-01

    A multiplex coherent Raman spectrometer (10) and spectroscopy method rapidly detects and identifies individual components of a chemical mixture separated by a separation technique, such as gas chromatography. The spectrometer (10) and method accurately identify a variety of compounds because they produce the entire gas phase vibrational Raman spectrum of the unknown gas. This is accomplished by tilting a Raman cell (20) to produce a high-intensity, backward-stimulated, coherent Raman beam of 683 nm, which drives a degenerate optical parametric oscillator (28) to produce a broadband beam of 1100-1700 nm covering a range of more than 3000 wavenumber. This broadband beam is combined with a narrowband beam of 532 nm having a bandwidth of 0.003 wavenumbers and focused into a heated windowless cell (38) that receives gases separated by a gas chromatograph (40). The Raman radiation scattered from these gases is filtered and sent to a monochromator (50) with multichannel detection.

  7. Multiplexed Microsphere Suspension Array-Based Immunoassays.

    PubMed

    Lin, Andrew; Salvador, Alexandra; Carter, J Mark

    2015-01-01

    ELISA is an extremely powerful tool to detect analytes because of its sensitivity, selectivity, reproducibility and ease of use. Here we describe sandwich immunoassays performed in suspension on spectrally unique microspheres developed by Luminex. Luminex assays offer the benefit of multiplex analysis of large numbers of analytes in a single reaction. Because the microspheres are spectrally unique, many microspheres, each attached to various antibodies, can be added to a single sample. Luminex instruments can distinguish each microsphere and detect the intensity of a reporter signal for each microsphere. Results are reported in Median Fluorescent Intensities for each analyte. Luminex assays can be used to detect up to 500 analytes in a high-throughput format. Luminex refers to this technology as xMAP(®). Here we describe a routine protocol for a Luminex immunoassay. Other Luminex assays would have to be optimized for specific conditions according to their use. PMID:26160569

  8. MULTIPLEXING IN THE PRIMATE MOTION PATHWAY

    PubMed Central

    Huk, Alexander C.

    2012-01-01

    This article begins by reviewing recent work on 3D motion processing in the primate visual system. Some of these results suggest that 3D motion signals may be processed in the same circuitry already known to compute 2D motion signals. Such “multiplexing” has implications for the study of visual cortical circuits and neural signals. A more explicit appreciation of multiplexing— and the computations required for demultiplexing— may enrich the study of the visual system by emphasizing the importance of a structured and balanced “encoding / decoding” framework. In addition to providing a fresh perspective on how successive stages of visual processing might be approached, multiplexing also raises caveats about the value of “neural correlates” for understanding neural computation. PMID:22811986

  9. Emergence of network features from multiplexity

    PubMed Central

    Cardillo, Alessio; Gómez-Gardeñes, Jesús; Zanin, Massimiliano; Romance, Miguel; Papo, David; Pozo, Francisco del; Boccaletti, Stefano

    2013-01-01

    Many biological and man-made networked systems are characterized by the simultaneous presence of different sub-networks organized in separate layers, with links and nodes of qualitatively different types. While during the past few years theoretical studies have examined a variety of structural features of complex networks, the outstanding question is whether such features are characterizing all single layers, or rather emerge as a result of coarse-graining, i.e. when going from the multilayered to the aggregate network representation. Here we address this issue with the help of real data. We analyze the structural properties of an intrinsically multilayered real network, the European Air Transportation Multiplex Network in which each commercial airline defines a network layer. We examine how several structural measures evolve as layers are progressively merged together. In particular, we discuss how the topology of each layer affects the emergence of structural properties in the aggregate network. PMID:23446838

  10. Multiplex coherent raman spectroscopy detector and method

    DOEpatents

    Chen, Peter; Joyner, Candace C.; Patrick, Sheena T.; Guyer, Dean R.

    2004-06-08

    A multiplex coherent Raman spectrometer (10) and spectroscopy method rapidly detects and identifies individual components of a chemical mixture separated by a separation technique, such as gas chromatography. The spectrometer (10) and method accurately identify a variety of compounds because they produce the entire gas phase vibrational Raman spectrum of the unknown gas. This is accomplished by tilting a Raman cell (20) to produce a high-intensity, backward-stimulated, coherent Raman beam of 683 nm, which drives a degenerate optical parametric oscillator (28) to produce a broadband beam of 1100-1700 nm covering a range of more than 3000 wavenumber. This broadband beam is combined with a narrowband beam of 532 nm having a bandwidth of 0.003 wavenumbers and focused into a heated windowless cell (38) that receives gases separated by a gas chromatograph (40). The Raman radiation scattered from these gases is filtered and sent to a monochromator (50) with multichannel detection.

  11. Weighted multiplex network of air transportation

    NASA Astrophysics Data System (ADS)

    Varga, Imre

    2016-06-01

    In several real networks large heterogeneity of links is present either in intensity or in the nature of relationships. Therefore, recent studies in network science indicate that more detailed topological information are available if weighted or multi-layer aspect is applied. In the age of globalization air transportation is a representative example of huge complex infrastructure systems, which has been analyzed from different points of view. In this paper a novel approach is applied to study the airport network as a weighted multiplex taking into account the fact that the rules and fashion of domestic and international flights differ. Restricting study to only topological features and their correlations in the system (disregarding traffic) one can see reasons why simple network approximation is not adequate.

  12. Pneumosinus dilatans multiplex associated with hormonal imbalance.

    PubMed

    Ushas, P; Ravi, V; Painatt, Jaeson Mohanan; Nair, Preeti P

    2013-01-01

    Pneumosinus dilatans describes an abnormal dilation of one or more paranasal sinuses without radiological evidence of localised bone destruction, hyperostosis or mucous membrane thickening. Dilation of mastoid air cells also occurs rarely along with involvement of paranasal sinuses. This rare combination of unknown aetiology was reported in two cases in the literature and termed 'Pneumosinus Dilatans Multiplex' (PSDM). It is usually asymptomatic, and is detected incidentally on plain radiography, CT or MRI. If left untreated, it can further erode the bone leading to complications such as facial asymmetry, neurological disorders and pathological fractures. The aetiology of the condition remains obscure. Various hypotheses proposed are the presence of gas-forming microorganisms, spontaneous drainage of a mucocele, the presence of a one-way valve, dysregulation of hormonal levels leading to a disturbance of osteoblastic and osteoclastic activity. This paper describes a case of PSDM possibly secondary to hormonal disturbance. PMID:23978497

  13. Adiabatically-tapered fiber mode multiplexers.

    PubMed

    Yerolatsitis, S; Gris-Sánchez, I; Birks, T A

    2014-01-13

    Simple all-fiber three-mode multiplexers were made by adiabatically merging three dissimilar single-mode cores into one multimode core. This was achieved by collapsing air holes in a photonic crystal fiber and (in a separate device) by fusing and tapering separate telecom fibers in a fluorine-doped silica capillary. In each case the LP01 mode and both LP11 modes were individually excited from three separate input cores, with losses below 0.3 and 0.7 dB respectively and mode purities exceeding 10 dB. Scaling to more modes is challenging, but would be assisted by using single-mode fibers with a smaller ratio of cladding to core diameter. PMID:24515021

  14. Development of a multiplex polymerase chain reaction for the simultaneous detection of microsporidians, nucleopolyhedrovirus, and densovirus infecting silkworms.

    PubMed

    Ravikumar, G; Raje Urs, S; Vijaya Prakash, N B; Rao, C G P; Vardhana, K V

    2011-07-01

    We have developed a novel PCR-based assay for individual and simultaneous detection of three major pathogens (microsporidians, nucleopolyhedrovirus (NPV) and densovirus (DNV)) infecting the silkworm, Bombyx mori. Multiplex PCR, using three primer pairs, two of which were designed from the conserved regions of 16S small subunit ribosomal RNA gene of microsporidians, and polyhedrin gene of NPVs respectively, and a third primer pair designed from the internal sequences of B. mori DNVs (BmDNV), showed discrete and pathogen specific PCR products. The assay showed high specificity and sensitivity for the pathogenic DNA. Under optimized PCR conditions, the assay yielded a 794bp DNA fragment from Nosema bombycis, 471bp fragment from B. mori NPV (BmNPV) and 391bp fragment from BmDNV. Further, this detection method was successfully applied to other silkworm species such as Antheraea mylitta and Samia cynthia ricini, in detecting same or similar pathogens infecting them. This method is a valuable supplement to the conventional microscopic diagnostic methods and can be used for the early detection of pathogens infecting silkworms. Furthermore it can assist research and extension centers for the safe supply of disease-free silkworms to farmers. PMID:21570404

  15. A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants.

    PubMed

    Ma, Xingliang; Zhang, Qunyu; Zhu, Qinlong; Liu, Wei; Chen, Yan; Qiu, Rong; Wang, Bin; Yang, Zhongfang; Li, Heying; Lin, Yuru; Xie, Yongyao; Shen, Rongxin; Chen, Shuifu; Wang, Zhi; Chen, Yuanling; Guo, Jingxin; Chen, Letian; Zhao, Xiucai; Dong, Zhicheng; Liu, Yao-Guang

    2015-08-01

    CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high-efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edited 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homologous end-joining mechanism followed by homologous recombination-based repair. We also obtained uniform biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mutations in T0 rice and T1 Arabidopsis plants by simultaneous targeting of multiple (up to eight) members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement. PMID:25917172

  16. Specific multiplex analysis of pathogens using a direct 16S rRNA hybridization in microarray system.

    PubMed

    Hwang, Byeong Hee; Shin, Hwa Hui; Seo, Jeong Hyun; Cha, Hyung Joon

    2012-06-01

    For the rapid multiplex analysis of pathogens, 16S rRNAs from cell lysates were directly applied onto a DNA microarray at room temperature (RT) for RNA-DNA hybridization. To eliminate the labeling step, seven fluorescent-labeled detector probes were cohybridized with 16S rRNA targets and adjacent specific capture probes. We found that eight pathogens were successfully discriminated by the 16S rRNA-based direct method, which showed greater specificity than the polymerase chain reaction (PCR)-labeled method due to chaperone and distance effects. A new specificity criterion for a perfect match between RNA and DNA was suggested to be 21-41% dissimilarity using correlation analysis between the mismatch and the sequence according to the guanine-cytosine (GC) percentage or the distribution of mismatches. Six categories of food matrix (egg, meat, milk, rice, vegetable, and mixed) were also tested, and the target pathogen was successfully discriminated within statistically significant levels. Finally, we found that the intrinsic abundance of 16S rRNA molecules successfully substituted PCR-based amplification with a low limit of detection of 10-10(3) cells mL(-1) and a high quantitative linear correlation. Collectively, our suggested 16S rRNA-based direct method enables the highly sensitive, specific, and quantitative analysis of selected pathogens at RT within 2 h, even in food samples. PMID:22551354

  17. Inter-layer synchronization in multiplex networks of identical layers.

    PubMed

    Sevilla-Escoboza, R; Sendiña-Nadal, I; Leyva, I; Gutiérrez, R; Buldú, J M; Boccaletti, S

    2016-06-01

    Inter-layer synchronization is a distinctive process of multiplex networks whereby each node in a given layer evolves synchronously with all its replicas in other layers, irrespective of whether or not it is synchronized with the other units of the same layer. We analytically derive the necessary conditions for the existence and stability of such a state, and verify numerically the analytical predictions in several cases where such a state emerges. We further inspect its robustness against a progressive de-multiplexing of the network, and provide experimental evidence by means of multiplexes of nonlinear electronic circuits affected by intrinsic noise and parameter mismatch. PMID:27368794

  18. Shift-multiplexed self-referential holographic data storage.

    PubMed

    Takabayashi, Masanori; Okamoto, Atsushi; Eto, Taisuke; Okamoto, Takashi

    2014-07-10

    The feasibility and the properties of shift-multiplexed self-referential holographic data storage (SR-HDS) were investigated. Although SR-HDS has attractive features as typified by referenceless holographic recording, its multiplexing properties, which are consummately important for holographic data storage, have not been clarified until now. The results of numerical and experimental evaluations of medium shift dependence in SR-HDS clarified that the shift selectivity is almost the same as in collinear holography. Furthermore, 25 datapages were successfully shift-multiplexed with the shift pitch of 8.3 μm by the numerical simulation. PMID:25090055

  19. Control of Angular Intervals for Angle-Multiplexed Holographic Memory

    NASA Astrophysics Data System (ADS)

    Kinoshita, Nobuhiro; Muroi, Tetsuhiko; Ishii, Norihiko; Kamijo, Koji; Shimidzu, Naoki

    2009-03-01

    In angle-multiplexed holographic memory, the full width at half maximum of the Bragg selectivity curves is dependent on the angle formed between the medium and incident laser beams. This indicates the possibility of high density and high multiplexing number by varying the angular intervals between adjacent holograms. We propose an angular interval scheduling for closely stacking holograms into medium even when the angle range is limited. We obtained bit error rates of the order of 10-4 under the following conditions: medium thickness of 1 mm, laser beam wavelength of 532 nm, and angular multiplexing number of 300.

  20. Multiplexing of discrete chaotic signals in presence of noise

    NASA Astrophysics Data System (ADS)

    Nagaraj, Nithin; Vaidya, Prabhakar G.

    2009-09-01

    Multiplexing of discrete chaotic signals in presence of noise is investigated. The existing methods are based on chaotic synchronization, which is susceptible to noise, precision limitations, and requires more iterates. Furthermore, most of these methods fail for multiplexing more than two discrete chaotic signals. We propose novel methods to multiplex multiple discrete chaotic signals based on the principle of symbolic sequence invariance in presence of noise and finite precision implementation of finding the initial condition of an arbitrarily long symbolic sequence of a chaotic map. Our methods work for single precision and as less as 35 iterates. For two signals, our method is robust up to 50% noise level.

  1. Multiplexed multi-longitudinal mode fiber laser sensor.

    PubMed

    Huang, Long; Wang, Peng; Gao, Liang; Zhang, Tingting; Chen, Xiangfei

    2014-10-20

    A multiplexed multi-longitudinal mode fiber laser sensor system is proposed and demonstrated. By incorporating two matched wavelength division multiplexers (WDMs) and a semiconductor optical amplifier (SOA) into a fiber laser cavity, multiwavelength oscillation is established. Each wavelength corresponding to one channel of WDMs contains multi-longitudinal modes. The multiwavelength output of the laser is directed to another WDM which functions as a demultiplexer. By monitoring the longitudinal mode beat frequency generated at photodetectors following the WDM, the sensing information can be demodulated. Preliminary results for multiplexing two sensors measuring strain and temperature are presented to verify the principle of the system. PMID:25401605

  2. Multiplex detection of protein-protein interactions using a next generation luciferase reporter.

    PubMed

    Verhoef, Lisette G G C; Mattioli, Michela; Ricci, Fernanda; Li, Yao-Cheng; Wade, Mark

    2016-02-01

    Cell-based assays of protein-protein interactions (PPIs) using split reporter proteins can be used to identify PPI agonists and antagonists. Generally, such assays measure one PPI at a time, and thus counterscreens for on-target activity must be run in parallel or at a subsequent stage; this increases both the cost and time during screening. Split luciferase systems offer advantages over those that use split fluorescent proteins (FPs). This is since split luciferase offers a greater signal:noise ratio and, unlike split FPs, the PPI can be reversed upon small molecule treatment. While multiplexed PPI assays using luciferase have been reported, they suffer from low signal:noise and require fairly complex spectral deconvolution during analysis. Furthermore, the luciferase enzymes used are large, which limits the range of PPIs that can be interrogated due to steric hindrance from the split luciferase fragments. Here, we report a multiplexed PPI assay based on split luciferases from Photinus pyralis (firefly luciferase, FLUC) and the deep-sea shrimp, Oplophorus gracilirostris (NanoLuc, NLUC). Specifically, we show that the binding of the p53 tumor suppressor to its two major negative regulators, MDM2 and MDM4, can be simultaneously measured within the same sample, without the requirement for complex filters or deconvolution. We provide chemical and genetic validation of this system using MDM2-targeted small molecules and mutagenesis, respectively. Combined with the superior signal:noise and smaller size of split NanoLuc, this multiplexed PPI assay format can be exploited to study the induction or disruption of pairwise interactions that are prominent in many cell signaling pathways. PMID:26646257

  3. Multiplexed chemiluminescent assays in ArrayPlates for high-throughput measurement of gene expression

    NASA Astrophysics Data System (ADS)

    Martel, Ralph R.; Rounseville, Matthew P.; Botros, Ihab W.; Seligmann, Bruce E.

    2002-06-01

    Multiplexed Molecular Profiling (MMP) assays for drug discovery are performed in ArrayPlates. ArrayPlates are 96- well microtiter plates that contain a 16-element array at the bottom of each well. Each element within an array measures one analyte in a sample. A CCD imager records the quantitative chemiluminescent readout of all 1,536 elements in a 96-well plate simultaneously. Since array elements are reagent modifiable by the end-user, ArrayPlates can be adapted to a broad range of nucleic acid- and protein-based assays. Such multiplexed assays are rapidly established, flexible, robust, automation-friendly and cost-effective. Nucleic acid assays in ArrayPlates can detect DNA and RNA, including SNPs and ESTs. A multiplexed mRNA assay to measure the expression of 16 genes is described. The assay combines a homogeneous nuclease protection assay with subsequent probe immobilization to the array by means of a sandwich hybridization followed with chemiluminescent detection. This assay was used to examine cells grown and treated in microplates and avoided cloning, transfection, RNA insolation, reverse transcription, amplification and fluorochrome labeling. Standard deviations for the measurement of 16 genes ranged from 3 percent to 13 percent in samples of 30,000 cells. Such ArrayPlates transcription assays are useful in drug discovery and development for target validation, screening, lead optimization, metabolism and toxicity profiling. Chemiluminescent detection provides ArrayPlates assays with high signal-to-noise readout and simplifies imager requirements. Imaging a 2D surface that contains arrays simplifies lens requirements relative to imaging columns of liquid in microtiter plate wells. The Omix imager for ArrayPlates is described.

  4. A MULTIPLEX REVERSE TRANSCIPTION-PCR METHOD FOR DETECTION OF HUMAN ENTERIC VIRUSES IN GROUNDWATER

    EPA Science Inventory

    Untreated groundwater is responsible for about half of the waterborne disease outbreaks in the United States. Human enteric viruses are thought to be leading etiological agents of many of these outbreaks, but there is relatively little information on the types and levels of viru...

  5. Digital image compression for a 2f multiplexing optical setup

    NASA Astrophysics Data System (ADS)

    Vargas, J.; Amaya, D.; Rueda, E.

    2016-07-01

    In this work a virtual 2f multiplexing system was implemented in combination with digital image compression techniques and redundant information elimination. Depending on the image type to be multiplexed, a memory-usage saving of as much as 99% was obtained. The feasibility of the system was tested using three types of images, binary characters, QR codes, and grey level images. A multiplexing step was implemented digitally, while a demultiplexing step was implemented in a virtual 2f optical setup following real experimental parameters. To avoid cross-talk noise, each image was codified with a specially designed phase diffraction carrier that would allow the separation and relocation of the multiplexed images on the observation plane by simple light propagation. A description of the system is presented together with simulations that corroborate the method. The present work may allow future experimental implementations that will make use of all the parallel processing capabilities of optical systems.

  6. Quantitative multiplexing with nano-self-assemblies in SERS

    PubMed Central

    Kasera, Setu; Herrmann, Lars O.; Barrio, Jesús del; Baumberg, Jeremy J.; Scherman, Oren A.

    2014-01-01

    Multiplexed or simultaneous detection of multiple analytes is a valuable tool in many analytical applications. However, complications caused by the presence of interfering compounds in a sample form a major drawback in existing molecular sensor technologies, particularly in multi-analyte systems. Although separating analytes through extraction or chromatography can partially address the problem of interferents, there remains a need for developing direct observational tools capable of multiplexing that can be applied in situ. Surface-enhanced Raman Spectroscopy (SERS) is an optical molecular finger-printing technique that has the ability to resolve analytes from within mixtures. SERS has attracted much attention for its potential in multiplexed sensing but it has been limited in its quantitative abilities. Here, we report a facile supramolecular SERS-based method for quantitative multiplex analysis of small organic molecules in aqueous environments such as human urine. PMID:25354650

  7. On-chip noninterference angular momentum multiplexing of broadband light

    NASA Astrophysics Data System (ADS)

    Ren, Haoran; Li, Xiangping; Zhang, Qiming; Gu, Min

    2016-05-01

    Angular momentum division has emerged as a physically orthogonal multiplexing method in high-capacity optical information technologies. However, the typical bulky elements used for information retrieval from the overall diffracted field, based on the interference method, impose a fundamental limit toward realizing on-chip multiplexing. We demonstrate noninterference angular momentum multiplexing by using a mode-sorting nanoring aperture with a chip-scale footprint as small as 4.2 micrometers by 4.2 micrometers, where nanoring slits exhibit a distinctive outcoupling efficiency on tightly confined plasmonic modes. The nonresonant mode-sorting sensitivity and scalability of our approach enable on-chip parallel multiplexing over a bandwidth of 150 nanometers in the visible wavelength range. The results offer the possibility of ultrahigh-capacity and miniaturized nanophotonic devices harnessing angular momentum division.

  8. Quantitative multiplexing with nano-self-assemblies in SERS.

    PubMed

    Kasera, Setu; Herrmann, Lars O; del Barrio, Jesús; Baumberg, Jeremy J; Scherman, Oren A

    2014-01-01

    Multiplexed or simultaneous detection of multiple analytes is a valuable tool in many analytical applications. However, complications caused by the presence of interfering compounds in a sample form a major drawback in existing molecular sensor technologies, particularly in multi-analyte systems. Although separating analytes through extraction or chromatography can partially address the problem of interferents, there remains a need for developing direct observational tools capable of multiplexing that can be applied in situ. Surface-enhanced Raman Spectroscopy (SERS) is an optical molecular finger-printing technique that has the ability to resolve analytes from within mixtures. SERS has attracted much attention for its potential in multiplexed sensing but it has been limited in its quantitative abilities. Here, we report a facile supramolecular SERS-based method for quantitative multiplex analysis of small organic molecules in aqueous environments such as human urine. PMID:25354650

  9. Quantitative multiplexing with nano-self-assemblies in SERS

    NASA Astrophysics Data System (ADS)

    Kasera, Setu; Herrmann, Lars O.; Barrio, Jesús Del; Baumberg, Jeremy J.; Scherman, Oren A.

    2014-10-01

    Multiplexed or simultaneous detection of multiple analytes is a valuable tool in many analytical applications. However, complications caused by the presence of interfering compounds in a sample form a major drawback in existing molecular sensor technologies, particularly in multi-analyte systems. Although separating analytes through extraction or chromatography can partially address the problem of interferents, there remains a need for developing direct observational tools capable of multiplexing that can be applied in situ. Surface-enhanced Raman Spectroscopy (SERS) is an optical molecular finger-printing technique that has the ability to resolve analytes from within mixtures. SERS has attracted much attention for its potential in multiplexed sensing but it has been limited in its quantitative abilities. Here, we report a facile supramolecular SERS-based method for quantitative multiplex analysis of small organic molecules in aqueous environments such as human urine.

  10. Communicability reveals a transition to coordinated behavior in multiplex networks.

    PubMed

    Estrada, Ernesto; Gómez-Gardeñes, Jesús

    2014-04-01

    We analyze the flow of information in multiplex networks by means of the communicability function. First, we generalize this measure from its definition from simple graphs to multiplex networks. Then, we study its relevance for the analysis of real-world systems by studying a social multiplex where information flows using formal-informal channels and an air transportation system where the layers represent different air companies. Accordingly, the communicability, which is essential for the good performance of these complex systems, emerges at a systemic operation point in the multiplex where the performance of the layers operates in a coordinated way very differently from the state represented by a collection of unconnected networks. PMID:24827305

  11. Integrated spatial multiplexing of heralded single-photon sources

    PubMed Central

    Collins, M.J.; Xiong, C.; Rey, I.H.; Vo, T.D.; He, J.; Shahnia, S.; Reardon, C.; Krauss, T.F.; Steel, M.J.; Clark, A.S.; Eggleton, B.J.

    2013-01-01

    The non-deterministic nature of photon sources is a key limitation for single-photon quantum processors. Spatial multiplexing overcomes this by enhancing the heralded single-photon yield without enhancing the output noise. Here the intrinsic statistical limit of an individual source is surpassed by spatially multiplexing two monolithic silicon-based correlated photon pair sources in the telecommunications band, demonstrating a 62.4% increase in the heralded single-photon output without an increase in unwanted multipair generation. We further demonstrate the scalability of this scheme by multiplexing photons generated in two waveguides pumped via an integrated coupler with a 63.1% increase in the heralded photon rate. This demonstration paves the way for a scalable architecture for multiplexing many photon sources in a compact integrated platform and achieving efficient two-photon interference, required at the core of optical quantum computing and quantum communication protocols. PMID:24107840

  12. Multiplexed Holographic Optical Data Storage In Thick Bacteriorhodopsin Films

    NASA Technical Reports Server (NTRS)

    Downie, John D.; Timucin, Dogan A.; Gary, Charles K.; Ozcan, Meric; Smithey, Daniel T.; Crew, Marshall

    1998-01-01

    The optical data storage capacity of photochromic bacteriorhodopsin films is investigated by means of theoretical calculations, numerical simulations, and experimental measurements on sequential recording of angularly multiplexed diffraction gratings inside a thick D85N BR film.

  13. Burst noise in the HAWAII-1RG multiplexer

    NASA Astrophysics Data System (ADS)

    Bacon, Candice M.; McMurtry, Craig W.; Pipher, Judith L.; Forrest, William J.; Garnett, James D.

    2005-08-01

    Burst noise (also known as popcorn noise and random telegraph signal/noise) is a phenomenon that is understood to be a result of defects in the vicinity of a p-n junction. It is characterized by rapid level shifts in both positive and negative directions and can have varying magnitudes. This noise has been seen in both HAWAII-1RG and HAWAII-2RG multiplexers and is under investigation. We have done extensive burst noise testing on a HAWAII-1RG multiplexer, where we have determined a significant percentage of pixels exhibit the phenomenon. In addition, the prevalence of small magnitude transitions make sensitivity of detection the main limiting factor. Since this is a noise source for the HAWAII-1RG multiplexer, its elimination would make the HAWAII-1RG and the HAWAII-2RG even lower noise multiplexers.

  14. Multiplex versus multiple taxonomy of paraphilia: case example.

    PubMed

    Lehne, Gregory K; Money, John

    2003-01-01

    Several different paraphilias are presently diagnosed in some individuals whereas a more parsimonious taxonomy would be that of one multiplex paraphilia. A multiplex paraphilia may be expressed by variations of content at different times in an individual's life or in different situations. The present case example shows the unfolding of a multiplex paraphilia over a lifetime. At age 7 the subject was dressed in public as a girl wearing a diaper as a humiliation for bed-wetting. This experience had 3 paraphilic components that were separately manifested at different times in his life: fetishistic transvestism, pedophilic incest, and infantilism. A multiplex paraphilia taxonomy may lead to improved identification of etiology, prognosis, and treatment of paraphilia. PMID:12616929

  15. On-chip noninterference angular momentum multiplexing of broadband light.

    PubMed

    Ren, Haoran; Li, Xiangping; Zhang, Qiming; Gu, Min

    2016-05-13

    Angular momentum division has emerged as a physically orthogonal multiplexing method in high-capacity optical information technologies. However, the typical bulky elements used for information retrieval from the overall diffracted field, based on the interference method, impose a fundamental limit toward realizing on-chip multiplexing. We demonstrate noninterference angular momentum multiplexing by using a mode-sorting nanoring aperture with a chip-scale footprint as small as 4.2 micrometers by 4.2 micrometers, where nanoring slits exhibit a distinctive outcoupling efficiency on tightly confined plasmonic modes. The nonresonant mode-sorting sensitivity and scalability of our approach enable on-chip parallel multiplexing over a bandwidth of 150 nanometers in the visible wavelength range. The results offer the possibility of ultrahigh-capacity and miniaturized nanophotonic devices harnessing angular momentum division. PMID:27056843

  16. Contact-based social contagion in multiplex networks

    NASA Astrophysics Data System (ADS)

    Cozzo, Emanuele; Baños, Raquel A.; Meloni, Sandro; Moreno, Yamir

    2013-11-01

    We develop a theoretical framework for the study of epidemiclike social contagion in large scale social systems. We consider the most general setting in which different communication platforms or categories form multiplex networks. Specifically, we propose a contact-based information spreading model, and show that the critical point of the multiplex system associated with the active phase is determined by the layer whose contact probability matrix has the largest eigenvalue. The framework is applied to a number of different situations, including a real multiplex system. Finally, we also show that when the system through which information is disseminating is inherently multiplex, working with the graph that results from the aggregation of the different layers is inaccurate.

  17. Optofluidic wavelength division multiplexing for single-virus detection.

    PubMed

    Ozcelik, Damla; Parks, Joshua W; Wall, Thomas A; Stott, Matthew A; Cai, Hong; Parks, Joseph W; Hawkins, Aaron R; Schmidt, Holger

    2015-10-20

    Optical waveguides simultaneously transport light at different colors, forming the basis of fiber-optic telecommunication networks that shuttle data in dozens of spectrally separated channels. Here, we reimagine this wavelength division multiplexing (WDM) paradigm in a novel context--the differentiated detection and identification of single influenza viruses on a chip. We use a single multimode interference (MMI) waveguide to create wavelength-dependent spot patterns across the entire visible spectrum and enable multiplexed single biomolecule detection on an optofluidic chip. Each target is identified by its time-dependent fluorescence signal without the need for spectral demultiplexing upon detection. We demonstrate detection of individual fluorescently labeled virus particles of three influenza A subtypes in two implementations: labeling of each virus using three different colors and two-color combinatorial labeling. By extending combinatorial multiplexing to three or more colors, MMI-based WDM provides the multiplexing power required for differentiated clinical tests and the growing field of personalized medicine. PMID:26438840

  18. A novel multiplex isothermal amplification method for rapid detection and identification of viruses

    PubMed Central

    Nyan, Dougbeh-Chris; Swinson, Kevin L.

    2015-01-01

    A rapid multiplex isothermal amplification assay has been developed for detection and identification of multiple blood-borne viruses that infect millions of people world-wide. These infections may lead to chronic diseases or death if not diagnosed and treated in a timely manner. Sets of virus-specific oligonucleotides and oligofluorophores were designed and used in a reverse-transcription loop-mediated multiplexed isothermal amplification reaction for detection and gel electrophoretic identification of human Immunodeficiency virus (HIV), hepatitis-B virus (HBV), hepatitis-C virus (HCV), hepatitis-E virus (HEV), dengue virus (DENV), and West Nile (WNV) virus infection in blood plasma. Amplification was catalyzed with two thermostable enzymes for 30–60 minutes under isothermal condition, utilizing a simple digital heat source. Electrophoretic analysis of amplified products demonstrated simultaneous detection of 6 viruses that were distinctly identified by unique ladder-like banding patterns. Naked-eye fluorescent visualization of amplicons revealed intensely fluorescing products that indicated positive detection. The test demonstrated a 97% sensitivity and a 100% specificity, with no cross-reaction with other viruses observed. This portable detection tool may have clinical and field utility in the developing and developed world settings. This may enable rapid diagnosis and identification of viruses for targeted therapeutic intervention and prevention of disease transmission. PMID:26643761

  19. Multiplexed Molecular Assays for Rapid Rule-Out of Foot-and-Mouth Disease

    SciTech Connect

    Lenhoff, R; Naraghi-Arani, P; Thissen, J; Olivas, J; Carillo, C; Chinn, C; Rasmussen, M; Messenger, S; Suer, L; Smith, S M; Tammero, L; Vitalis, E; Slezak, T R; Hullinger, P J; Hindson, B J; Hietala, S; Crossley, B; Mcbride, M

    2007-06-26

    A nucleic acid-based multiplexed assay was developed that combines detection of foot-and-mouth disease virus (FMDV) with rule-out assays for two other foreign animal diseases and four domestic animal diseases that cause vesicular or ulcerative lesions indistinguishable from FMDV infection in cattle, sheep and swine. The FMDV 'look-alike' diagnostic assay panel contains five PCR and twelve reverse transcriptase PCR (RT-PCR) signatures for a total of seventeen simultaneous PCR amplifications for seven diseases plus incorporating four internal assay controls. It was developed and optimized to amplify both DNA and RNA viruses simultaneously in a single tube and employs Luminex{trademark} liquid array technology. Assay development including selection of appropriate controls, a comparison of signature performance in single and multiplex testing against target nucleic acids, as well of limits of detection for each of the individual signatures is presented. While this assay is a prototype and by no means a comprehensive test for FMDV 'look-alike' viruses, an assay of this type is envisioned to have benefit to a laboratory network in routine surveillance and possibly for post-outbreak proof of freedom from foot-and-mouth disease.

  20. The physiologic state of Escherichia coli O157:H7 does not affect its detection in two commercial real-time PCR-based tests.

    PubMed

    Wang, Rong; Schmidt, John W; Arthur, Terrance M; Bosilevac, Joseph M

    2013-04-01

    Multiplex real-time PCR detection of Escherichia coli O157:H7 is an efficient molecular tool with high sensitivity and specificity for meat safety assurance. The Biocontrol GDS(®) and DuPont Qualicon BAX(®)-RT rapid detection systems are two commercial tests based on real-time PCR amplification with potential applications for quantification of specific E. coli O157:H7 gene targets in enriched meat samples. However, there are arguments surrounding the use of these tests to predict pre-enrichment concentrations of E. coli O157:H7, as well as arguments pertaining to the influence of non-viable cells causing false positive results. The present study attempts to illustrate the effects of different bacterial physiologic states and the presence of non-viable cells on the ability of these systems to accurately measure contamination levels of E. coli O157:H7 in ground beef. While the PCR threshold cycle (C(T)) values of these assays showed a direct correlation with the number of bacteria present in pure cultures, this was not the case for ground beef samples spiked with various levels of injured or healthy cells. Furthermore, comparison of post-enrichment cell densities of bacteria did not correlate with injured or healthy cell numbers inoculated before enrichment process. Ground beef samples spiked with injured or healthy cells at different doses could not be distinguished by C(T) values from either assay. In addition, the contribution of nonviable cells in generating positive real-time PCR signals was investigated using both assays on pre-enriched and post-enriched beef samples, but only if inoculated at levels of 10(6) cells/sample or higher, which are levels not typically seen in ground beef. PMID:23200653

  1. Computational tradeoffs in multiplex PCR assay design for SNP genotyping

    PubMed Central

    Rachlin, John; Ding, Chunming; Cantor, Charles; Kasif, Simon

    2005-01-01

    Background Multiplex PCR is a key technology for detecting infectious microorganisms, whole-genome sequencing, forensic analysis, and for enabling flexible yet low-cost genotyping. However, the design of a multiplex PCR assays requires the consideration of multiple competing objectives and physical constraints, and extensive computational analysis must be performed in order to identify the possible formation of primer-dimers that can negatively impact product yield. Results This paper examines the computational design limits of multiplex PCR in the context of SNP genotyping and examines tradeoffs associated with several key design factors including multiplexing level (the number of primer pairs per tube), coverage (the % of SNP whose associated primers are actually assigned to one of several available tube), and tube-size uniformity. We also examine how design performance depends on the total number of available SNPs from which to choose, and primer stringency criterial. We show that finding high-multiplexing/high-coverage designs is subject to a computational phase transition, becoming dramatically more difficult when the probability of primer pair interaction exceeds a critical threshold. The precise location of this critical transition point depends on the number of available SNPs and the level of multiplexing required. We also demonstrate how coverage performance is impacted by the number of available snps, primer selection criteria, and target multiplexing levels. Conclusion The presence of a phase transition suggests limits to scaling Multiplex PCR performance for high-throughput genomics applications. Achieving broad SNP coverage rapidly transitions from being very easy to very hard as the target multiplexing level (# of primer pairs per tube) increases. The onset of a phase transition can be "delayed" by having a larger pool of SNPs, or loosening primer selection constraints so as to increase the number of candidate primer pairs per SNP, though the latter

  2. Multiplexed dispersive spectrometers using reduced background infrared detectors.

    PubMed

    Wyatt, C L; Esplin, R W

    1974-11-01

    The application of multiplex spectrometry to cryogenically cooled LWIR extrinsic photodetectors is limited by system noise. This noise limitation results in a detector NEP that is directly proportional to bandwidth. Therefore, multiplex schemes that require increased bandwidth are not productive of real advantage. However, doubly encoded systems that are based on 2n - 1 or n + N - 1 measurements have the potential to provide a real throughput gain proportional to the number of elements used on the throughput matrix. PMID:20134749

  3. Wavelength division multiplexed fiber optic absolute position encoder

    NASA Technical Reports Server (NTRS)

    Park, Eric D.; Gat, Erann

    1989-01-01

    A wavelength division multiplexing (WDM) method for fiber optic sensors is proposed which uses a broadband light source and narrow bandpass thin film optical filter coatings on cylindrical graded index lenses. In the WDM system described here, all bits are multiplexed onto a single signal return fiber by assigning each bit a unique wavelength. A multielement photodetector array is used as the encoded position information is in parallel. Preliminary prototype test results are presented.

  4. Multiplex Holography For The Display Of Three-Dimensional Information

    NASA Astrophysics Data System (ADS)

    Drinkwater, John; Hart, Stephen

    1987-10-01

    A system based on multiplex holography has been developed for the display and storage of three-dimensional information. Volume ('stack') multiplex holograms have been produced for viewing on a novel white light display device based on dispersion compensation. The image processing and holographic techniques developed to optimise these results are described. The demonstration of potential applications of the displays for data from medical imaging, analytical techniques such as electron microscopy and scientific data analysis is detailed.

  5. Simultaneous detection of five notifiable viral diseases of cattle by single-tube multiplex real-time RT-PCR.

    PubMed

    Wernike, Kerstin; Hoffmann, Bernd; Beer, Martin

    2015-06-01

    Multiplexed real-time PCR (qPCR) assays enable the detection of several target genes in a single reaction, which is applicable for simultaneous testing for the most important viral diseases in samples obtained from ruminants with unspecific clinical symptoms. Here, reverse transcription qPCR (RT-qPCR) systems for the detection of bovine viral diarrhoea virus (BVDV) and bluetongue virus (BTV) were combined with an internal control system based on the beta-actin gene. Additionally, a background screening for three further major pathogens of cloven-hoofed animals reportable to the World Organisation for Animal Health, namely foot-and-mouth disease virus, epizootic haemorrhagic disease virus, and Rift Valley fever virus, was integrated using the identical fluorophore for the respective RT-qPCR assays. Every pathogen-specific assay had an analytical sensitivity of at least 100 genome copies per reaction within the multiplex approach, and a series of reference samples and clinical specimens obtained from cattle, but also from small ruminants, were detected reliably. The qPCR systems integrated in the background screening were even not influenced by the simultaneous amplification of very high BVDV and BTV genome copy numbers. The newly developed multiplex qPCR allows the specific and sensitive detection of five of the most important diseases of ruminants and could be used in the context of monitoring programs or for differential diagnostics. PMID:25746154

  6. Demonstration of Time Domain Multiplexed Readout for Magnetically Coupled Calorimeters

    NASA Technical Reports Server (NTRS)

    Porst, J.-P.; Adams, J. S.; Balvin, M.; Bandler, S.; Beyer, J.; Busch, S. E.; Drung, D.; Seidel, G. M.; Smith, S. J.; Stevenson, T. R.

    2012-01-01

    Magnetically coupled calorimeters (MCC) have extremely high potential for x-ray applications due to the inherent high energy resolution capability and being non-dissipative. Although very high energy-resolution has been demonstrated, until now there has been no demonstration of multiplexed read-out. We report on the first realization of a time domain multiplexed (TDM) read-out. While this has many similarities with TDM of transition-edge-sensors (TES), for MGGs the energy resolution is limited by the SQUID read-out noise and requires the well established scheme to be altered in order to minimize degradation due to noise aliasing effects. In cur approach, each pixel is read out by a single first stage SQUID (SQ1) that is operated in open loop. The outputs of the SQ1 s are low-pass filtered with an array of low cross-talk inductors, then fed into a single-stage SQUID TD multiplexer. The multiplexer is addressed from room temperature and read out through a single amplifier channel. We present results achieved with a new detector platform. Noise performance is presented and compared to expectations. We have demonstrated multiplexed X-ray spectroscopy at 5.9keV with delta_FWHM=10eV. In an optimized setup, we show it is possible to multiplex 32 detectors without significantly degrading the Intrinsic detector resolution.

  7. Multiplex Detection and SNP Genotyping in a Single Fluorescence Channel

    PubMed Central

    Fu, Guoliang; Miles, Andrea; Alphey, Luke

    2012-01-01

    Probe-based PCR is widely used for SNP (single nucleotide polymorphism) genotyping and pathogen nucleic acid detection due to its simplicity, sensitivity and cost-effectiveness. However, the multiplex capability of hydrolysis probe-based PCR is normally limited to one target (pathogen or allele) per fluorescence channel. Current fluorescence PCR machines typically have 4–6 channels. We present a strategy permitting the multiplex detection of multiple targets in a single detection channel. The technique is named Multiplex Probe Amplification (MPA). Polymorphisms of the CYP2C9 gene (cytochrome P450, family 2, subfamily C, polypeptide 9, CYP2C9*2) and human papillomavirus sequences HPV16, 18, 31, 52 and 59 were chosen as model targets for testing MPA. The allele status of the CYP2C9*2 determined by MPA was entirely concordant with the reference TaqMan® SNP Genotyping Assays. The four HPV strain sequences could be independently detected in a single fluorescence detection channel. The results validate the multiplex capacity, the simplicity and accuracy of MPA for SNP genotyping and multiplex detection using different probes labeled with the same fluorophore. The technique offers a new way to multiplex in a single detection channel of a closed-tube PCR. PMID:22272339

  8. Multiplex detection and SNP genotyping in a single fluorescence channel.

    PubMed

    Fu, Guoliang; Miles, Andrea; Alphey, Luke

    2012-01-01

    Probe-based PCR is widely used for SNP (single nucleotide polymorphism) genotyping and pathogen nucleic acid detection due to its simplicity, sensitivity and cost-effectiveness. However, the multiplex capability of hydrolysis probe-based PCR is normally limited to one target (pathogen or allele) per fluorescence channel. Current fluorescence PCR machines typically have 4-6 channels. We present a strategy permitting the multiplex detection of multiple targets in a single detection channel. The technique is named Multiplex Probe Amplification (MPA). Polymorphisms of the CYP2C9 gene (cytochrome P450, family 2, subfamily C, polypeptide 9, CYP2C9*2) and human papillomavirus sequences HPV16, 18, 31, 52 and 59 were chosen as model targets for testing MPA. The allele status of the CYP2C9*2 determined by MPA was entirely concordant with the reference TaqMan® SNP Genotyping Assays. The four HPV strain sequences could be independently detected in a single fluorescence detection channel. The results validate the multiplex capacity, the simplicity and accuracy of MPA for SNP genotyping and multiplex detection using different probes labeled with the same fluorophore. The technique offers a new way to multiplex in a single detection channel of a closed-tube PCR. PMID:22272339

  9. Multiplexed neurochemical signaling by neurons of the ventral tegmental area.

    PubMed

    Barker, David J; Root, David H; Zhang, Shiliang; Morales, Marisela

    2016-04-01

    The ventral tegmental area (VTA) is an evolutionarily conserved structure that has roles in reward-seeking, safety-seeking, learning, motivation, and neuropsychiatric disorders such as addiction and depression. The involvement of the VTA in these various behaviors and disorders is paralleled by its diverse signaling mechanisms. Here we review recent advances in our understanding of neuronal diversity in the VTA with a focus on cell phenotypes that participate in 'multiplexed' neurotransmission involving distinct signaling mechanisms. First, we describe the cellular diversity within the VTA, including neurons capable of transmitting dopamine, glutamate or GABA as well as neurons capable of multiplexing combinations of these neurotransmitters. Next, we describe the complex synaptic architecture used by VTA neurons in order to accommodate the transmission of multiple transmitters. We specifically cover recent findings showing that VTA multiplexed neurotransmission may be mediated by either the segregation of dopamine and glutamate into distinct microdomains within a single axon or by the integration of glutamate and GABA into a single axon terminal. In addition, we discuss our current understanding of the functional role that these multiplexed signaling pathways have in the lateral habenula and the nucleus accumbens. Finally, we consider the putative roles of VTA multiplexed neurotransmission in synaptic plasticity and discuss how changes in VTA multiplexed neurons may relate to various psychopathologies including drug addiction and depression. PMID:26763116

  10. Growing multiplex networks with arbitrary number of layers

    NASA Astrophysics Data System (ADS)

    Momeni, Naghmeh; Fotouhi, Babak

    2015-12-01

    This paper focuses on the problem of growing multiplex networks. Currently, the results on the joint degree distribution of growing multiplex networks present in the literature pertain to the case of two layers and are confined to the special case of homogeneous growth and are limited to the state state (that is, the limit of infinite size). In the present paper, we first obtain closed-form solutions for the joint degree distribution of heterogeneously growing multiplex networks with arbitrary number of layers in the steady state. Heterogeneous growth means that each incoming node establishes different numbers of links in different layers. We consider both uniform and preferential growth. We then extend the analysis of the uniform growth mechanism to arbitrary times. We obtain a closed-form solution for the time-dependent joint degree distribution of a growing multiplex network with arbitrary initial conditions. Throughout, theoretical findings are corroborated with Monte Carlo simulations. The results shed light on the effects of the initial network on the transient dynamics of growing multiplex networks and takes a step towards characterizing the temporal variations of the connectivity of growing multiplex networks, as well as predicting their future structural properties.

  11. Shift-multiplexing complex spectral-domain optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Huang, Haochong; Jiang, Zhuqing; Wang, Dayong; Cai, Wenyuan; Man, Tianlong; Wang, Zhe; Panezai, Spozmai

    2014-01-01

    We propose and experimentally demonstrate a shift-multiplexing complex spectral-domain optical coherence tomography (shift-multiplexing CSD-OCT) method, in which the maximum detection depth of SD-OCT can be greatly extended by incorporating the shift-multiplexing of detection positions with CSD-OCT. The tomographic imaging with twofold or threefold microscopic slides as the target sample is performed. The experimental results show that the tomographic imaging with more uniform brightness and clarity for the different depth regions in a thick sample can be achieved by the shift-multiplexing CSD-OCT system. In particular, even while the sample's depth is beyond the maximum imaging depth of CSD-OCT system, the tomographic imaging of this sample can still be realized by using the shift-multiplexing CSD-OCT method without the need for any replacement of the equipment, such as high spectral capacity grating or high resolution of CCD. The shift-multiplexing CSD-OCT system can perform the imaging with the optimization and less reduction of sensitivity for the deeper detection position in the sample.

  12. Performance Analysis of Wavelength Multiplexed Sac Ocdma Codes in Beat Noise Mitigation in Sac Ocdma Systems

    NASA Astrophysics Data System (ADS)

    Alhassan, A. M.; Badruddin, N.; Saad, N. M.; Aljunid, S. A.

    2013-07-01

    In this paper we investigate the use of wavelength multiplexed spectral amplitude coding (WM SAC) codes in beat noise mitigation in coherent source SAC OCDMA systems. A WM SAC code is a low weight SAC code, where the whole code structure is repeated diagonally (once or more) in the wavelength domain to achieve the same cardinality as a higher weight SAC code. Results show that for highly populated networks, the WM SAC codes provide better performance than SAC codes. However, for small number of active users the situation is reversed. Apart from their promising improvement in performance, these codes are more flexible and impose less complexity on the system design than their SAC counterparts.

  13. High Throughput Flow Cytometry Bead-based Multiplex Assay for Identification of Rho GTPase Inhibitors

    PubMed Central

    Surviladze, Zurab; Young, Susan M; Sklar, Larry A

    2015-01-01

    Summary Rho family GTPases and their effector proteins regulate a wide range of cell signaling pathways. In normal physiological conditions their activity is tightly controlled and it is not surprising that their aberrant activation contributes to tumorigenesis or other diseases. For this reason, the identification of small, cell permeable molecules capable of inhibition of Rho GTPases can be extraordinarily useful, particularly if they are specific and act reversibly. Herein we describe a flow cytometric assay, which allows us to measure the activity of six small GTPases simultaneously. GST-tagged small GTPases are bound to six glutathione bead sets each set having a different intensity of red fluorescence at a fixed wavelength. The coated bead sets were washed, combined, and dispensed into 384-well plates with test compounds, and fluorescent-GTP binding was used as the read-out. This multiplex bead-based assay was successfully used for to identify both general and selective inhibitors of Rho family GTPases. PMID:22144280

  14. Reverse Transfer in Australia

    ERIC Educational Resources Information Center

    Moodie, Gavin

    2004-01-01

    This article considers national Australian data on reverse transfer--the transfer of students from bachelor programs or higher to sub baccalaureate programs, institutions and sectors. It finds that previous studies have overstated the prevalence and perhaps also the significance of reverse transfer. The data are not good, but the best conclusion…

  15. Quantum reverse hypercontractivity

    SciTech Connect

    Cubitt, Toby; Kastoryano, Michael; Montanaro, Ashley; Temme, Kristan

    2015-10-15

    We develop reverse versions of hypercontractive inequalities for quantum channels. By generalizing classical techniques, we prove a reverse hypercontractive inequality for tensor products of qubit depolarizing channels. We apply this to obtain a rapid mixing result for depolarizing noise applied to large subspaces and to prove bounds on a quantum generalization of non-interactive correlation distillation.

  16. Justice and Reverse Discrimination.

    ERIC Educational Resources Information Center

    Goldman, Alan H.

    Defining reverse discrimination as hiring or admissions decisions based on normally irrelevant criteria, this book develops principles of rights, compensation, and equal opportunity applicable to the reverse discrimination issue. The introduction defines the issue and discusses deductive and inductive methodology as applied to reverse…

  17. Reverse Discrimination: Recent Cases.

    ERIC Educational Resources Information Center

    Steinhilber, August W.

    This paper discusses reverse discrimination cases with particular emphasis on Bakke v. Regents of University of California and those cases which preceded it. A brief history is given of court cases used by opponents and proponents in the discussion of reverse discrimination. Legal theory and a discussion of court cases that preceded Bakke follow.…

  18. Ultrasonic Time Reversal Mirrors

    NASA Astrophysics Data System (ADS)

    Fink, Mathias; Montaldo, Gabriel; Tanter, Mickael

    2004-11-01

    For more than ten years, time reversal techniques have been developed in many different fields of applications including detection of defects in solids, underwater acoustics, room acoustics and also ultrasound medical imaging and therapy. The essential property that makes time reversed acoustics possible is that the underlying physical process of wave propagation would be unchanged if time were reversed. In a non dissipative medium, the equations governing the waves guarantee that for every burst of sound that diverges from a source there exists in theory a set of waves that would precisely retrace the path of the sound back to the source. If the source is pointlike, this allows focusing back on the source whatever the medium complexity. For this reason, time reversal represents a very powerful adaptive focusing technique for complex media. The generation of this reconverging wave can be achieved by using Time Reversal Mirrors (TRM). It is made of arrays of ultrasonic reversible piezoelectric transducers that can record the wavefield coming from the sources and send back its time-reversed version in the medium. It relies on the use of fully programmable multi-channel electronics. In this paper we present some applications of iterative time reversal mirrors to target detection in medical applications.

  19. Reversible Shape Memory

    NASA Astrophysics Data System (ADS)

    Zhou, Jing; Li, Qiaoxi; Turner, Sara; Brosnan, Sarah; Tippets, Cary; Carrillo, Jan-Michael; Nykypnachuk, Dmytro; Gang, Oleg; Dobrynin, Andrey; Lopez, Rene; Ashby, Valerie; Sheiko, Sergei

    2014-03-01

    Reversible shape memory has been achieved on various shapes, e.g. hairpin, origami, coil, robotic gripper and flow rate control device, allowing for multiple switching between encoded shapes without applying any external force. Also, the reversible photonic structure molded in dielectric elastomers has been designed. Maximum reversibility can be achieved by tuning the crosslinking density and the degree of crystallinity of semi-crystalline elastomers. Different crystallization protocols including isothermal and cooling crystallization have been applied to develop a universal picture integrating different shape memory (SM) behaviors: conventional one-way SM, two-way reversible SM, and one-way reversible SM. Acknowledge financial support from the NSF DMR-1122483, DMR- 1004576, and DMR-1206957.

  20. Silicon nanophotonic integrated devices enabling multiplexed on-chip optical interconnects

    NASA Astrophysics Data System (ADS)

    Dai, Daoxin; Wang, Jian; Chen, Sitao

    2015-05-01

    Advanced multiplexing technologies including wavelength-division-multiplexing (WDM), polarization-division multiplexing (PDM), and mode-division multiplexing (MDM) have been utilized as a cost-effective solution to enhance the capacity of an optical-interconnect link. The on-chip (de)multiplexers, including WDM filters, PDM devices, and MDM devices, are the most important key components in a multi-channel multiplexed optical interconnect system. Hybrid (de)multiplexer to enable various multiplexing technologies simultaneously are becoming more and more important to achieve many channels. In this paper we give a review for our recent work on silicon photonic integrated devices for realizing multi-channel multiplexed on-chip optical interconnects.

  1. Contextual factors multiplex to control multisensory processes.

    PubMed

    Sarmiento, Beatriz R; Matusz, Pawel J; Sanabria, Daniel; Murray, Micah M

    2016-01-01

    This study analyzed high-density event-related potentials (ERPs) within an electrical neuroimaging framework to provide insights regarding the interaction between multisensory processes and stimulus probabilities. Specifically, we identified the spatiotemporal brain mechanisms by which the proportion of temporally congruent and task-irrelevant auditory information influences stimulus processing during a visual duration discrimination task. The spatial position (top/bottom) of the visual stimulus was indicative of how frequently the visual and auditory stimuli would be congruent in their duration (i.e., context of congruence). Stronger influences of irrelevant sound were observed when contexts associated with a high proportion of auditory-visual congruence repeated and also when contexts associated with a low proportion of congruence switched. Context of congruence and context transition resulted in weaker brain responses at 228 to 257 ms poststimulus to conditions giving rise to larger behavioral cross-modal interactions. Importantly, a control oddball task revealed that both congruent and incongruent audiovisual stimuli triggered equivalent non-linear multisensory interactions when congruence was not a relevant dimension. Collectively, these results are well explained by statistical learning, which links a particular context (here: a spatial location) with a certain level of top-down attentional control that further modulates cross-modal interactions based on whether a particular context repeated or changed. The current findings shed new light on the importance of context-based control over multisensory processing, whose influences multiplex across finer and broader time scales. PMID:26466522

  2. (Multiplex mapping of human cDNAs)

    SciTech Connect

    Nierman, W.C.

    1991-01-01

    J. Craig Venter, National Institute of Neurological Disorders and Stroke, has begun to identify genes expressed in the human brain by partially sequences cDNA clones. We are collaborating with the Venter group and using their sequence data to develop methods for rapid localization of newly identified cDNAs to human chromosomes. We are applying the ABI automated DNA sequencer to the analysis of fluorescently-tagged PCR products for assigning sequences to individual human chromosomes. The steps in our mapping protocol are (1) to design PCR primers from the Venter laboratory-generated sequence data, (2) to test the primers for specific amplification from human genomic DNA, (3) to use the primers for PCR amplification from a somatic cell hybrid cell mapping panel, (4) to determine the presence or absence of the specific amplification products from each cell line DNA by electrophoretic analysis using the ABI sequencer, and (5) to analyze the pattern of amplification results from the hybrid panel to identify the chromosomal origin of the cDNA sequence. We have demonstrated the principle by mapping 12 sequences or Expressed Sequence Tags'' (ESTs), providing primer sequence data for subsequent subchromosomal localizations. We will now concentrate on developing methodology to allow multiplexing the amplification reactions and analysis of the reaction products, to achieve a high throughput with a minimum allocation of resources. This project will generate a data set from which to evaluate strategies to identify functional primer sequences from cDNA sequence data.

  3. Multiplexed microfluidic quantification of proteins in serum

    NASA Astrophysics Data System (ADS)

    Rajan, Nitin; Rajauria, Sukumar; Cleland, Andrew

    2015-03-01

    Rapid and low cost immunoassays targeting proteins in blood or other bodily fluids are highly sought after for point-of-care devices and early screening of patients. Immunoturbidimetric assays utilize latex particles functionalized with antibodies, with particle aggregation in the presence of the analyte detected by a change in absorbance. Using a high throughput micro-fluidic particle analyzer based solely on electrical signals (resistive pulse sensing), we are able to accurately quantify the degree of aggregation by analyzing the changes in the particle size distribution. Thus we study the aggregation of streptavidin (SAv) coated beads in the presence of biotinylated bovine serum albumin as a proof-of-principle assay and extract the binding capacity of the SAv beads from the dose-response curve. We also use our aggregation measurement platform to characterize a commercial C-reactive protein (CRP) immunoturbidimetric assay (hsCRP, Diazyme Inc.). We obtain a linear calibration curve as well as a better limit of detection of CRP than that obtained by absorbance measurements. By using different bead sizes functionalized with different antibodies, multiplexed analyte detection is also possible. We demonstrate this by combining the commercial anti-CRP functionalized beads (0.4 microns) with biotin coated beads (1.0 microns), and carry out the simultaneous detection of SAv and CRP in a single sample.

  4. Space-division multiplexing optical coherence tomography

    PubMed Central

    Zhou, Chao; Alex, Aneesh; Rasakanthan, Janarthanan; Ma, Yutao

    2013-01-01

    High speed, high resolution and high sensitivity are desirable for optical coherence tomography (OCT). Here, we demonstrate a space-division multiplexing (SDM) technology that translates long coherence length of a commercially available wavelength tunable laser into high OCT imaging speed. We achieved an effective 800,000 A-scans/s imaging speed using a 100,000 Hz tunable vertical cavity surface-emitting laser (VCSEL). A sensitivity of 94.6 dB and a roll-off of < 2 dB over ~30 mm imaging depth were measured from a single channel in the prototype SDM-OCT system. An axial resolution of ~11 μm in air (or ~8.3 μm in tissue) was achieved throughout the entire depth range. An in vivo, 3D SDM-OCT volume of an entire Drosophila larva consisting of 400 x 605 A-scans was acquired in 0.37 seconds. Synchronized cross-sectional OCT imaging of three different segments of a beating Drosophila larva heart is demonstrated. The SDM technology provides a new orthogonal dimension for further speed improvement for OCT with favorable cost scaling. SDM-OCT also preserves image resolution and allows synchronized cross-sectional and three-dimensional (3D) imaging of biological samples, enabling new biomedical applications. PMID:23938839

  5. Multiplexed Holographic Data Storage in Bacteriorhodopsin

    NASA Technical Reports Server (NTRS)

    Mehrl, David J.; Krile, Thomas F.

    1999-01-01

    Biochrome photosensitive films in particular Bacteriorhodopsin exhibit features which make these materials an attractive recording medium for optical data storage and processing. Bacteriorhodopsin films find numerous applications in a wide range of optical data processing applications; however the short-term memory characteristics of BR limits their applications for holographic data storage. The life-time of the BR can be extended using cryogenic temperatures [1], although this method makes the system overly complicated and unstable. Longer life-times can be provided in one modification of BR - the "blue" membrane BR [2], however currently available films are characterized by both low diffraction efficiency and difficulties in providing photoreversible recording. In addition, as a dynamic recording material, the BR requires different wavelengths for recording and reconstructing of optical data in order to prevent the information erasure during its readout. This fact also put constraints on a BR-based Optical Memory, due to information loss in holographic memory systems employing the two-lambda technique for reading-writing thick multiplexed holograms.

  6. (Multiplex mapping of human cDNAs)

    SciTech Connect

    Nierman, W.C.

    1992-01-01

    We have tested and implemented several protocols to increase productivity for mapping expressed sequence tags EST sequences to human chromosomes. These protocols include adopting PRIMER which permits utilization of batch files, as the standard software for PCR primer design; adding a human 21-only cell line to the NIGMS panel No. 1 to improve discrimination in discordancy analyses involving chromosome 21, adding a monochromosomal hybrid panel to facilitate chromosome assignment of sequences that are amplified from more than 1 chromosome; combining the products of multiple PCR reactions for electrophoretic analysis (pseudoplexing); routinely multiplexing PCR reactions; and automating data entry and analysis as much as possible. We have applied these protocols to assign an overall total of 132 human brain CDNA sequences to individual human chromosomes. PCR primers were designed from ESTS and tested for specific amplification from human genomic DNA. DNA was then amplified using DNA from somatic cell hybrid mapping panels as templates. The amplification products were identified using an automated fluorescence detection system. Chromosomal assignments were made by discordancy analysis. The localized cDNAs include 2 for known human genes, 2 that map to 2 different human chromosomes, and 25 for cDNAs matching existing database records.

  7. Multiplexed coding in the human basal ganglia

    NASA Astrophysics Data System (ADS)

    Andres, D. S.; Cerquetti, D.; Merello, M.

    2016-04-01

    A classic controversy in neuroscience is whether information carried by spike trains is encoded by a time averaged measure (e.g. a rate code), or by complex time patterns (i.e. a time code). Here we apply a tool to quantitatively analyze the neural code. We make use of an algorithm based on the calculation of the temporal structure function, which permits to distinguish what scales of a signal are dominated by a complex temporal organization or a randomly generated process. In terms of the neural code, this kind of analysis makes it possible to detect temporal scales at which a time patterns coding scheme or alternatively a rate code are present. Additionally, finding the temporal scale at which the correlation between interspike intervals fades, the length of the basic information unit of the code can be established, and hence the word length of the code can be found. We apply this algorithm to neuronal recordings obtained from the Globus Pallidus pars interna from a human patient with Parkinson’s disease, and show that a time pattern coding and a rate coding scheme co-exist at different temporal scales, offering a new example of multiplexed neuronal coding.

  8. Nanoscale Test Strips for Multiplexed Blood Analysis

    NASA Technical Reports Server (NTRS)

    Chan, Eugene

    2015-01-01

    A critical component of the DNA Medicine Institute's Reusable Handheld Electrolyte and Lab Technology for Humans (rHEALTH) sensor are nanoscale test strips, or nanostrips, that enable multiplexed blood analysis. Nanostrips are conceptually similar to the standard urinalysis test strip, but the strips are shrunk down a billionfold to the microscale. Each nanostrip can have several sensor pads that fluoresce in response to different targets in a sample. The strips carry identification tags that permit differentiation of a specific panel from hundreds of other nanostrip panels during a single measurement session. In Phase I of the project, the company fabricated, tested, and demonstrated functional parathyroid hormone and vitamin D nanostrips for bone metabolism, and thrombin aptamer and immunoglobulin G antibody nanostrips. In Phase II, numerous nanostrips were developed to address key space flight-based medical needs: assessment of bone metabolism, immune response, cardiac status, liver metabolism, and lipid profiles. This unique approach holds genuine promise for space-based portable biodiagnostics and for point-of-care (POC) health monitoring and diagnostics here on Earth.

  9. Multiplexed coding by cerebellar Purkinje neurons

    PubMed Central

    Hong, Sungho; Negrello, Mario; Junker, Marc; Smilgin, Aleksandra; Thier, Peter; De Schutter, Erik

    2016-01-01

    Purkinje cells (PC), the sole output neurons of the cerebellar cortex, encode sensorimotor information, but how they do it remains a matter of debate. Here we show that PCs use a multiplexed spike code. Synchrony/spike time and firing rate encode different information in behaving monkeys during saccadic eye motion tasks. Using the local field potential (LFP) as a probe of local network activity, we found that infrequent pause spikes, which initiated or terminated intermittent pauses in simple spike trains, provide a temporally reliable signal for eye motion onset, with strong phase-coupling to the β/γ band LFP. Concurrently, regularly firing, non-pause spikes were weakly correlated with the LFP, but were crucial to linear encoding of eye movement kinematics by firing rate. Therefore, PC spike trains can simultaneously convey information necessary to achieve precision in both timing and continuous control of motion. DOI: http://dx.doi.org/10.7554/eLife.13810.001 PMID:27458803

  10. Multiplex lateral flow immunoassay for mycotoxin determination.

    PubMed

    Song, Suquan; Liu, Na; Zhao, Zhiyong; Njumbe Ediage, Emmanuel; Wu, Songling; Sun, Changpo; De Saeger, Sarah; Wu, Aibo

    2014-05-20

    A new lateral flow immunoassay (LFA) is proposed for qualitative and/or semiquantitative determination of aflatoxin B1 (AFB1), zearalenone (ZEA), deoxynivalenol (DON), and their analogues (AFs, ZEAs, DONs) in cereal samples. Each of the mycotoxin specific antibody was class specific and there was no cross reactivity to other groups of compounds. The visual limits of detection (vLOD) of the strip were 0.03, 1.6, and 10 μg/kg for AFB1, ZEA and DON, respectively. The calculated limits of detection (cLOD) were 0.05, 1, and 3 μg/kg, respectively. Meanwhile the cutoff values were achieved at 1, 50, and 60 μg/kg for AFB1, ZEA and DON, respectively. Recoveries ranged from 80% to 122% and RSD from 5% to 20%. Both the vLOD and cLOD for the three mycotoxins were lower than the EU maximum levels. Analysis of naturally contaminated maize samples resulted in a good agreement between the multiplex LFA and LC-MS/MS (100% for DONs and AFs, and 81% for ZEAs). Careful analysis of the results further explained the general overestimation of LFA compared to chromatographic methods for quantification of mycotoxins. PMID:24745689

  11. Comparison study of time reversal OFDM acoustic communication with vector and scalar sensors

    NASA Astrophysics Data System (ADS)

    Wang, Zhongkang; Zhang, Hongtao; Xie, Zhe

    2012-11-01

    To compare the performance of time reversal orthogonal frequency division multiplexing (OFDM) acoustic communication on vector and scalar sensors, the vector and scalar acoustic fields were modeled. Time reversal OFDM acoustic communication was then simulated for each sensor type. These results are compared with data from the CAPEx'09 experiment. The abilityof particle velocity channels to achieve reliable acoustic communication, as predicted by the model, is confirmed with the experiment data. Experimental results show that vector receivers can reduce the required array size, in comparisonto hydrophone arrays, whileproviding comparable communication performance.

  12. Sexing a sex-role-reversed species based on plumage: potential challenges in the red phalarope

    PubMed Central

    Ditlecadet, Delphine; Martin, Luc J.; Lanctot, Richard B.; Lecomte, Nicolas

    2016-01-01

    Sex-role reversal, in which males care for offspring, can occur when mate competition is stronger between females than males. Secondary sex traits and mate attracting displays in sex-role-reversed species are usually more pronounced in females than in males. The red phalarope (Phalaropus fulicarius) is a textbook example of a sex-role-reversed species. It is generally agreed that males are responsible for all incubation and parental care duties, whereas females typically desert males after having completed a clutch and may pair with new males to lay additional clutches. The breeding plumage of female red phalaropes is usually more brightly colored than male plumage, a reversed sexual dichromatism usually associated with sex-role reversal. Here, we confirm with PCR-based sexing that male red phalaropes can exhibit both the red body plumage typical of a female and the incubation behavior typical of a male. Our result, combined with previous observations of brightly colored red phalaropes incubating nests at the same arctic location (Igloolik Island, Nunavut, Canada), suggests that plumage dichromatism alone may not be sufficient to distinguish males from females in this breeding population of red phalaropes. This stresses the need for more systematic genetic sexing combined with standardized description of intersexual differences in red phalarope plumages. Determining whether such female-like plumage on males is a result of phenotypic plasticity or genetic variation could contribute to further understanding sex-role reversal strategies in the short Arctic summer. PMID:27168992

  13. Sexing a sex-role-reversed species based on plumage: potential challenges in the red phalarope.

    PubMed

    Giroux, Marie-Andrée; Ditlecadet, Delphine; Martin, Luc J; Lanctot, Richard B; Lecomte, Nicolas

    2016-01-01

    Sex-role reversal, in which males care for offspring, can occur when mate competition is stronger between females than males. Secondary sex traits and mate attracting displays in sex-role-reversed species are usually more pronounced in females than in males. The red phalarope (Phalaropus fulicarius) is a textbook example of a sex-role-reversed species. It is generally agreed that males are responsible for all incubation and parental care duties, whereas females typically desert males after having completed a clutch and may pair with new males to lay additional clutches. The breeding plumage of female red phalaropes is usually more brightly colored than male plumage, a reversed sexual dichromatism usually associated with sex-role reversal. Here, we confirm with PCR-based sexing that male red phalaropes can exhibit both the red body plumage typical of a female and the incubation behavior typical of a male. Our result, combined with previous observations of brightly colored red phalaropes incubating nests at the same arctic location (Igloolik Island, Nunavut, Canada), suggests that plumage dichromatism alone may not be sufficient to distinguish males from females in this breeding population of red phalaropes. This stresses the need for more systematic genetic sexing combined with standardized description of intersexual differences in red phalarope plumages. Determining whether such female-like plumage on males is a result of phenotypic plasticity or genetic variation could contribute to further understanding sex-role reversal strategies in the short Arctic summer. PMID:27168992

  14. Bayesian estimation of the diagnostic accuracy of a multiplex real-time PCR assay and bacteriological culture for 4 common bovine intramammary pathogens.

    PubMed

    Paradis, M-È; Haine, D; Gillespie, B; Oliver, S P; Messier, S; Comeau, J; Scholl, D T

    2012-11-01

    -PCR did not concur with any apparent differences between Se estimates of the 2 tests. Further work should be done to extend these results to other PCR-based tests for detecting bacterial species in milk samples, for which presented results could be used as prior parameter distributions. Limits of sample handling and storage and the potential existence of substances in clinical case samples that may interfere with PCR reactions also are worth further investigation. PMID:22981579

  15. Reversible shape memory

    NASA Astrophysics Data System (ADS)

    Sheiko, Sergei; Zhou, Jing; White, Sarah; Ashby, Valerie

    2012-02-01

    An ``Achilles' heel'' of shape memory materials is that shape transformations triggered by an external stimulus are usually irreversible. Here we present a new concept of reversible transitions between two well-defined shapes by controlling hierarchic crystallization of a dual-network elastomer. The reversibility was demonstrated for different types of shape transformations including rod bending, winding of a helical coil, and widening an aperture. The distinct feature of the reversible shape alterations is that both counter-shapes are infinitely stable at a temperature of exploitation. Shape reversibility is highly desirable property in many practical applications such as non-surgical removal of a previously inserted catheter and handfree wrapping up of an earlier unraveled solar sail on a space shuttle.

  16. Tubal ligation reversal

    MedlinePlus

    ... Fernandez H, Gervaise A. Tubal anastomosis after tubal sterilization: a review. Arch Gynecol Obstet . 2011 May;283( ... Berger GS, Zerden ML. Pregnancy success after hysteroscopic sterilization reversal. Obstet Gynecol . 2014 Dec;124(6):1183- ...

  17. Reverse genetics in rice using Tos17.

    PubMed

    Mieulet, Delphine; Diévart, Anne; Droc, Gaëtan; Lanau, Nadège; Guiderdoni, Emmanuel

    2013-01-01

    Transposon of Oryza sativa 17 (Tos17), a Ty1-Copia Class I retroelement, is one of the few active retroelements identified in rice, the main cereal crop of human consumption and the model genome for cereals. Tos17 exists in two copies in the standard Nipponbare japonica genome (n = 12 and 379 Mb). Tos17 copies are inactive in the plant grown under normal conditions. However, the copy located on chromosome 7 can be activated upon tissue culture. Plants regenerated from 3- and 5-month-old tissue cultures harbor, respectively, an average of 3.5 and 8 newly transposed copies that are stably inserted at new positions in the genome. Due to its favorable features, Tos17 has been extensively used for insertion mutagenesis of the model genome and 31,403 sequence indexed inserts harbored by regenerants/T-DNA plants are available in the databases. The corresponding seed stocks can be ordered from the laboratories which generated them. Both forward genetics and reverse genetics approaches using these lines have allowed the deciphering of gene function in rice. We report here two protocols for ascertaining the presence of a Tos17 insertion in a gene of interest among R2/T2 seeds received from Tos17 mutant stock centers: The first protocol is PCR-based and allows the identification of azygous, heterozygous and homozygous plants among progenies segregating the insertion. The second protocol is based on DNA blot analysis and can be used to identify homozygous plants carrying the Tos17 copy responsible for gene disruption while cleaning the mutant background from other unwitting mutagen inserts. PMID:23918431

  18. Multiplexing Technology for Acoustic Emission Monitoring of Aerospace Vehicles

    NASA Technical Reports Server (NTRS)

    Prosser, William; Percy, Daniel

    2003-01-01

    The initiation and propagation of damage mechanisms such as cracks and delaminations generate acoustic waves, which propagate through a structure. These waves can be detected and analyzed to provide the location and severity of damage as part of a structural health monitoring (SHM) system. This methodology of damage detection is commonly known as acoustic emission (AE) monitoring, and is widely used on a variety of applications on civil structures. AE has been widely considered for SHM of aerospace vehicles. Numerous successful ground and flight test demonstrations have been performed, which show the viability of the technology for damage monitoring in aerospace structures. However, one significant current limitation for application of AE techniques on aerospace vehicles is the large size, mass, and power requirements for the necessary monitoring instrumentation. To address this issue, a prototype multiplexing approach has been developed and demonstrated in this study, which reduces the amount of AE monitoring instrumentation required. Typical time division multiplexing techniques that are commonly used to monitor strain, pressure and temperature sensors are not applicable to AE monitoring because of the asynchronous and widely varying rates of AE signal occurrence. Thus, an event based multiplexing technique was developed. In the initial prototype circuit, inputs from eight sensors in a linear array were multiplexed into two data acquisition channels. The multiplexer rapidly switches, in less than one microsecond, allowing the signals from two sensors to be acquired by a digitizer. The two acquired signals are from the sensors on either side of the trigger sensor. This enables the capture of the first arrival of the waves, which cannot be accomplished with the signal from the trigger sensor. The propagation delay to the slightly more distant neighboring sensors makes this possible. The arrival time from this first arrival provides a more accurate source location

  19. Giant rodlike reversed micelles

    SciTech Connect

    Yu, Z.J.; Neuman, R.D. )

    1994-05-04

    Herein we report that sodium bis(2-ethylhexyl)phosphate, which is similar in structure to the classical surfactant sodium bis(2-ethylhexyl)sulfosuccinate (AOT), forms very large rodlike reversed micelles and that their size can be even much larger if water is removed from the apolar solution. We further suggest that long-range electrostatic interactions are the primary driving force for the formation of giant reversed micelles. 19 refs., 3 figs.

  20. SBE primer : multiplexing minisequencing-based genotyping

    SciTech Connect

    Kaderali, L.; Deshpande, A.; Uribe-Romeo, F. J.; Schliep, A.; Torney, D. C.

    2002-01-01

    Single-nucleotide polymorphism (SNP) analysis is a powerful tool for mapping and diagnosing disease-related alleles. Most of the known genetic diseases are caused by point mutations, and a growing number of SNPs will be routinely analyzed to diagnose genetic disorders. Mutation analysis by polymerase mediated single-base primer extension (minisequencing) can be massively parallelized using for example DNA microchips or flow cytometry with microspheres as solid support. By adding a unique oligonucleotide tag to the 5-inch end of the minisequencing primer and attaching the complementary anti-tag to the array or bead surface, the assay can be 'demultiplexed'. However, such high-throughput scoring of SNPs requires a high level of primer multiplexing in order to analyze multiple loci in one assay, thus enabling inexpensive and fast polymorphism scoring. Primers can be chosen from either the plus or the minus strand, and primers used in the same experiment must not bind to one another. To genotype a given number of polymorphic sites, the question is which primer to use for each SNP, and which primers to group into the same experiment. Furthermore, a crosshybridization-free tag/anti-tag code is required in order to sort the extended primers to the corresponding microspheres or chip spots. These problems pose challenging algorithmic questions. We present a computer program lo automate the design process for the assay. Oligonucleotide primers for the reaction are automatically selected by the software, a unique DNA tag/anti-tag system is generated, and the pairing of primers and DNA-Tags is automatically done in a way to avoid any crossreactivity. We report first results on a 45-plex genotyping assay, indicating that minisequencing can be adapted to be a powerful tool for high-throughput, massively parallel genotyping.