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Sample records for acid lna taqman

  1. Evaluation of LNA, MGB and non-modified DNA probes to improve the detection limit of TaqMan real-time PCR assay for Pantoea stewartii subsp. stewartii

    USDA-ARS?s Scientific Manuscript database

    The goal of this study was to compare the sensitivity and amplification efficiency of the TaqMan assay using locked nucleic acid (LNA), minor groove binder (MGB) ligands and non-modified DNA probes. In monoplex or single target TaqMan assays for P. stewartii subsp. stewartii, LNA and MGB probes impr...

  2. Detection and quantification of genetically modified organisms using very short, locked nucleic acid TaqMan probes.

    PubMed

    Salvi, Sergio; D'Orso, Fabio; Morelli, Giorgio

    2008-06-25

    Many countries have introduced mandatory labeling requirements on foods derived from genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (PCR) based upon the TaqMan probe chemistry has become the method mostly used to support these regulations; moreover, event-specific PCR is the preferred method in GMO detection because of its high specificity based on the flanking sequence of the exogenous integrant. The aim of this study was to evaluate the use of very short (eight-nucleotide long), locked nucleic acid (LNA) TaqMan probes in 5'-nuclease PCR assays for the detection and quantification of GMOs. Classic TaqMan and LNA TaqMan probes were compared for the analysis of the maize MON810 transgene. The performance of the two types of probes was tested on the maize endogenous reference gene hmga, the CaMV 35S promoter, and the hsp70/cryIA(b) construct as well as for the event-specific 5'-integration junction of MON810, using plasmids as standard reference molecules. The results of our study demonstrate that the LNA 5'-nuclease PCR assays represent a valid and reliable analytical system for the detection and quantification of transgenes. Application of very short LNA TaqMan probes to GMO quantification can simplify the design of 5'-nuclease assays.

  3. X-ray Crystal Structure of a Locked Nucleic Acid (LNA) Duplex Composed of a Palindromic 10-mer DNA Strand Containing One LNA Thymine Monomer

    SciTech Connect

    Egli, M.; Minasov, G.; Teplova, M.; Kumar, R.; Wengel, J.

    2010-03-05

    Locked nucleic acid (LNA), a recently introduced nucleic acid analogue with a bicyclic 2'-O,4'-C-methylene linked furanose sugar, exhibits enhanced affinities for DNA and RNA relative to the corresponding oligodeoxyribonucleotides and oligoribonucleotides; we report the first crystal structure of an LNA unit incorporated in an oligonucleotide duplex. The structure at 1.4 {angstrom} resolution of the DNA-LNA decamer duplex with one LNA thymine monomer per strand provides a detailed view of the conformation and hydration of locked nucleic acid residues in a duplex A-form. Our study provides a first look at the conformational properties of LNA in a crystal structure at relatively high resolution. The main characteristics of the structure are the standard A-type conformation induced by LNA residues and the capacity of the 2'-oxygen that is part of the bicyclic sugar framework to engage in a least two hydrogen bonds to water molecules. Circular dichroism spectra (CD) of LNA-LNA duplexes in solution indicated that such duplexes appear to adopt a conformation that closely resembles the A-form geometry of RNA-RNA duplexes. However, these spectra also manifested subtle differences between the two species (data not shown). The present analysis of a duplex with only a single LNA residue per strand does not provide any insight into potential conformational differences between LNA and RNA duplexes. Attempts to determine a crystal structure of a completely modified LNA-LNA duplex are underway.

  4. C5-amino acid functionalized LNA: positively poised for antisense applications.

    PubMed

    Guenther, Dale C; Kumar, Pawan; Anderson, Brooke A; Hrdlicka, Patrick J

    2014-08-18

    Incorporation of positively charged C5-amino acid functionalized LNA uridines into oligodeoxyribonucleotides (ONs) results in extraordinary RNA affinity, binding specificity and stability towards 3'-exonucleases.

  5. Position-dependent effects of locked nucleic acid (LNA) on DNA sequencing and PCR primers

    PubMed Central

    Levin, Joshua D.; Fiala, Dean; Samala, Meinrado F.; Kahn, Jason D.; Peterson, Raymond J.

    2006-01-01

    Genomes are becoming heavily annotated with important features. Analysis of these features often employs oligonucleotides that hybridize at defined locations. When the defined location lies in a poor sequence context, traditional design strategies may fail. Locked Nucleic Acid (LNA) can enhance oligonucleotide affinity and specificity. Though LNA has been used in many applications, formal design rules are still being defined. To further this effort we have investigated the effect of LNA on the performance of sequencing and PCR primers in AT-rich regions, where short primers yield poor sequencing reads or PCR yields. LNA was used in three positional patterns: near the 5′ end (LNA-5′), near the 3′ end (LNA-3′) and distributed throughout (LNA-Even). Quantitative measures of sequencing read length (Phred Q30 count) and real-time PCR signal (cycle threshold, CT) were characterized using two-way ANOVA. LNA-5′ increased the average Phred Q30 score by 60% and it was never observed to decrease performance. LNA-5′ generated cycle thresholds in quantitative PCR that were comparable to high-yielding conventional primers. In contrast, LNA-3′ and LNA-Even did not improve read lengths or CT. ANOVA demonstrated the statistical significance of these results and identified significant interaction between the positional design rule and primer sequence. PMID:17071964

  6. Detection and measurement of benzimidazole resistance alleles in Haemonchus contortus using real-time PCR with locked nucleic acid Taqman probes.

    PubMed

    Walsh, Thomas K; Donnan, Alison A; Jackson, Frank; Skuce, Philip; Wolstenholme, Adrian J

    2007-03-31

    Benzimidazole resistance is a common problem in parasitic nematodes of ruminants and early detection is vital if its spread is to be monitored and controlled. Real time PCR offers a fast and reliable method for rapid detection and measurement of resistance allele frequencies. In Haemonchus contortus a single nucleotide polymorphism at codon 200 of the beta-tubulin gene (TTC to TAC), causing a phenylalanine to tyrosine amino acid substitution, has been shown to be involved in many cases of resistance. Locked nucleic acid (LNA) Taqman probes have been used in this work to detect and measure the frequency of resistance alleles in individual and multiple H. contortus. Detection of resistant genotypes using LNA Taqman probes in individual H. contortus is simpler and more reliable than with previously described assays. Measurement of the frequency of resistant alleles in populations of H. contortus was achieved by using the cycle threshold (C(t)) values and a standard curve derived from populations with known allele frequencies. Results using the LNA probes on individual and multiple worms gave similar results to the allele specific PCR. The sensitivity of the LNA assay on multiple nematodes allowed reliable detection of > or = 10% resistance allele frequency. Using the final fluorescence method, it was possible to differentiate populations with approximately 0, 5 and 10% resistance allele frequencies.

  7. Discrimination between mild and severe Citrus tristeza virus isolates with a rapid and highly specific real-time reverse transcription-polymerase chain reaction method using TaqMan LNA probes.

    PubMed

    Ruiz-Ruiz, S; Moreno, P; Guerri, J; Ambrós, S

    2009-03-01

    Severe isolates of Citrus tristeza virus (CTV) inducing seedling yellows (SY) and/or stem pitting (SP) in grapefruit or sweet orange are a major threat for the citrus industry worldwide. Identification of these CTV variants was achieved by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) using a general primer set and three TaqMan locked nucleic acids (LNA) probes targeting sequences characteristic of severe, mild (non-SY, non-SP), and T36-like isolates. Successful amplification was achieved from fresh or silica-desiccated CTV-infected samples and all isolates but one reacted with one or more probes. Standard curves using RNA transcripts homologous to the three probes allowed a reproducible quantitative assay, with a wide dynamic range of detection starting with 10(2) copies. RT-PCR assays with homologous and heterologous transcript RNA mixes demonstrated that each probe reacted only with its cognate sequence which was detected even at ratios below 2.5%. Analysis of 56 pathogenically distinct CTV isolates from 20 countries showed that mild isolates reacted only with the mild probe, whereas severe SP and SY isolates reacted with the severe-SP or the T36-like probes, respectively, and often with a second probe. This procedure can be useful to identify and control potentially dangerous CTV isolates in areas affected only by mild isolates.

  8. Locked nucleic acid (LNA): Based single-stranded oligonucleotides are not genotoxic.

    PubMed

    Guérard, Melanie; Andreas, Zeller; Erich, Koller; Christine, Marchand; Martina, Müller B; Christian, Weile; Franz, Schuler; Thomas, Singer; Yann, Tessier

    2017-04-01

    Over the last decade, single stranded oligonucleotides (ON) have gained increased attention as a new drug modality. Because the assessment of genotoxicity risk during early development of pharmaceuticals is essential, we evaluated the potential of locked nucleic acids (LNA)-ONs to induce DNA damage in L5178Y tk(+/-) cells both with the mouse lymphoma assay (MLA) and the micronucleus test (MNT). Further, the MLA was performed to assess gene and chromosome mutation over 3 and 24h (± metabolic activation). In addition, the MNT was performed to assess, in addition, a potential aneugenic liability. None of the experiments demonstrated a genotoxic effect for the five tested LNA-ONs. We further show data from four proprietary LNA-ONs tested in standard genotoxicity assays in vitro and partially in vivo, which were all negative. In addition, cellular and nuclear uptake of LNA-ONs in L5178Y tk(+/-) cells was demonstrated. Based on the results presented here as well as in the literature about other representatives of this class, we consider LNA-ONs as generally not DNA reactive and question whether genotoxicity testing of this class of ONs should be required. This is in line with recent recommendation made by the OSWG that extensively assessed the genotoxicity of oligonucleotides. Environ. Mol. Mutagen. 58:112-121, 2017. © 2017 Wiley Periodicals, Inc.

  9. Single and double stranded DNA detection using locked nucleic acid (LNA) functionalized nanoparticles

    NASA Astrophysics Data System (ADS)

    McKenzie, Fiona; Stokes, Robert; Faulds, Karen; Graham, Duncan

    2008-08-01

    Gold and silver nanoparticles functionalized with oligonucleotides can be used for the detection of specific sequences of DNA. We show that gold nanoparticles modified with locked nucleic acid (LNA) form stronger duplexes with a single stranded DNA target and offer better discrimination against single base pair mismatches than analogous DNA probes. Our LNA nanoparticle probes have also been used to detect double stranded DNA through triplex formation, whilst still maintaining selectivity for only complementary targets. Nanoparticle conjugates embedded with suitable surface enhanced resonance Raman scattering (SERRS) labels have been synthesized enabling simultaneous detection and identification of multiple DNA targets.

  10. Optimization of an AMBER Force Field for the Artificial Nucleic Acid, LNA, and Benchmarking with NMR of L(CAAU)

    PubMed Central

    2013-01-01

    Locked Nucleic Acids (LNAs) are RNA analogues with an O2′-C4′ methylene bridge which locks the sugar into a C3′-endo conformation. This enhances hybridization to DNA and RNA, making LNAs useful in microarrays and potential therapeutics. Here, the LNA, L(CAAU), provides a simplified benchmark for testing the ability of molecular dynamics (MD) to approximate nucleic acid properties. LNA χ torsions and partial charges were parametrized to create AMBER parm99_LNA. The revisions were tested by comparing MD predictions with AMBER parm99 and parm99_LNA against a 200 ms NOESY NMR spectrum of L(CAAU). NMR indicates an A-Form equilibrium ensemble. In 3000 ns simulations starting with an A-form structure, parm99_LNA and parm99 provide 66% and 35% agreement, respectively, with NMR NOE volumes and 3J-couplings. In simulations of L(CAAU) starting with all χ torsions in a syn conformation, only parm99_LNA is able to repair the structure. This implies methods for parametrizing force fields for nucleic acid mimics can reasonably approximate key interactions and that parm99_LNA will improve reliability of MD studies for systems with LNA. A method for approximating χ population distribution on the basis of base to sugar NOEs is also introduced. PMID:24377321

  11. Configuration of the 5'-methyl group modulates the biophysical and biological properties of locked nucleic acid (LNA) oligonucleotides.

    PubMed

    Seth, Punit P; Allerson, Charles R; Siwkowski, Andrew; Vasquez, Guillermo; Berdeja, Andres; Migawa, Michael T; Gaus, Hans; Prakash, Thazha P; Bhat, Balkrishen; Swayze, Eric E

    2010-12-09

    As part of a program aimed at exploring the structure- activity relationships of 2',4'-bridged nucleic acid (BNA) containing antisense oligonucleotides (ASOs), we report the synthesis and biophysical and biological properties of R- and S-5'-Me LNA modified oligonucleotides. We show that introduction of a methyl group in the (S) configuration at the 5'-position is compatible with the high affinity recognition of complementary nucleic acids observed with LNA. In contrast, introduction of a methyl group in the (R) configuration reversed the stabilization effect of LNA. NMR studies indicated that the R-5'-Me group changes the orientation around torsion angle γ from the +sc to the ap range at the nucleoside level, and this may in part be responsible for the poor hybridization behavior exhibited by this modification. In animal experiments, S-5'-Me-LNA modified gapmer antisense olignucleotides showed slightly reduced potency relative to the sequence matched LNA ASOs while improving the therapeutic profile.

  12. Locked Nucleic Acid Probes (LNA) for Enhanced Detection of Low-Level, Clinically Significant Mutations.

    PubMed

    Nafa, Khedoudja; Hameed, Meera; Arcila, Marie E

    2016-01-01

    The detection of clinically significant somatic mutations present at low level in a tissue sample represents a challenge in any laboratory. While several high sensitivity methods are described, the incorporation of these new techniques in a clinical lab may be difficult if the technology is not readily available or requires major changes in the workflow of the laboratory. Techniques that are robust and easily adapted to existing laboratory protocols are highly advantageous. In this chapter we describe the use of locked nucleic acid (LNA) probes to modify existing polymerase chain reaction (PCR)-based protocols which can then be sequenced by Sanger sequencing. LNA probes are used to enhance the sensitivity of Sanger sequencing to mutation frequencies below 1 %. The method is robust and is easily incorporated for assessment of any sample with low tumor content or low mutant allele burden.

  13. Application of Locked Nucleic Acid (LNA) Primer and PCR Clamping by LNA Oligonucleotide to Enhance the Amplification of Internal Transcribed Spacer (ITS) Regions in Investigating the Community Structures of Plant–Associated Fungi

    PubMed Central

    Ikenaga, Makoto; Tabuchi, Masakazu; Kawauchi, Tomohiro; Sakai, Masao

    2016-01-01

    The simultaneous extraction of host plant DNA severely limits investigations of the community structures of plant–associated fungi due to the similar homologies of sequences in primer–annealing positions between fungi and host plants. Although fungal-specific primers have been designed, plant DNA continues to be excessively amplified by PCR, resulting in the underestimation of community structures. In order to overcome this limitation, locked nucleic acid (LNA) primers and PCR clamping by LNA oligonucleotides have been applied to enhance the amplification of fungal internal transcribed spacer (ITS) regions. LNA primers were designed by converting DNA into LNA, which is specific to fungi, at the forward primer side. LNA oligonucleotides, the sequences of which are complementary to the host plants, were designed by overlapping a few bases with the annealing position of the reverse primer. Plant-specific DNA was then converted into LNA at the shifted position from the 3′ end of the primer–binding position. PCR using the LNA technique enhanced the amplification of fungal ITS regions, whereas those of the host plants were more likely to be amplified without the LNA technique. A denaturing gradient gel electrophoresis (DGGE) analysis displayed patterns that reached an acceptable level for investigating the community structures of plant–associated fungi using the LNA technique. The sequences of the bands detected using the LNA technique were mostly affiliated with known isolates. However, some sequences showed low similarities, indicating the potential to identify novel fungi. Thus, the application of the LNA technique is considered effective for widening the scope of community analyses of plant–associated fungi. PMID:27600711

  14. Application of Locked Nucleic Acid (LNA) Primer and PCR Clamping by LNA Oligonucleotide to Enhance the Amplification of Internal Transcribed Spacer (ITS) Regions in Investigating the Community Structures of Plant-Associated Fungi.

    PubMed

    Ikenaga, Makoto; Tabuchi, Masakazu; Kawauchi, Tomohiro; Sakai, Masao

    2016-09-29

    The simultaneous extraction of host plant DNA severely limits investigations of the community structures of plant-associated fungi due to the similar homologies of sequences in primer-annealing positions between fungi and host plants. Although fungal-specific primers have been designed, plant DNA continues to be excessively amplified by PCR, resulting in the underestimation of community structures. In order to overcome this limitation, locked nucleic acid (LNA) primers and PCR clamping by LNA oligonucleotides have been applied to enhance the amplification of fungal internal transcribed spacer (ITS) regions. LNA primers were designed by converting DNA into LNA, which is specific to fungi, at the forward primer side. LNA oligonucleotides, the sequences of which are complementary to the host plants, were designed by overlapping a few bases with the annealing position of the reverse primer. Plant-specific DNA was then converted into LNA at the shifted position from the 3' end of the primer-binding position. PCR using the LNA technique enhanced the amplification of fungal ITS regions, whereas those of the host plants were more likely to be amplified without the LNA technique. A denaturing gradient gel electrophoresis (DGGE) analysis displayed patterns that reached an acceptable level for investigating the community structures of plant-associated fungi using the LNA technique. The sequences of the bands detected using the LNA technique were mostly affiliated with known isolates. However, some sequences showed low similarities, indicating the potential to identify novel fungi. Thus, the application of the LNA technique is considered effective for widening the scope of community analyses of plant-associated fungi.

  15. Quantification of Staphylococcus aureus in white cheese by the improved DNA extraction strategy combined with TaqMan and LNA probe-based qPCR.

    PubMed

    Kadiroğlu, Pınar; Korel, Figen; Ceylan, Cagatay

    2014-10-01

    Four different bacterial DNA extraction strategies and two different qPCR probe chemistries were studied for detection of Stapylococcus aureus from white cheeses. Method employing trypsin treatment followed by a commercial kit application and TaqMan probe-based qPCR was the most sensitive one detecting higher counts than standards in naturally contaminated samples. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Novel multiplex PCR assay using locked nucleic acid (LNA)-based universal primers for the simultaneous detection of five swine viruses.

    PubMed

    Chen, Ru; Gao, Xiao-Bo; Yu, Xiao-Lu; Song, Chang-Xu; Qiu, Yang

    2016-02-01

    A novel multiplex PCR assay using non-homologous oligonucleotides with locked nucleic acid (LNA) modifications as universal primers was developed and validated for the simultaneous detection of five swine viruses. The assay utilizes five virus-specific primer pairs modified at the 5' end through the addition of the universal primer sequence. In the reaction, small amounts of target templates with the 5' tail were generated and subsequently amplified through the extension of a LNA universal primer set. To validate the specificity of this assay, 27 viral target strains and 12 non-target pathogens were tested. The lower limit of detection of viral nucleic acids was 1.1-1.9 pg per reaction or 11-32 pg in a five-plex viral nucleic acid mixture. The LNA mPCR assay displayed higher analytical sensitivity and efficiency for the detection of plasmid standards compared with the conventional assay, which uses standard primers without the 5' tail. A total of 207 field samples were tested using both assays. The LNA mPCR assay provided numerically higher detection rates for all pathogens in independent samples. Moreover, the LNA mPCR assay had significantly higher detection rates in independent samples compared with the conventional assay.

  17. Hybridization-based detection of Helicobacter pylori at human body temperature using advanced locked nucleic acid (LNA) probes.

    PubMed

    Fontenete, Sílvia; Guimarães, Nuno; Leite, Marina; Figueiredo, Céu; Wengel, Jesper; Filipe Azevedo, Nuno

    2013-01-01

    The understanding of the human microbiome and its influence upon human life has long been a subject of study. Hence, methods that allow the direct detection and visualization of microorganisms and microbial consortia (e.g. biofilms) within the human body would be invaluable. In here, we assessed the possibility of developing a variant of fluorescence in situ hybridization (FISH), named fluorescence in vivo hybridization (FIVH), for the detection of Helicobacter pylori. Using oligonucleotide variations comprising locked nucleic acids (LNA) and 2'-O-methyl RNAs (2'OMe) with two types of backbone linkages (phosphate or phosphorothioate), we were able to successfully identify two probes that hybridize at 37 °C with high specificity and sensitivity for H. pylori, both in pure cultures and in gastric biopsies. Furthermore, the use of this type of probes implied that toxic compounds typically used in FISH were either found to be unnecessary or could be replaced by a non-toxic substitute. We show here for the first time that the use of advanced LNA probes in FIVH conditions provides an accurate, simple and fast method for H. pylori detection and location, which could be used in the future for potential in vivo applications either for this microorganism or for others.

  18. Hybridization-Based Detection of Helicobacter pylori at Human Body Temperature Using Advanced Locked Nucleic Acid (LNA) Probes

    PubMed Central

    Fontenete, Sílvia; Guimarães, Nuno; Leite, Marina; Figueiredo, Céu; Wengel, Jesper; Filipe Azevedo, Nuno

    2013-01-01

    The understanding of the human microbiome and its influence upon human life has long been a subject of study. Hence, methods that allow the direct detection and visualization of microorganisms and microbial consortia (e.g. biofilms) within the human body would be invaluable. In here, we assessed the possibility of developing a variant of fluorescence in situ hybridization (FISH), named fluorescence in vivo hybridization (FIVH), for the detection of Helicobacter pylori. Using oligonucleotide variations comprising locked nucleic acids (LNA) and 2’-O-methyl RNAs (2’OMe) with two types of backbone linkages (phosphate or phosphorothioate), we were able to successfully identify two probes that hybridize at 37 °C with high specificity and sensitivity for H. pylori, both in pure cultures and in gastric biopsies. Furthermore, the use of this type of probes implied that toxic compounds typically used in FISH were either found to be unnecessary or could be replaced by a non-toxic substitute. We show here for the first time that the use of advanced LNA probes in FIVH conditions provides an accurate, simple and fast method for H. pylori detection and location, which could be used in the future for potential in vivo applications either for this microorganism or for others. PMID:24278398

  19. Increased stability and specificity through combined hybridization of peptide nucleic acid (PNA) and locked nucleic acid (LNA) to supercoiled plasmids for PNA-anchored "Bioplex" formation.

    PubMed

    Lundin, Karin E; Hasan, Maroof; Moreno, Pedro M; Törnquist, Elisabeth; Oprea, Iulian; Svahn, Mathias G; Simonson, E Oscar; Smith, C I Edvard

    2005-12-01

    Low cellular uptake and poor nuclear transfer hamper the use of non-viral vectors in gene therapy. Addition of functional entities to plasmids using the Bioplex technology has the potential to improve the efficiency of transfer considerably. We have investigated the possibility of stabilizing sequence-specific binding of peptide nucleic acid (PNA) anchored functional peptides to plasmid DNA by hybridizing PNA and locked nucleic acid (LNA) oligomers as "openers" to partially overlapping sites on the opposite DNA strand. The PNA "opener" stabilized the binding of "linear" PNA anchors to mixed-base supercoiled DNA in saline. For higher stability under physiological conditions, bisPNA anchors were used. To reduce nonspecific interactions when hybridizing highly cationic constructs and to accommodate the need for increased amounts of bisPNA when the molecules are uncharged, or negatively charged, we used both PNA and LNA oligomers as "openers" to increase binding kinetics. To our knowledge, this is the first time that LNA has been used together with PNA to facilitate strand invasion. This procedure allows hybridization at reduced PNA-to-plasmid ratios, allowing greater than 80% hybridization even at ratios as low as 2:1. Using significantly lower amounts of PNA-peptides combined with shorter incubation times reduces unspecific binding and facilitates purification.

  20. A Locked Nucleic Acid (LNA)-Based Real-Time PCR Assay for the Rapid Detection of Multiple Bacterial Antibiotic Resistance Genes Directly from Positive Blood Culture

    PubMed Central

    Zhu, Lingxiang; Shen, Dingxia; Zhou, Qiming; Li, Zexia; Fang, Xiangdong; Li, Quan-Zhen

    2015-01-01

    Bacterial strains resistant to various antibiotic drugs are frequently encountered in clinical infections, and the rapid identification of drug-resistant strains is highly essential for clinical treatment. We developed a locked nucleic acid (LNA)-based quantitative real-time PCR (LNA-qPCR) method for the rapid detection of 13 antibiotic resistance genes and successfully used it to distinguish drug-resistant bacterial strains from positive blood culture samples. A sequence-specific primer-probe set was designed, and the specificity of the assays was assessed using 27 ATCC bacterial strains and 77 negative blood culture samples. No cross-reaction was identified among bacterial strains and in negative samples, indicating 100% specificity. The sensitivity of the assays was determined by spiking each bacterial strain into negative blood samples, and the detection limit was 1–10 colony forming units (CFU) per reaction. The LNA-qPCR assays were first applied to 72 clinical bacterial isolates for the identification of known drug resistance genes, and the results were verified by the direct sequencing of PCR products. Finally, the LNA-qPCR assays were used for the detection in 47 positive blood culture samples, 19 of which (40.4%) were positive for antibiotic resistance genes, showing 91.5% consistency with phenotypic susceptibility results. In conclusion, LNA-qPCR is a reliable method for the rapid detection of bacterial antibiotic resistance genes and can be used as a supplement to phenotypic susceptibility testing for the early detection of antimicrobial resistance to allow the selection of appropriate antimicrobial treatment and to prevent the spread of resistant isolates. PMID:25775001

  1. A locked nucleic acid (LNA)-based real-time PCR assay for the rapid detection of multiple bacterial antibiotic resistance genes directly from positive blood culture.

    PubMed

    Zhu, Lingxiang; Shen, Dingxia; Zhou, Qiming; Li, Zexia; Fang, Xiangdong; Li, Quan-Zhen

    2015-01-01

    Bacterial strains resistant to various antibiotic drugs are frequently encountered in clinical infections, and the rapid identification of drug-resistant strains is highly essential for clinical treatment. We developed a locked nucleic acid (LNA)-based quantitative real-time PCR (LNA-qPCR) method for the rapid detection of 13 antibiotic resistance genes and successfully used it to distinguish drug-resistant bacterial strains from positive blood culture samples. A sequence-specific primer-probe set was designed, and the specificity of the assays was assessed using 27 ATCC bacterial strains and 77 negative blood culture samples. No cross-reaction was identified among bacterial strains and in negative samples, indicating 100% specificity. The sensitivity of the assays was determined by spiking each bacterial strain into negative blood samples, and the detection limit was 1-10 colony forming units (CFU) per reaction. The LNA-qPCR assays were first applied to 72 clinical bacterial isolates for the identification of known drug resistance genes, and the results were verified by the direct sequencing of PCR products. Finally, the LNA-qPCR assays were used for the detection in 47 positive blood culture samples, 19 of which (40.4%) were positive for antibiotic resistance genes, showing 91.5% consistency with phenotypic susceptibility results. In conclusion, LNA-qPCR is a reliable method for the rapid detection of bacterial antibiotic resistance genes and can be used as a supplement to phenotypic susceptibility testing for the early detection of antimicrobial resistance to allow the selection of appropriate antimicrobial treatment and to prevent the spread of resistant isolates.

  2. In vitro inhibition of promyelocytic leukemia/retinoic acid receptor-alpha (PML/RARalpha) expression and leukemogenic activity by DNA/LNA chimeric antisense oligos.

    PubMed

    Caprodossi, Sara; Galluzzi, Luca; Biagetti, Simona; Della Chiara, Giulia; Pelicci, Pier Giuseppe; Magnani, Mauro; Fanelli, Mirco

    2005-01-01

    Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by the chromosomal translocation t(15:17) that leads to the expression of promyelocytic leukemia/retinoic acid receptor-alpha (PML/ RARalpha) oncofusion protein. The block of differentiation at the promyelocytic stage of the blasts and their increased survival induced by PML/RARalpha are the principal biological features of the disease. Therapies based on pharmacological doses of retinoic acid (RA, 10(-6) M) are able to restore APL cell differentiation in most cases, but not to achieve complete hematological remission because retinoic acid resistance occurs in many patients. In order to elaborate alternative therapeutic approaches, we focused our attention on the use of antisense oligonucleotides as gene-specific drug directed to PML/RARalpha mRNA target. We used antisense molecules containing multiple locked nucleic acid (LNA) modifications. The LNAs are nucleotide analogues that are able to form duplexes with complementary DNA or RNA sequences with highly increased thermal stability and are resistant to 3'-exonuclease degradation in vitro. The DNA/LNA chimeric molecules were designed on the fusion sequence of PML and RARalpha genes to specifically target the oncofusion protein. Cell-free and in vitro experiments using U937-PR9-inducible cell line showed that DNA/LNA oligonucleotides were able to interfere with PML/RARalpha expression more efficiently than the corresponding unmodified DNA oligo. Moreover, the treatment of U937-PR9 cells with these chimeric antisense molecules was able to abrogate the block of differentiation induced by PML/RARalpha oncoprotein. These data suggest a possible application of oligonucleotides containing LNA in an antisense therapeutic strategy for APL.

  3. Validation of the TaqMan Influenza A Detection Kit and a rapid automated total nucleic acid extraction method to detect influenza A virus in nasopharyngeal specimens.

    PubMed

    Bolotin, Shelly; De Lima, Cedric; Choi, Kam-Wing; Lombos, Ernesto; Burton, Laura; Mazzulli, Tony; Drews, Steven J

    2009-01-01

    This study describes the validation of the TaqMan Influenza A Detection Kit v2.0 combined with an automated nucleic acid extraction method. The limit of detection of this assay was determined by probit regression (95% confidence interval) to be 2 influenza A/PR/8/34 (H1N1) virus particles per microlitre. One hundred and eleven specimens previously tested using the Seeplex RV assay and viral culture methods were tested using the TaqMan Influenza A Detection Kit. Compared to the aggregate gold-standard, the sensitivity and specificity of the TaqMan Influenza A Detection Kit were 100% (35/35) and 97% (74/76), respectively. Because of its accuracy, quick turn-around-time and lyophilized bead form, the TaqMan Influenza A Detection Kit, combined with the NucliSense easyMAG automated extraction method, constitutes a reliable protocol for influenza A diagnosis.

  4. Kinetics of membrane damage to high (HNA) and low (LNA) nucleic acid bacterial clusters in drinking water by ozone, chlorine, chlorine dioxide, monochloramine, ferrate(VI), and permanganate.

    PubMed

    Ramseier, Maaike K; von Gunten, Urs; Freihofer, Pietro; Hammes, Frederik

    2011-01-01

    Drinking water was treated with ozone, chlorine, chlorine dioxide, monochloramine, ferrate(VI), and permanganate to investigate the kinetics of membrane damage of native drinking water bacterial cells. Membrane damage was measured by flow cytometry using a combination of SYBR Green I and propidium iodide (SGI+PI) staining as indicator for cells with permeabilized membranes and SGI alone to measure total cell concentration. SGI+PI staining revealed that the cells were permeabilized upon relatively low oxidant exposures of all tested oxidants without a detectable lag phase. However, only ozonation resulted in a decrease of the total cell concentrations for the investigated reaction times. Rate constants for the membrane damage reaction varied over seven orders of magnitude in the following order: ozone > chlorine > chlorine dioxide ≈ ferrate > permanganate > chloramine. The rate constants were compared to literature data and were in general smaller than previously measured rate constants. This confirmed that membrane integrity is a conservative and therefore safe parameter for disinfection control. Interestingly, the cell membranes of high nucleic acid (HNA) content bacteria were damaged much faster than those of low nucleic acid (LNA) content bacteria during treatment with chlorine dioxide and permanganate. However, only small differences were observed during treatment with chlorine and chloramine, and no difference was observed for ferrate treatment. Based on the different reactivity of these oxidants it was suggested that HNA and LNA bacterial cell membranes have a different chemical constitution.

  5. Evaluation of TaqMan qPCR System Integrating Two Identically Labelled Hydrolysis Probes in Single Assay.

    PubMed

    Nagy, Alexander; Vitásková, Eliška; Černíková, Lenka; Křivda, Vlastimil; Jiřincová, Helena; Sedlák, Kamil; Horníčková, Jitka; Havlíčková, Martina

    2017-01-25

    Ongoing evolution of viral pathogens is a significant issue in diagnostic virology employing TaqMan qPCR/RT-qPCR. Specific concerns are related to false negativity due to probe binding failure. One option for compensating for such deficiency is to integrate a second identically labelled probe in the assay. However, how this alteration influences the reaction parameters has not been comprehensively demonstrated. In the present study, we evaluate a TaqMan protocol using two identically labelled hydrolysis probes (simple, LNA (locked-nucleic-acid)) and MGB (minor-groove-binder) modified probes and combinations thereof in a single assay. Our results based on a synthetic amplicon suggest that the second probe does not compromise the TaqMan qPCR/RT-qPCR parameters, which repeatedly and reproducibly remained comparable to those of the corresponding single-probe assays, irrespective of the relative probe orientation, whether opposite or tandem, and probe modifications or combinations thereof. On the other hand, the second probe additively contributed to the overall fluorescence signal. The utility of the dual-probe approach was demonstrated on practical examples by using field specimens. We hope that the present study might serve as a theoretical basis for the development or improvement of TaqMan qPCR/RT-qPCR assays for the detection of highly variable nucleic acid templates.

  6. Evaluation of TaqMan qPCR System Integrating Two Identically Labelled Hydrolysis Probes in Single Assay

    PubMed Central

    Nagy, Alexander; Vitásková, Eliška; Černíková, Lenka; Křivda, Vlastimil; Jiřincová, Helena; Sedlák, Kamil; Horníčková, Jitka; Havlíčková, Martina

    2017-01-01

    Ongoing evolution of viral pathogens is a significant issue in diagnostic virology employing TaqMan qPCR/RT-qPCR. Specific concerns are related to false negativity due to probe binding failure. One option for compensating for such deficiency is to integrate a second identically labelled probe in the assay. However, how this alteration influences the reaction parameters has not been comprehensively demonstrated. In the present study, we evaluate a TaqMan protocol using two identically labelled hydrolysis probes (simple, LNA (locked-nucleic-acid)) and MGB (minor-groove-binder) modified probes and combinations thereof in a single assay. Our results based on a synthetic amplicon suggest that the second probe does not compromise the TaqMan qPCR/RT-qPCR parameters, which repeatedly and reproducibly remained comparable to those of the corresponding single-probe assays, irrespective of the relative probe orientation, whether opposite or tandem, and probe modifications or combinations thereof. On the other hand, the second probe additively contributed to the overall fluorescence signal. The utility of the dual-probe approach was demonstrated on practical examples by using field specimens. We hope that the present study might serve as a theoretical basis for the development or improvement of TaqMan qPCR/RT-qPCR assays for the detection of highly variable nucleic acid templates. PMID:28120891

  7. Locked nucleic acid (LNA) induced effect on the hybridization and fluorescence properties of oligodeoxyribonucleotides modified with nucleobase-functionalized DNA monomers.

    PubMed

    Kaura, Mamta; Hrdlicka, Patrick J

    2015-07-14

    LNA and nucleobase-modified DNA monomers are two types of building blocks that are used extensively in oligonucleotide chemistry. However, there are only very few reports in which these two monomer families are used alongside each other. In the present study we set out to characterize the biophysical properties of oligodeoxyribonucleotides in which C5-modified 2'-deoxyuridine or C8-modified 2'-deoxyadenosine monomers are flanked by LNA nucleotides. We hypothesized that the LNA monomers would alter the sugar rings of the modified DNA monomers toward more RNA-like North-type conformations for maximal DNA/RNA affinity and specificity. Indeed, the incorporation of LNA monomers almost invariably results in increased target affinity and specificity relative to the corresponding LNA-free ONs, but the magnitude of the stabilization varies greatly. Introduction of LNA nucleotides as direct neighbors into C5-pyrene-functionalized pyrimidine DNA monomers yields oligonucleotide probes with more desirable photophysical properties as compared to the corresponding LNA-free probes, including more intense fluorescence emission upon target binding and improved discrimination of single nucleotide polymorphisms (SNPs). These hybrid oligonucleotides are therefore promising probes for diagnostic applications.

  8. Real-Time PCR Genotyping using Taqman Probes to Detect High Oleic Acid Peanuts

    USDA-ARS?s Scientific Manuscript database

    Oleic acid, a monounsaturated, omega-9 fatty acid is an important agronomic trait in peanut cultivars because it provides increased shelf life, improved flavor, enhanced fatty acid composition, and a beneficial effect on human health. Currently, most high oleic peanuts confer limited resistance to ...

  9. Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): A Method for Bacterial Small RNA Detection

    DTIC Science & Technology

    2012-01-10

    LNA oligonucleotide. The biotinylation is necessary for post-hybridization staining with a streptavidin-dye conjugate. It is possible to add this...which the LNA probe is hybridized to the target sRNA but the hybridization event is not detected due to the absence of the fluorescent stain , and (iii) a... peptidoglycan present in bacterial cell walls. 2. Prepare a 3.0 μg/mL solution of proteinase K in TE buffer. This solution should be prepared fresh from

  10. Potent and sustained cellular inhibition of miR-122 by lysine-derivatized peptide nucleic acids (PNA) and phosphorothioate locked nucleic acid (LNA)/2'-O-methyl (OMe) mixmer anti-miRs in the absence of transfection agents

    PubMed Central

    Torres, Adrian G.; Threlfall, Richard N.

    2011-01-01

    Efficient cell delivery of antisense oligonucleotides (ONs) is a key issue for their potential therapeutic use. It has been shown recently that some ONs can be delivered into cells without the use of transfection agents (gymnosis), but this generally requires cell incubation over several days and high amounts of ONs (micromolar concentrations). Here we have targeted microRNA 122 (miR-122), a small non-coding RNA involved in regulation of lipid metabolism and in the replication of hepatitis C virus, with ONs of different chemistries (anti-miRs) by gymnotic delivery in cell culture. Using a sensitive dual-luciferase reporter assay, anti-miRs were screened for their ability to enter liver cells gymnotically and inhibit miR-122 activity. Efficient miR-122 inhibition was obtained with cationic PNAs and 2'-O-methyl (OMe) and Locked Nucleic Acids (LNA)/OMe mixmers containing either phosphodiester (PO) or phosphorothioate (PS) linkages at sub-micromolar concentrations when incubated with cells for just 4 hours. Furthermore, PNA and PS-containing anti-miRs were able to sustain miR-122 inhibitory effects for at least 4 days. LNA/OMe PS anti-miRs were the most potent anti-miR chemistry tested in this study, an ON chemistry that has been little exploited so far as anti-miR agents towards therapeutics. PMID:22567190

  11. Why Carba-LNA-modified oligonucleotides show considerably improved 3'-exonuclease stability compared to that of the LNA modified or the native counterparts: A Michaelis-Menten kinetic analysis.

    PubMed

    Zhou, Chuanzheng; Chattopadhyaya, Jyoti

    2010-04-02

    In this study, 12 different native or LNA, carba-LNA-modified dinucleoside phosphates were designed as simple chemical models to study how carba-LNA modifications improve the 3'-exonuclease (SVPDE in this study) resistance of internucleotidic phosphate compared to those exhibited by LNA-modified and the native counterparts. Michaelis-Menten kinetic studies for dimers 3 - 7, in which the LNA or carba-LNA modifications are located at the 5'-end, showed that (i) increased 3'-exonuclease resistance of (5')[LNA-T](p)T (3) compared to the native (5')T(p)T (1) was mainly attributed to steric hindrance imposed by the LNA modification that retards the nuclease binding (K(M)) and (ii) digestion of (5')[carba-LNA-dT](p)T (4) and (5')[LNA-T](p)T (3), however, exhibit similar K(M)s, whereas the former shows a 100x decrease in K(cat) and is hence more stable than the latter. By studying the correlation between log k(cat) and pK(a) of the departing 3'(or 6')-OHs for 3-7, we found the pK(a) of 3'-OH of carba-LNA-T was 1.4 pK(a) units higher than that of LNA-T, and this relatively less acidic character of the 3'-OH in the former leads to the 100x decrease in the catalytic efficiency for the digestion of (5')[carba-LNA-T](p)T (4). In contrast, Michaelis-Menten kinetic studies for dimers 9-12, with the LNA or carba-LNA modifications at the 3'-end, showed that the digestion of (5')T(p)[LNA-T] (9) exhibited similar K(M) but k(cat) decreased around 40 times compared to that of the native (5')T(p)T (1). Similar k(cat) values have been observed for digestion of (5')T(p)[carba-LNA-T] (10) and (5')T(p)[LNA-T] (9). The higher stability of carba-LNA modified dimer 10 compared with LNA modified dimer 9 comes solely from the increased K(M).

  12. Comparative detection of rotavirus RNA by conventional RT-PCR, TaqMan RT-PCR and real-time nucleic acid sequence-based amplification.

    PubMed

    Mo, Qiu-Hua; Wang, Hai-Bo; Tan, Hua; Wu, Bi-Mei; Feng, Zi-Li; Wang, Qi; Lin, Ji-Can; Yang, Ze

    2015-03-01

    Rotavirus is one of the major viral pathogens leading to diarrhea. Diagnosis has been conducted by either traditional cultural, serological methods or molecular biology techniques, which include RT-PCR and nucleic acid sequence-based amplification (NASBA). However, their differences regarding accuracy and sensitivity remain unknown. In this study, an in-house conventional RT-PCR assay and more importantly, an in-house real-time NASBA (RT-NASBA) were established, and compared with a commercial TaqMan RT-PCR assay. The results showed that all of these methods were able to detect and distinguish rotavirus from other diarrhea viruses with a 100% concordance rate during the course of an evaluation on 20 clinical stool samples. However, RT-NASBA was much quicker than the other two methods. More importantly, the limit of detection of RT-NASBA could reach seven copies per reaction and was one to two logs lower than that of conventional RT-PCR and TaqMan RT-PCR. These results indicate that this in-house assay was more sensitive, and thus could be used as an efficient diagnosis tool for rotavirus. To the best of our knowledge, this is the first direct comparison among three different assays for the detection of rotavirus. These findings would provide implication for the rational selection of diagnosis tool for rotavirus.

  13. Development of LNA oligonucleotide-PCR clamping technique in investigating the community structures of plant-associated bacteria.

    PubMed

    Ikenaga, Makoto; Tabuchi, Masakazu; Oyama, Takuya; Akagi, Isao; Sakai, Masao

    2015-01-01

    Simultaneous extraction of plant organelle (mitochondria and plastid) genes during the DNA extraction step is major limitation in investigating the community structures of plant-associated bacteria. Although locked nucleic acid (LNA) oligonucleotides was designed to selectively amplify the bacterial small subunit rRNA genes by applying the PCR clamping technique, those for plastids were applicable only for particular plants, while those for mitochondria were available throughout most plants. To widen the applicable range, new LNA oligonucleotides specific for plastids were designed, and the efficacy was investigated. PCR without LNA oligonucleotides predominantly amplified the organelle genes, while bacterial genes were predominantly observed in having applied the LNA oligonucleotides. Denaturing gradient gel electrophoresis (DGGE) analysis displayed additional bacterial DGGE bands, the amplicons of which were prepared using the LNA oligonucleotides. Thus, new designed LNA oligonucleotides specific for plastids were effective and have widened the scope in investigating the community structures of plant-associated bacteria.

  14. Molecularly resolved label-free sensing of single nucleobase mismatches by interfacial LNA probes

    PubMed Central

    Mishra, Sourav; Lahiri, Hiya; Banerjee, Siddhartha; Mukhopadhyay, Rupa

    2016-01-01

    So far, there has been no report on molecularly resolved discrimination of single nucleobase mismatches using surface-confined single stranded locked nucleic acid (ssLNA) probes. Herein, it is exemplified using a label-independent force-sensing approach that an optimal coverage of 12-mer ssLNA sensor probes formed onto gold(111) surface allows recognition of ssDNA targets with twice stronger force sensitivity than 12-mer ssDNA sensor probes. The force distributions are reproducible and the molecule-by-molecule force measurements are largely in agreement with ensemble on-surface melting temperature data. Importantly, the molecularly resolved detection is responsive to the presence of single nucleobase mismatches in target sequences. Since the labelling steps can be eliminated from protocol, and each force-based detection event occurs within milliseconds' time scale, the force-sensing assay is potentially capable of rapid detection. The LNA probe performance is indicative of versatility in terms of substrate choice - be it gold (for basic research and array-based applications) or silicon (for ‘lab-on-a-chip’ type devices). The nucleic acid microarray technologies could therefore be generally benefited by adopting the LNA films, in place of DNA. Since LNA is nuclease-resistant, unlike DNA, and the LNA-based assay is sensitive to single nucleobase mismatches, the possibilities for label-free in vitro rapid diagnostics based on the LNA probes may be explored. PMID:27025649

  15. C5-Alkynyl-Functionalized α-L-LNA: Synthesis, Thermal Denaturation Experiments and Enzymatic Stability

    PubMed Central

    2015-01-01

    Major efforts are currently being devoted to improving the binding affinity, target specificity, and enzymatic stability of oligonucleotides used for nucleic acid targeting applications in molecular biology, biotechnology, and medicinal chemistry. One of the most popular strategies toward this end has been to introduce additional modifications to the sugar ring of affinity-inducing conformationally restricted nucleotide building blocks such as locked nucleic acid (LNA). In the preceding article in this issue, we introduced a different strategy toward this end, i.e., C5-functionalization of LNA uridines. In the present article, we extend this strategy to α-L-LNA: i.e., one of the most interesting diastereomers of LNA. α-L-LNA uridine monomers that are conjugated to small C5-alkynyl substituents induce significant improvements in target affinity, binding specificity, and enzymatic stability relative to conventional α-L-LNA. The results from the back-to-back articles therefore suggest that C5-functionalization of pyrimidines is a general and synthetically straightforward approach to modulate biophysical properties of oligonucleotides modified with LNA or other conformationally restricted monomers. PMID:24797769

  16. An Exocyclic Methylene Group Acts As A Bio-isostere of the 2’-Oxygen Atom in LNA

    PubMed Central

    Seth, Punit P; Allerson, Charles R.; Berdeja, Andres; Siwkowski, Andrew; Pallan, Pradeep S.; Gaus, Hans; Prakash, Thazha P.; Watt, Andrew T.; Egli, Martin; Swayze, Eric E.

    2010-01-01

    We show for the first time that it is possible to obtain LNA (Locked Nucleic Acid 1) like binding affinity and biological activity with carbocyclic LNA (cLNA) analogs by replacing the 2’-oxygen atom in LNA with an exocyclic methylene group. Synthesis of the methylene-cLNA nucleoside was accomplished by an intramolecular cyclization reaction between a radical at the 2’-position and a propynyl group at C-4’ position. Only methylene-cLNA modified oligonucleotides showed similar thermal stability and mismatch discrimination properties for complementary nucleic acids as LNA. In contrast, the close structurally related methyl-cLNA analogs showed diminished hybridization properties. Analysis of crystal structures of cLNA modified self-complementary DNA decamer duplexes revealed that the methylene group participates in a tight interaction with a 2’-deoxyribose residue of the 5’-terminal G of a neighboring duplex, resulting in the formation of a CH…O type hydrogen bond. This indicates that the methylene group retains a negative polarization at the edge of the minor groove in the absence of a hydrophilic 2’-substituent and provides a rationale for the superior thermal stability of this modification. In animal experiments, methylene-cLNA ASOs showed similar in vivo activity but reduced toxicity as compared to LNA ASOs. Our work highlights the interchangeable role of oxygen and unsaturated moeities in nucleic acid structure and emphasizes greater use of this bio-isostere to improve the properties of nucleic acids for therapeutic and diagnostic applications. PMID:20886816

  17. An Exocyclic Methylene Group Acts As a Bioisostere of the 2′-Oxygen Atom in LNA

    SciTech Connect

    Seth, Punit P.; Allerson, Charles R.; Berdeja, Andres; Siwkowski, Andrew; Pallan, Pradeep S.; Gaus, Hans; Prakash, Thazha P.; Watt, Andrew T.; Egli, Martin; Swayze, Eric E.

    2010-12-07

    We show for the first time that it is possible to obtain LNA-like (Locked Nucleic Acid 1) binding affinity and biological activity with carbocyclic LNA (cLNA) analogs by replacing the 2{prime}-oxygen atom in LNA with an exocyclic methylene group. Synthesis of the methylene-cLNA nucleoside was accomplished by an intramolecular cyclization reaction between a radical at the 2{prime}-position and a propynyl group at the C-4{prime} position. Only methylene-cLNA modified oligonucleotides showed similar thermal stability and mismatch discrimination properties for complementary nucleic acids as LNA. In contrast, the close structurally related methyl-cLNA analogs showed diminished hybridization properties. Analysis of crystal structures of cLNA modified self-complementary DNA decamer duplexes revealed that the methylene group participates in a tight interaction with a 2{prime}-deoxyribose residue of the 5{prime}-terminal G of a neighboring duplex, resulting in the formation of a CH...O type hydrogen bond. This indicates that the methylene group retains a negative polarization at the edge of the minor groove in the absence of a hydrophilic 2{prime}-substituent and provides a rationale for the superior thermal stability of this modification. In animal experiments, methylene-cLNA antisense oligonucleotides (ASOs) showed similar in vivo activity but reduced toxicity as compared to LNA ASOs. Our work highlights the interchangeable role of oxygen and unsaturated moieties in nucleic acid structure and emphasizes greater use of this bioisostere to improve the properties of nucleic acids for therapeutic and diagnostic applications.

  18. Oligonucleotides Containing Aminated 2'-Amino-LNA Nucleotides: Synthesis and Strong Binding to Complementary DNA and RNA.

    PubMed

    Lou, Chenguang; Samuelsen, Simone V; Christensen, Niels Johan; Vester, Birte; Wengel, Jesper

    2017-04-19

    Mono- and diaminated 2'-amino-LNA monomers were synthesized and introduced into oligonucleotides. Each modification imparts significant stabilization of nucleic acid duplexes and triplexes, excellent sequence selectivity, and significant nuclease resistance. Molecular modeling suggested that structural stabilization occurs via intrastrand electrostatic attraction between the protonated amino groups of the aminated 2'-amino-LNA monomers and the host oligonucleotide backbone.

  19. Quantum mechanical studies of DNA and LNA.

    PubMed

    Koch, Troels; Shim, Irene; Lindow, Morten; Ørum, Henrik; Bohr, Henrik G

    2014-04-01

    Quantum mechanical (QM) methodology has been employed to study the structure activity relations of DNA and locked nucleic acid (LNA). The QM calculations provide the basis for construction of molecular structure and electrostatic surface potentials from molecular orbitals. The topologies of the electrostatic potentials were compared among model oligonucleotides, and it was observed that small structural modifications induce global changes in the molecular structure and surface potentials. Since ligand structure and electrostatic potential complementarity with a receptor is a determinant for the bonding pattern between molecules, minor chemical modifications may have profound changes in the interaction profiles of oligonucleotides, possibly leading to changes in pharmacological properties. The QM modeling data can be used to understand earlier observations of antisense oligonucleotide properties, that is, the observation that small structural changes in oligonucleotide composition may lead to dramatic shifts in phenotypes. These observations should be taken into account in future oligonucleotide drug discovery, and by focusing more on non RNA target interactions it should be possible to utilize the exhibited property diversity of oligonucleotides to produce improved antisense drugs.

  20. In vivo efficacy and off-target effects of locked nucleic acid (LNA) and unlocked nucleic acid (UNA) modified siRNA and small internally segmented interfering RNA (sisiRNA) in mice bearing human tumor xenografts.

    PubMed

    Mook, Orf; Vreijling, Jeroen; Wengel, Suzy L; Wengel, Jesper; Zhou, Chuanzheng; Chattopadhyaya, Jyoti; Baas, Frank; Fluiter, Kees

    2010-07-01

    The clinical use of small interfering RNA (siRNA) is hampered by poor uptake by tissues and instability in circulation. In addition, off-target effects pose a significant additional problem for therapeutic use of siRNA. Chemical modifications of siRNA have been reported to increase stability and reduce off-target effects enabling possible therapeutic use of siRNA. Recently a large scale direct comparison of the impact of 21 different types of novel chemical modifications on siRNA efficiency and cell viability was published.1 It was found that several types of chemical modifications could enhance siRNA activity beyond that of an unmodified siRNA in vitro. In addition, a novel siRNA design, termed small internally segmented interfering RNA (sisiRNA), composed of an intact antisense strand and segmented guide strand stabilized using LNA was shown to be effective in cell based assays. In the present study we examined the in vivo efficacy of the LNA and UNA modified siRNA and sisiRNA in a mouse model bearing human tumor xenografts. We studied the biodistribution and efficacy of target knockdown in the mouse model. In addition we used whole genome profiling to assess the off-target effects in the liver of the mouse and the tumor xenografts. We report that LNA and UNA modified siRNA and sisiRNA improve the efficacy in target knockdown as compared with unmodified siRNA in the tumor xenografts without formulation. However, the level of off-target gene regulation in both the tumor and the liver correlated with the increase in efficacy in target knockdown, unless the seed region of the siRNA was modified.

  1. LNA effects on DNA binding and conformation: from single strand to duplex and triplex structures.

    PubMed

    Pabon-Martinez, Y Vladimir; Xu, You; Villa, Alessandra; Lundin, Karin E; Geny, Sylvain; Nguyen, Chi-Hung; Pedersen, Erik B; Jørgensen, Per T; Wengel, Jesper; Nilsson, Lennart; Smith, C I Edvard; Zain, Rula

    2017-09-08

    The anti-gene strategy is based on sequence-specific recognition of double-strand DNA by triplex forming (TFOs) or DNA strand invading oligonucleotides to modulate gene expression. To be efficient, the oligonucleotides (ONs) should target DNA selectively, with high affinity. Here we combined hybridization analysis and electrophoretic mobility shift assay with molecular dynamics (MD) simulations to better understand the underlying structural features of modified ONs in stabilizing duplex- and triplex structures. Particularly, we investigated the role played by the position and number of locked nucleic acid (LNA) substitutions in the ON when targeting a c-MYC or FXN (Frataxin) sequence. We found that LNA-containing single strand TFOs are conformationally pre-organized for major groove binding. Reduced content of LNA at consecutive positions at the 3'-end of a TFO destabilizes the triplex structure, whereas the presence of Twisted Intercalating Nucleic Acid (TINA) at the 3'-end of the TFO increases the rate and extent of triplex formation. A triplex-specific intercalating benzoquinoquinoxaline (BQQ) compound highly stabilizes LNA-containing triplex structures. Moreover, LNA-substitution in the duplex pyrimidine strand alters the double helix structure, affecting x-displacement, slide and twist favoring triplex formation through enhanced TFO major groove accommodation. Collectively, these findings should facilitate the design of potent anti-gene ONs.

  2. Inhibition of hepatitis B virus (HBV) by LNA-mediated nuclear interference with HBV DNA transcription

    SciTech Connect

    Sun, Zhen; Xiang, Wenqing; Guo, Yajuan; Chen, Zhi; Liu, Wei; Lu, Daru

    2011-06-10

    Highlights: {yields} LNA-modified oligonucleotides can pass through the plasma membrane of cultured cells even without using transfection machinery. {yields} LNA-modified oligonucleotides passed efficiently across the cell membrane, and lipid-coating facilitated translocation from the cytoplasm to the nucleus. {yields} LNA-oligonucleotide designed to target nuclear HBV DNA efficiently suppresses HBV replication and transcription in cultured hepatic cells. -- Abstract: Silencing target genes with small regulatory RNAs is widely used to investigate gene function and therapeutic drug development. Recently, triplex-based approaches have provided another attractive means to achieve targeted gene regulation and gene manipulation at the molecular and cellular levels. Nuclear entry of oligonucleotides and enhancement of their affinity to the DNA targets are key points of such approaches. In this study, we developed lipid-based transport of a locked-nucleic-acid (LNA)-modified oligonucleotide for hepatitis B virus (HBV) DNA interference in human hepatocytes expressing HBV genomic DNA. In these cells, the LNA-modified oligonucleotides passed efficiently across the cell membrane, and lipid-coating facilitated translocation from the cytoplasm to the nucleus. The oligonucleotide specifically targeting HBV DNA clearly interfered with HBV DNA transcription as shown by a block in pregenomic RNA (pgRNA) production. The HBV DNA-targeted oligonucleotide suppressed HBV DNA replication and HBV protein production more efficiently than small interfering RNAs directed to the pgRNA. These results demonstrate that fusion with lipid can carry LNA-modified oligonucleotides to the nucleus where they regulate gene expression. Interfering with HBV DNA transcription by LNA-modified oligonucleotides has strong potential as a new strategy for HBV inhibition.

  3. Functionalized 2′-Amino-α-L-LNA - Directed Positioning of Intercalators for DNA Targeting

    PubMed Central

    Kumar, T. Santhosh; Madsen, Andreas S.; Østergaard, Michael E.; Sau, Sujay P.; Wengel, Jesper; Hrdlicka, Patrick J.

    2010-01-01

    Chemically modified oligonucleotides are increasingly applied in nucleic acid based therapeutics and diagnostics. LNA (Locked Nucleic Acid) and its diastereomer α-L-LNA are two promising examples hereof that exhibit increased thermal and enzymatic stability. Herein, the synthesis, biophysical characterization and molecular modeling of N2′-functionalized 2′-amino-α-L-LNA is described. Chemoselective N2′-functionalization of protected amino alcohol 1 followed by phosphitylation afforded a structurally varied set of target phosphoramidites, which were incorporated into oligodeoxyribonucleotides. Incorporation of pyrene-functionalized building blocks such as 2′-N-(pyren-1-yl)carbonyl-2′-amino-α-L-LNA (monomer X) led to extraordinary increases in thermal affinity of up to +19.5 °C per modification against DNA targets in particular. In contrast, incorporation of building blocks with small non-aromatic N2′-functionalities such as 2′-N-acetyl-2′-amino-α-L-LNA (monomer V) had detrimental effects on thermal affinity toward DNA/RNA complements with decreases of as much as −16.5 °C per modification. Extensive thermal DNA selectivity, favorable entropic contributions upon duplex formation, hybridization-induced bathochromic shifts of pyrene absorption maxima and increases of circular dichroism signals, and molecular modeling studies suggest that pyrene functionalized 2′-amino-α-L-LNA monomers W-Y having short linkers between the bicyclic skeleton and the pyrene moiety, allow high-affinity hybridization with DNA complements and precise positioning of intercalators in nucleic acid duplexes. This rigorous positional control has been utilized for the development probes for emerging therapeutic and diagnostic applications focusing on DNA-targeting. PMID:19108636

  4. Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification.

    PubMed

    Itonaga, Masahiro; Matsuzaki, Ibu; Warigaya, Kenji; Tamura, Takaaki; Shimizu, Yuki; Fujimoto, Masakazu; Kojima, Fumiyoshi; Ichinose, Masao; Murata, Shin-Ichi

    2016-01-01

    Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid-locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP), for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD) of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V) of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation.

  5. Novel application of Locked Nucleic Acid chemistry for a TaqMan assay for measuring diverse Human Immunodeficiency Virus type 1 subtypes

    PubMed Central

    Li, Peilin; Ruel, Theodore; Fujimoto, Katsuya; Hatano, Hiroyu; Yukl, Steven; Eller, Leigh Anne; Liegler, Teri; Kamya, Moses; Gassasira, Anne; Dorsey, Grant; Rosenthal, Phil; Havlir, Diane; Wong, Joseph K.

    2010-01-01

    Summary There remains a need for sensitive and cost-effective assays to monitor therapy in human immunodeficiency virus type-1 (HIV-1) infection. However, the genetic diversity of HIV poses difficulties for traditional real-time PCR assays that require long oligonucleotides probes. LNA™ probes may be useful in overcoming these limits to traditional probe design. A new application of LNA™ chemistry in a Taqman assay applicable to a wide range of HIV-1 subtypes is described. This assay, based on a 13-mer LNA™ probe that matches the majority of HIV-1 sequences in the Los Alamos database, exhibited a wide dynamic range (101 to 107 copies of HIV DNA), high sensitivity (limit of detection of 1 copy of HIV DNA in 105 cells), and broad applicability to a range of HIV-1 subtypes (including A, B, C, D, F, H, B/C, and A/E CRFs). Using the LNA™ probe assay, HIV-1 DNA was detected in all dried blood spots (DBS) from treatment naïve HIV-1 positive Ugandan children, and HIV DNA levels significantly correlated with viral RNA levels in plasma (r=0.765, p<0.0001). This approach to Taqman probe design should be explored further for use in diagnosis and monitoring of HIV in resource-limited settings, especially where several subtypes co-circulate. PMID:20863855

  6. Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification

    PubMed Central

    Warigaya, Kenji; Tamura, Takaaki; Shimizu, Yuki; Fujimoto, Masakazu; Kojima, Fumiyoshi; Ichinose, Masao; Murata, Shin-ichi

    2016-01-01

    Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid—locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP), for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD) of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V) of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation. PMID:26999437

  7. Fast quantitative PCR, locked nucleic acid probes and reduced volume reactions are effective tools for detecting Batrachochytrium dendrobatidis DNA.

    PubMed

    Ruthig, Gregory R; Deridder, Benjamin P

    2012-01-24

    The fungal pathogen Batrachochytrium dendrobatidis threatens amphibian populations around the world. The ability to detect this pathogen on infected animals and in the environment is critical for understanding and controlling this pandemic. We tested several advances in quantitative PCR (qPCR) techniques to detect B. dendrobatidis DNA. We used a fast PCR thermocycler and enzymes that reduced the volume and the duration of the reaction. We also compared a conventional TaqMan minor groove binding (MGB) probe to an identical locked nucleic acid (LNA) counterpart. The fast qPCR reaction had a high degree of sensitivity to B. dendrobatidis DNA. The LNA probe was effective for detecting B. dendrobatidis DNA and produced results -similar to those of the MGB probe. The modifications that we tested can improve the cost, time efficiency and specificity of quantitative PCR as a tool for detecting pathogen DNA.

  8. Synthesis and Biophysical Investigations of Oligonucleotides Containing Galactose-Modified DNA, LNA, and 2'-Amino-LNA Monomers.

    PubMed

    Ries, Annika; Kumar, Rajesh; Lou, Chenguang; Kosbar, Tamer; Vengut-Climent, Empar; Jørgensen, Per T; Morales, Juan C; Wengel, Jesper

    2016-11-18

    Galactose-modified thymidine, LNA-T, and 2'-amino-LNA-T nucleosides were synthesized, converted into the corresponding phosphoramidite derivatives and introduced into short oligonucleotides. Compared to the unmodified control strands, the galactose-modified oligonucleotides in general, and the N2'-functionalized 2'-amino-LNA derivatives in particular, showed improved duplex thermal stability against DNA and RNA complements and increased ability to discriminate mismatches. In addition, the 2'-amino-LNA-T derivatives induced remarkable 3'-exonuclease resistance. These results were further investigated using molecular modeling studies.

  9. A novel noise optimization technique for inductively degenerated CMOS LNA

    NASA Astrophysics Data System (ADS)

    Zhiqing, Geng; Haiyong, Wang; Nanjian, Wu

    2009-10-01

    This paper proposes a novel noise optimization technique. The technique gives analytical formulae for the noise performance of inductively degenerated CMOS low noise amplifier (LNA) circuits with an ideal gate inductor for a fixed bias voltage and nonideal gate inductor for a fixed power dissipation, respectively, by mathematical analysis and reasonable approximation methods. LNA circuits with required noise figure can be designed effectively and rapidly just by using hand calculations of the proposed formulae. We design a 1.8 GHz LNA in a TSMC 0.25 μm CMOS process. The measured results show a noise figure of 1.6 dB with a forward gain of 14.4 dB at a power consumption of 5 mW, demonstrating that the designed LNA circuits can achieve low noise figure levels at low power dissipation.

  10. Evaluation of Cobas TaqMan MTB PCR for detection of Mycobacterium tuberculosis.

    PubMed

    Kim, Jeong Hyun; Kim, Young Jae; Ki, Chang-Seok; Kim, Ji-Youn; Lee, Nam Yong

    2011-01-01

    Nucleic acid-based amplification tests allow the rapid detection of Mycobacterium tuberculosis. Recently, a real-time PCR assay for M. tuberculosis complex, the Cobas TaqMan MTB test (Roche Diagnostics, Basel, Switzerland), was introduced. We performed a prospective study to evaluate the diagnostic performance of the Cobas TaqMan MTB test system. A total of 406 specimens collected from 247 patients were simultaneously tested by conventional culture, Cobas Amplicor MTB PCR, and TaqMan MTB PCR. The cross-reactivity with other Mycobacterium species and the detection limit were also evaluated. Among 406 specimens, a total of 24 specimens (5.9%) were culture positive: 14 specimens were positive by both TaqMan and Amplicor MTB PCRs, while 5 specimens were positive by only TaqMan PCR. The remaining five specimens were negative by both PCR methods. Seven specimens with negative culture results were positive by TaqMan PCR, but five of these were negative by Amplicor MTB PCR. The sensitivity, specificity, and positive (PPV) and negative (NPV) predictive values were 79.1%, 98.2%, 73.1%, and 98.7% for TaqMan and 58.3%, 99.5%, 87.5%, and 97.4% for the Amplicor MTB PCR test, respectively. There was no cross-reactivity with M. tuberculosis and nontuberculous mycobacterial species. The detection limit for the Cobas TaqMan MTB PCR test was 4.0 copies/μl. The Cobas TaqMan MTB PCR test showed higher sensitivity for detection of the M. tuberculosis complex without disturbing the specificity and NPV than the Amplicor MTB PCR test.

  11. Specific RNP capture with antisense LNA/DNA mixmers

    PubMed Central

    Rogell, Birgit; Fischer, Bernd; Rettel, Mandy; Krijgsveld, Jeroen; Castello, Alfredo; Hentze, Matthias W.

    2017-01-01

    RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe “specific ribonucleoprotein (RNP) capture,” a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein–RNA interactions taking place at “zero distance.” Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins. PMID:28476952

  12. Synthesis and Characterization of Oligodeoxyribonucleotides Modified with 2′-Amino-α-L-LNA Adenine Monomers: High-affinity Targeting of Single-Stranded DNA

    PubMed Central

    Andersen, Nicolai K.; Anderson, Brooke A.; Wengel, Jesper

    2014-01-01

    Development of conformationally restricted nucleotide building blocks continues to attract considerable interest due to their successful use within antisense, antigene and other gene-targeting strategies. Locked nucleic acid (LNA) and its diastereomer α-L-LNA are two interesting examples hereof. Oligonucleotides modified with these units display greatly increased affinity toward nucleic acid targets, improved binding specificity and enhanced enzymatic stability relative to unmodified strands. Here, we present the synthesis and biophysical characterization of oligodeoxyribonucleotides (ONs) modified with 2′-amino-α-L-LNA adenine monomers W–Z. The synthesis of target phosphoramidites 1–4 initiates from pentafuranose 5, which upon Vorbrüggen glycosylation, O2′-deacylation, O2′-activation and C2′-azide introduction yields nucleoside 8. A one-pot tandem Staudinger/intramolecular nucleophilic substitution converts 8 into 2′-amino-α-L-LNA adenine intermediate 9, which after a series of non-trivial protecting group manipulations affords key intermediate 15. Subsequent chemoselective N2′-functionalization and O3′-phosphitylation gives targets 1–4 in ~1–3% overall yield over eleven steps from 5. ONs modified with pyrene-functionalized 2′-amino-α-L-LNA adenine monomers X-Z display greatly increased affinity toward DNA targets (ΔTm/modification up to +14 °C). Results from absorption and fluorescence spectroscopy suggest that the duplex stabilization is a result of pyrene intercalation. These characteristics render N2′-pyrene-functionalized 2′-amino-α-L-LNA of considerable interest for DNA-targeting applications. PMID:24304240

  13. Dietary ALA, but not LNA, increase growth, reduce inflammatory processes, and increase anti-oxidant capacity in the marine finfish Larimichthys crocea: dietary ALA, but not LNA, increase growth, reduce inflammatory processes, and increase anti-oxidant capacity in the large yellow croaker.

    PubMed

    Zuo, Rantao; Mai, Kangsen; Xu, Wei; Turchini, Giovanni M; Ai, Qinghui

    2015-02-01

    Whilst aquaculture feed is increasingly formulated with the inclusion of plant oils replacing fish oil, and increasing research effort has been invested in understanding the metabolic effects of reduced dietary n-3 long chain poly unsaturated fatty acids (n-3 LC-PUFA), relatively little information is available on the potential direct metabolic roles of dietary alpha-linolenic acid (ALA, 18:3n-3) and alpha-linolenic acid/linoleic acid (LNA, 18:2n-6) ratio in cultured marine finfish species. In this study, four plant oil based diets, with varying ALA/LNA ratio (0.0, 0.5, 1.0 and 1.5) were fed to juvenile large yellow croakers (Larimichthys crocea) and compared to a fish oil-based control diet (CD) to evaluate the resulting effects on growth, nonspecific immunity, anti-oxidant capacity and related gene expression. High dietary LNA negatively impacted fish growth performance, nonspecific immunity and antioxidant capacity, but growth and immunity were maintained to levels comparable to CD by increasing the ratio of dietary ALA/LNA. The over-expression of genes associated with inflammation (cyclooxygenase-2 and interleukin-1β) and fatty acid oxidation (carnitine palmitoyl transferase I and acyl CoA oxidase) in croakers fed high concentrations of LNA were reduced to levels comparable to those fed CD by increasing dietary ALA/LNA. This study showed that dietary ALA, by increasing the overall n-3/n-6 PUFA ratio, exerts direct anti-inflammatory and antioxidant effects, similar to those exerted by dietary n-3 LC-PUFA.

  14. Crystallization and preliminary X-ray diffraction data of an LNA 7-mer duplex derived from a ricin aptamer

    PubMed Central

    Förster, Charlotte; Oberthuer, Dominik; Gao, Jiang; Eichert, André; Quast, Frederick G.; Betzel, Christian; Nitsche, Andreas; Erdmann, Volker A.; Fürste, Jens P.

    2009-01-01

    Locked nucleic acids (LNAs) are modified nucleic acids which contain a modified sugar such as β-d-2′-O,4′-C methylene-bridged ribofuranose or other sugar derivatives in LNA analogues. The β-d-2′-O,4′-C methylene ribofuranose LNAs in particular possess high stability and melting temperatures, which makes them of interest for stabilizing the structure of different nucleic acids. Aptamers, which are DNAs or RNAs targeted against specific ligands, are candidates for substitution with LNAs in order to increase their stability. A 7-­mer helix derived from the terminal part of an aptamer that was targeted against ricin was chosen. The ricin aptamer originally consisted of natural RNA building blocks and showed high affinity in ricin binding. For future stabilization of the aptamer, the terminal helix has been constructed as an ‘all-locked’ LNA and was successfully crystallized in order to investigate its structural properties. Optimization of crystal growth succeeded by the use of different metal salts as additives, such as CuCl2, MgCl2, MnCl2, CaCl2, CoCl2 and ZnSO4. Preliminary X-ray diffraction data were collected and processed to 2.8 Å resolution. The LNA crystallized in space group P65, with unit-cell parameters a = 50.11, b = 50.11, c = 40.72 Å. The crystals contained one LNA helix per asymmetric unit with a Matthews coefficient of 3.17 Å3 Da−1, which implies a solvent content of 70.15%. PMID:19724123

  15. A new wideband LNA using a gm-boosting technique

    NASA Astrophysics Data System (ADS)

    Liang, Chen; Zhiqun, Li; Jia, Cao; Chenjian, Wu; Meng, Zhang

    2014-01-01

    A new broadband low-noise amplifier (LNA) is proposed. The conventional common gate (CG) LNA exhibits a relatively high noise figure, so active gm-boosting technology is utilized to restrain the noise generated by the input transistors and reduce the noise figure. Theory, simulation and measurement are shown. An implemented prototype using 0.13 μm CMOS technology is evaluated using on-wafer probing. S11 and S22 are below -10 dB across 0.1-5 GHz. Measurements also show a gain of 18.3 dB with a 3 dB bandwidth from 100 MHz to 2.1 GHz and an IIP3 of -7 dBm at 2 GHz. The measured noise figure is better than 2.5 dB below 2.1 GHz, is better than 4.5 dB below 5 GHz, and at 500 MHz, it gets its minimum value 1.8 dB. The LNA consumes 9 mA from 1.5 V supply and occupies an area of 0.04 mm2.

  16. The effect of linoleic acid on the whole body synthesis rates of polyunsaturated fatty acids from α-linolenic acid and linoleic acid in free-living rats.

    PubMed

    Domenichiello, Anthony F; Kitson, Alex P; Chen, Chuck T; Trépanier, Marc-Olivier; Stavro, P Mark; Bazinet, Richard P

    2016-04-01

    Docosahexaenoic acid (DHA) is thought to be important for brain function. The main dietary source of DHA is fish, however, DHA can also be synthesized from precursor omega-3 polyunsaturated fatty acids (n-3 PUFA), the most abundantly consumed being α-linolenic acid (ALA). The enzymes required to synthesize DHA from ALA are also used to synthesize longer chain omega-6 (n-6) PUFA from linoleic acid (LNA). The large increase in LNA consumption that has occurred over the last century has led to concern that LNA and other n-6 PUFA outcompete n-3 PUFA for enzymes involved in DHA synthesis, and therefore, decrease overall DHA synthesis. To assess this, rats were fed diets containing LNA at 53 (high LNA diet), 11 (medium LNA diet) or 1.5% (low LNA diet) of the fatty acids with ALA being constant across all diets (approximately 4% of the fatty acids). Rats were maintained on these diets from weaning for 8 weeks, at which point they were subjected to a steady-state infusion of labeled ALA and LNA to measure DHA and arachidonic acid (ARA) synthesis rates. DHA and ARA synthesis rates were generally highest in rats fed the medium and high LNA diets, while the plasma half-life of DHA was longer in rats fed the low LNA diet. Therefore, increasing dietary LNA, in rats, did not impair DHA synthesis; however, low dietary LNA led to a decrease in DHA synthesis with tissue concentrations of DHA possibly being maintained by a longer DHA half-life. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  17. LNA/DNA mixmer-based antisense oligonucleotides correct alternative splicing of the SMN2 gene and restore SMN protein expression in type 1 SMA fibroblasts.

    PubMed

    Touznik, Aleksander; Maruyama, Rika; Hosoki, Kana; Echigoya, Yusuke; Yokota, Toshifumi

    2017-06-16

    Spinal muscular atrophy (SMA) is an autosomal recessive disorder affecting motor neurons, and is currently the most frequent genetic cause of infant mortality. SMA is caused by a loss-of-function mutation in the survival motor neuron 1 (SMN1) gene. SMN2 is an SMN1 paralogue, but cannot compensate for the loss of SMN1 since exon 7 in SMN2 mRNA is excluded (spliced out) due to a single C-to-T nucleotide transition in the exon 7. One of the most promising strategies to treat SMA is antisense oligonucleotide (AON)-mediated therapy. AONs are utilized to block intronic splicing silencer number 1 (ISS-N1) on intron 7 of SMN2, which causes exon 7 inclusion of the mRNA and the recovery of the expression of functional SMN protein from the endogenous SMN2 gene. We developed novel locked nucleic acid (LNA)-based antisense oligonucleotides (LNA/DNA mixmers), which efficiently induce exon 7 inclusion in SMN2 and restore the SMN protein production in SMA patient fibroblasts. The mixmers are highly specific to the targeted sequence, and showed significantly higher efficacy than an all-LNA oligonucleotide with the equivalent sequence. These data suggest that use of LNA/DNA mixmer-based AONs may be an attractive therapeutic strategy to treat SMA.

  18. Disruption of Higher Order DNA Structures in Friedreich’s Ataxia (GAA)n Repeats by PNA or LNA Targeting

    PubMed Central

    Bergquist, Helen; Rocha, Cristina S. J.; Álvarez-Asencio, Rubén; Nguyen, Chi-Hung; Rutland, Mark. W.; Smith, C. I. Edvard; Good, Liam; Nielsen, Peter E.; Zain, Rula

    2016-01-01

    Expansion of (GAA)n repeats in the first intron of the Frataxin gene is associated with reduced mRNA and protein levels and the development of Friedreich’s ataxia. (GAA)n expansions form non-canonical structures, including intramolecular triplex (H-DNA), and R-loops and are associated with epigenetic modifications. With the aim of interfering with higher order H-DNA (like) DNA structures within pathological (GAA)n expansions, we examined sequence-specific interaction of peptide nucleic acid (PNA) with (GAA)n repeats of different lengths (short: n=9, medium: n=75 or long: n=115) by chemical probing of triple helical and single stranded regions. We found that a triplex structure (H-DNA) forms at GAA repeats of different lengths; however, single stranded regions were not detected within the medium size pathological repeat, suggesting the presence of a more complex structure. Furthermore, (GAA)4-PNA binding of the repeat abolished all detectable triplex DNA structures, whereas (CTT)5-PNA did not. We present evidence that (GAA)4-PNA can invade the DNA at the repeat region by binding the DNA CTT strand, thereby preventing non-canonical-DNA formation, and that triplex invasion complexes by (CTT)5-PNA form at the GAA repeats. Locked nucleic acid (LNA) oligonucleotides also inhibited triplex formation at GAA repeat expansions, and atomic force microscopy analysis showed significant relaxation of plasmid morphology in the presence of GAA-LNA. Thus, by inhibiting disease related higher order DNA structures in the Frataxin gene, such PNA and LNA oligomers may have potential for discovery of drugs aiming at recovering Frataxin expression. PMID:27846236

  19. Applications of peptide nucleic acids (PNAs) and locked nucleic acids (LNAs) in biosensor development.

    PubMed

    Briones, Carlos; Moreno, Miguel

    2012-04-01

    Nucleic acid biosensors have a growing number of applications in genetics and biomedicine. This contribution is a critical review of the current state of the art concerning the use of nucleic acid analogues, in particular peptide nucleic acids (PNA) and locked nucleic acids (LNA), for the development of high-performance affinity biosensors. Both PNA and LNA have outstanding affinity for natural nucleic acids, and the destabilizing effect of base mismatches in PNA- or LNA-containing heterodimers is much higher than in double-stranded DNA or RNA. Therefore, PNA- and LNA-based biosensors have unprecedented sensitivity and specificity, with special applicability in DNA genotyping. Herein, the most relevant PNA- and LNA-based biosensors are presented, and their advantages and their current limitations are discussed. Some of the reviewed technology, while promising, still needs to bridge the gap between experimental status and the harder reality of biotechnological or biomedical applications.

  20. Design and evaluation of locked nucleic acid-based splice-switching oligonucleotides in vitro

    PubMed Central

    Shimo, Takenori; Tachibana, Keisuke; Saito, Kiwamu; Yoshida, Tokuyuki; Tomita, Erisa; Waki, Reiko; Yamamoto, Tsuyoshi; Doi, Takefumi; Inoue, Takao; Kawakami, Junji; Obika, Satoshi

    2014-01-01

    Antisense-mediated modulation of pre-mRNA splicing is an attractive therapeutic strategy for genetic diseases. Currently, there are few examples of modulation of pre-mRNA splicing using locked nucleic acid (LNA) antisense oligonucleotides, and, in particular, no systematic study has addressed the optimal design of LNA-based splice-switching oligonucleotides (LNA SSOs). Here, we designed a series of LNA SSOs complementary to the human dystrophin exon 58 sequence and evaluated their ability to induce exon skipping in vitro using reverse transcription-polymerase chain reaction. We demonstrated that the number of LNAs in the SSO sequence and the melting temperature of the SSOs play important roles in inducing exon skipping and seem to be key factors for designing efficient LNA SSOs. LNA SSO length was an important determinant of activity: a 13-mer with six LNA modifications had the highest efficacy, and a 7-mer was the minimal length required to induce exon skipping. Evaluation of exon skipping activity using mismatched LNA/DNA mixmers revealed that 9-mer LNA SSO allowed a better mismatch discrimination. LNA SSOs also induced exon skipping of endogenous human dystrophin in primary human skeletal muscle cells. Taken together, our findings indicate that LNA SSOs are powerful tools for modulating pre-mRNA splicing. PMID:24935206

  1. A 13 mer LNA-i-miR-221 Inhibitor Restores Drug Sensitivity in Melphalan-Refractory Multiple Myeloma Cells.

    PubMed

    Gullà, Annamaria; Di Martino, Maria Teresa; Gallo Cantafio, Maria Eugenia; Morelli, Eugenio; Amodio, Nicola; Botta, Cirino; Pitari, Maria Rita; Lio, Santo Giovanni; Britti, Domenico; Stamato, Maria Angelica; Hideshima, Teru; Munshi, Nikhil C; Anderson, Kenneth C; Tagliaferri, Pierosandro; Tassone, Pierfrancesco

    2016-03-01

    The onset of drug resistance is a major cause of treatment failure in multiple myeloma. Although increasing evidence is defining the role of miRNAs in mediating drug resistance, their potential activity as drug-sensitizing agents has not yet been investigated in multiple myeloma. Here we studied the potential utility of miR-221/222 inhibition in sensitizing refractory multiple myeloma cells to melphalan. miR-221/222 expression inversely correlated with melphalan sensitivity of multiple myeloma cells. Inhibition of miR-221/222 overcame melphalan resistance and triggered apoptosis of multiple myeloma cells in vitro, in the presence or absence of human bone marrow (BM) stromal cells. Decreased multiple myeloma cell growth induced by inhibition of miR-221/222 plus melphalan was associated with a marked upregulation of pro-apoptotic BBC3/PUMA protein, a miR-221/222 target, as well as with modulation of drug influx-efflux transporters SLC7A5/LAT1 and the ABC transporter ABCC1/MRP1. Finally, in vivo treatment of SCID/NOD mice bearing human melphalan-refractory multiple myeloma xenografts with systemic locked nucleic acid (LNA) inhibitors of miR-221 (LNA-i-miR-221) plus melphalan overcame drug resistance, evidenced by growth inhibition with significant antitumor effects together with modulation of PUMA and ABCC1 in tumors retrieved from treated mice. Taken together, our findings provide the proof of concept that LNA-i-miR-221 can reverse melphalan resistance in preclinical models of multiple myeloma, providing the framework for clinical trials to overcome drug resistance, and improve patient outcome in multiple myeloma. ©2015 American Association for Cancer Research.

  2. How RNase HI (Escherichia coli) promoted site-selective hydrolysis works on RNA in duplex with carba-LNA and LNA substituted antisense strands in an antisense strategy context?

    PubMed

    Plashkevych, Oleksandr; Li, Qing; Chattopadhyaya, Jyoti

    2017-03-29

    A detailed kinetic study of 36 single modified AON-RNA heteroduplexes shows that substitution of a single native nucleotide in the antisense strand (AON) by locked nucleic acid (LNA) or by diastereomerically pure carba-LNA results in site-dependent modulation of RNase H promoted cleavage of complementary mRNA strands by 2 to 5 fold at 5'-GpN-3' cleavage sites, giving up to 70% of the RNA cleavage products. The experiments have been performed using RNase HI of Escherichia coli. The 2nd best cleavage site, being the 5'-ApN-3' sites, cleaves up to 23%, depending upon the substitution site in 36 isosequential complementary AONs. A comparison of the modified AON promoted RNA cleavage rates with that of the native AON shows that sequence-specificity is considerably enhanced as a result of modification. Clearly, relatively weaker 5'-purine (Pu)-pyrimidine (Py)-3' stacking in the complementary RNA strand is preferred (giving ∼90% of total cleavage products), which plays an important role in RNase H promoted RNA cleavage. A plausible mechanism of RNase H mediated cleavage of the RNA has been proposed to be two-fold, dictated by the balancing effect of the aromatic character of the purine aglycone: first, the locally formed 9-guanylate ion (pKa 9.3, ∼18-20% N1 ionized at pH 8) alters the adjoining sugar-phosphate backbone around the scissile phosphate, transforming its sugar N/S conformational equilibrium, to preferential S-type, causing preferential cleavage at 5'-GpN-3' sites around the center of 20 mer complementary mRNA. Second, the weaker nearest-neighbor strength of 5'-Pu-p-Py-3' stacking promotes preferential 5'-GpN-3' and 5'-ApN-3' cleavage, providing ∼90% of the total products, compared to ∼50% in that of the native one, because of the cLNA/LNA substituent effect on the neighboring 5'-Pu-p-Py-3' sites, providing both local steric flexibility and additional hydration. This facilitates both the water and water/Mg(2+) ion availability at the cleavage site

  3. miRNA detection at single-cell resolution using microfluidic LNA flow-FISH

    DOE PAGES

    Wu, Meiye; Piccini, Matthew Ernest; Koh, Chung -Yan; ...

    2014-08-20

    Flow cytometry in combination with fluorescent in situ hybridization (flow-FISH) is a powerful technique that can be utilized to rapidly detect nucleic acids at single-cell resolution without the need for homogenization or nucleic acid extraction. Here, we describe a microfluidic-based method which enables the detection of microRNAs or miRNAs in single intact cells by flow-FISH using locked nucleic acid (LNA)-containing probes. Our method can be applied to all RNA species including mRNA and small noncoding RNA and is suitable for multiplexing with protein immunostaining in the same cell. For demonstration of our method, this chapter details the detection of miR155more » and CD69 protein in PMA and ionomycin-stimulated Jurkat cells. Here, we also include instructions on how to set up a microfluidic chip sample preparation station to prepare cells for imaging and analysis on a commercial flow cytometer or a custom-built micro-flow cytometer.« less

  4. miRNA detection at single-cell resolution using microfluidic LNA flow-FISH

    SciTech Connect

    Wu, Meiye; Piccini, Matthew Ernest; Koh, Chung -Yan; Singh, Anup K.

    2014-08-20

    Flow cytometry in combination with fluorescent in situ hybridization (flow-FISH) is a powerful technique that can be utilized to rapidly detect nucleic acids at single-cell resolution without the need for homogenization or nucleic acid extraction. Here, we describe a microfluidic-based method which enables the detection of microRNAs or miRNAs in single intact cells by flow-FISH using locked nucleic acid (LNA)-containing probes. Our method can be applied to all RNA species including mRNA and small noncoding RNA and is suitable for multiplexing with protein immunostaining in the same cell. For demonstration of our method, this chapter details the detection of miR155 and CD69 protein in PMA and ionomycin-stimulated Jurkat cells. Here, we also include instructions on how to set up a microfluidic chip sample preparation station to prepare cells for imaging and analysis on a commercial flow cytometer or a custom-built micro-flow cytometer.

  5. Threshold changes in rat brain docosahexaenoic acid incorporation and concentration following graded reductions in dietary alpha-linolenic acid

    PubMed Central

    Taha, Ameer Y.; Chang, Lisa; Chen, Mei

    2016-01-01

    Background This study tested the dietary level of alpha-linolenic acid (α-LNA, 18:3n-3) sufficient to maintain brain 14C-Docosahexaenoic acid (DHA, 22:6n-3) metabolism and concentration following graded α-LNA reduction. Methods 18–21 day male Fischer-344 (CDF) rats were randomized to the AIN-93G diet containing as a % of total fatty acids, 4.6% (“n-3 adequate”), 3.6%, 2.7%, 0.9% or 0.2% (“n-3 deficient”) α-LNA for 15 weeks. Rats were intravenously infused with 14C-DHA to steady state for 5 minutes, serial blood samples collected to obtain plasma and brains excised following microwave fixation. Labeled and unlabeled DHA concentrations were measured in plasma and brain to calculate the incorporation coefficient, k*, and incorporation rate, Jin. Results Compared to 4.6% α-LNA controls, k* was significantly increased in ethanolamine glycerophospholipids in the 0.2% α-LNA group. Circulating unesterified DHA and brain incorporation rates (Jin) were significantly reduced at 0.2% α-LNA. Brain total lipid and phospholipid DHA concentrations were reduced at or below 0.9% α-LNA. Conclusion Threshold changes for brain DHA metabolism and concentration were maintained at or below 0.9% dietary α-LNA, suggesting the presence of homeostatic mechanisms to maintain brain DHA metabolism when dietary α-LNA intake is low. PMID:26869088

  6. Superior Silencing by 2',4'-BNA(NC)-Based Short Antisense Oligonucleotides Compared to 2',4'-BNA/LNA-Based Apolipoprotein B Antisense Inhibitors.

    PubMed

    Yamamoto, Tsuyoshi; Yasuhara, Hidenori; Wada, Fumito; Harada-Shiba, Mariko; Imanishi, Takeshi; Obika, Satoshi

    2012-01-01

    The duplex stability with target mRNA and the gene silencing potential of a novel bridged nucleic acid analogue are described. The analogue, 2',4'-BNA(NC) antisense oligonucleotides (AONs) ranging from 10- to 20-nt-long, targeted apolipoprotein B. 2',4'-BNA(NC) was directly compared to its conventional bridged (or locked) nucleic acid (2',4'-BNA/LNA)-based counterparts. Melting temperatures of duplexes formed between 2',4'-BNA(NC)-based antisense oligonucleotides and the target mRNA surpassed those of 2',4'-BNA/LNA-based counterparts at all lengths. An in vitro transfection study revealed that when compared to the identical length 2',4'-BNA/LNA-based counterpart, the corresponding 2',4'-BNA(NC)-based antisense oligonucleotide showed significantly stronger inhibitory activity. This inhibitory activity was more pronounced in shorter (13-, 14-, and 16-mer) oligonucleotides. On the other hand, the 2',4'-BNA(NC)-based 20-mer AON exhibited the highest affinity but the worst IC(50) value, indicating that very high affinity may undermine antisense potency. These results suggest that the potency of AONs requires a balance between reward term and penalty term. Balance of these two parameters would depend on affinity, length, and the specific chemistry of the AON, and fine-tuning of this balance could lead to improved potency. We demonstrate that 2',4'-BNA(NC) may be a better alternative to conventional 2',4'-BNA/LNA, even for "short" antisense oligonucleotides, which are attractive in terms of drug-likeness and cost-effective bulk production.

  7. LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells.

    PubMed

    van Ravesteyn, Thomas W; Dekker, Marleen; Fish, Alexander; Sixma, Titia K; Wolters, Astrid; Dekker, Rob J; Te Riele, Hein P J

    2016-04-12

    Synthetic single-stranded DNA oligonucleotides (ssODNs) can be used to generate subtle genetic modifications in eukaryotic and prokaryotic cells without the requirement for prior generation of DNA double-stranded breaks. However, DNA mismatch repair (MMR) suppresses the efficiency of gene modification by >100-fold. Here we present a commercially available ssODN design that evades MMR and enables subtle gene modification in MMR-proficient cells. The presence of locked nucleic acids (LNAs) in the ssODNs at mismatching bases, or also at directly adjacent bases, allowed 1-, 2-, or 3-bp substitutions in MMR-proficient mouse embryonic stem cells as effectively as in MMR-deficient cells. Additionally, in MMR-proficient Escherichia coli, LNA modification of the ssODNs enabled effective single-base-pair substitution. In vitro, LNA modification of mismatches precluded binding of purified E. coli MMR protein MutS. These findings make ssODN-directed gene modification particularly well suited for applications that require the evaluation of a large number of sequence variants with an easy selectable phenotype.

  8. LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells

    PubMed Central

    van Ravesteyn, Thomas W.; Dekker, Marleen; Fish, Alexander; Sixma, Titia K.; Wolters, Astrid; Dekker, Rob J.; te Riele, Hein P. J.

    2016-01-01

    Synthetic single-stranded DNA oligonucleotides (ssODNs) can be used to generate subtle genetic modifications in eukaryotic and prokaryotic cells without the requirement for prior generation of DNA double-stranded breaks. However, DNA mismatch repair (MMR) suppresses the efficiency of gene modification by >100-fold. Here we present a commercially available ssODN design that evades MMR and enables subtle gene modification in MMR-proficient cells. The presence of locked nucleic acids (LNAs) in the ssODNs at mismatching bases, or also at directly adjacent bases, allowed 1-, 2-, or 3-bp substitutions in MMR-proficient mouse embryonic stem cells as effectively as in MMR-deficient cells. Additionally, in MMR-proficient Escherichia coli, LNA modification of the ssODNs enabled effective single-base-pair substitution. In vitro, LNA modification of mismatches precluded binding of purified E. coli MMR protein MutS. These findings make ssODN-directed gene modification particularly well suited for applications that require the evaluation of a large number of sequence variants with an easy selectable phenotype. PMID:26951689

  9. Evaluation of the Cobas TaqMan MTB real-time PCR assay for direct detection of Mycobacterium tuberculosis in respiratory specimens.

    PubMed

    Lee, Meng-Rui; Chung, Kuei-Pin; Wang, Hao-Chien; Lin, Chih-Bin; Yu, Chong-Jen; Lee, Jen-Jyh; Hsueh, Po-Ren

    2013-08-01

    The Cobas TaqMan MTB assay is a real-time PCR (qPCR) kit for rapid detection of Mycobacterium tuberculosis from clinical specimens. There are, however, limited studies validating its performance. We performed a prospective study in two hospitals in Taiwan on 586 respiratory specimens. By using culture as the reference method, the sensitivity and specificity of the Cobas TaqMan MTB assay were found to be 82.7 and 96.5 %, respectively. The sensitivity of the Cobas TaqMan MTB assay in acid-fast stain-negative respiratory specimens was only 34.9 %. Five specimens from five patients were positive for M. tuberculosis by the Cobas TaqMan MTB assay but were negative for M. tuberculosis by conventional culture methods. A diagnosis of pulmonary tuberculosis (TB) was made based on clinical and radiological findings as well as the response to anti-TB treatment in these five patients. Addition of data from these five specimens with discrepant results (PCR vs culture) from patients with symptoms clinically compatible with TB increased the sensitivity of the Cobas TaqMan MTB assay to 83.1 %. The Cobas TaqMan MTB assay is a rapid identification tool with a high degree of specificity for the direct detection of M. tuberculosis in respiratory specimens. The sensitivity for detecting acid-fast smear-negative respiratory specimens, however, is low.

  10. LNA with wide range of gain control and wideband interference rejection

    NASA Astrophysics Data System (ADS)

    Wang, Jhen-Ji; Chen, Duan-Yu

    2016-10-01

    This work presents a low-noise amplifier (LNA) design with a wide-range gain control characteristic that integrates adjustable current distribution and output impedance techniques. For a given gain characteristic, the proposed LNA provides better wideband interference rejection performance than conventional LNA. Moreover, the proposed LNA also has a wider gain control range than conventional LNA. Therefore, it is suitable for satellite communications systems. The simulation results demonstrate that the voltage gain control range is between 14.5 and 34.2 dB for such applications (2600 MHz); the input reflection coefficient is less than -18.9 dB; the noise figure (NF) is 1.25 dB; and the third-order intercept point (IIP3) is 4.52 dBm. The proposed LNA consumes 23.85-28.17 mW at a supply voltage of 1.8 V. It is implemented by using TSMC 0.18-um RF CMOS process technology.

  11. Stability and free energy calculation of LNA modified quadruplex: a molecular dynamics study

    NASA Astrophysics Data System (ADS)

    Chaubey, Amit Kumar; Dubey, Kshatresh Dutta; Ojha, Rajendra Prasad

    2012-03-01

    Telomeric ends of chromosomes, which comprise noncoding repeat sequences of guanine-rich DNA, which are the fundamental in protecting the cell from recombination and degradation. Telomeric DNA sequences can form four stranded quadruplex structures, which are involved in the structure of telomere ends. The formation and stabilization of telomeric quadruplexes has been shown to inhibit the activity of telomerase, thus establishing telomeric DNA quadrulex as an attractive target for cancer therapeutic intervention. Molecular dynamic simulation offers the prospects of detailed description of the dynamical structure with ion and water at molecular level. In this work we have taken a oligomeric part of human telomeric DNA, d(TAGGGT) to form different monomeric quadruplex structures d(TAGGGT)4. Here we report the relative stabilities of these structures under K+ ion conditions and binding interaction between the strands, as determined by molecular dynamic simulations followed by energy calculation. We have taken locked nucleic acid (LNA) in this study. The free energy molecular mechanics Poission Boltzman surface area calculations are performed for the determination of most stable complex structure between all modified structures. We calculated binding free energy for the combination of different strands as the ligand and receptor for all structures. The energetic study shows that, a mixed hybrid type quadruplex conformation in which two parallel strands are bind with other two antiparallel strands, are more stable than other conformations. The possible mechanism for the inhibition of the cancerous growth has been discussed. Such studies may be helpful for the rational drug designing.

  12. Kinetics of DNA Strand Displacement Systems with Locked Nucleic Acids.

    PubMed

    Olson, Xiaoping; Kotani, Shohei; Yurke, Bernard; Graugnard, Elton; Hughes, William L

    2017-03-30

    Locked nucleic acids (LNAs) are conformationally restricted RNA nucleotides. Their increased thermal stability and selectivity toward their complements make them well-suited for diagnostic and therapeutic applications. Although the structural and thermodynamic properties of LNA-LNA, LNA-RNA, and LNA-DNA hybridizations are known, the kinetic effects of incorporating LNA nucleotides into DNA strand displacement systems are not. Here, we thoroughly studied the strand displacement kinetics as a function of the number and position of LNA nucleotides in DNA oligonucleotides. When compared to that of an all-DNA control, with an identical sequence, the leakage rate constant was reduced more than 50-fold, to an undetectable level, and the invasion rate was preserved for a hybrid DNA/LNA system. The total performance enhancement ratio also increased more than 70-fold when calculating the ratio of the invading rate to the leakage rate constants for a hybrid system. The rational substitution of LNA nucleotides for DNA nucleotides preserves sequence space while improving the signal-to-noise ratio of strand displacement systems. Hybrid DNA/LNA systems offer great potential for high-performance chemical reaction networks that include catalyzed hairpin assemblies, hairpin chain reactions, motors, walkers, and seesaw gates.

  13. A Single-To-Differential LNA using Differential Active Inductor for GPS Applications

    NASA Astrophysics Data System (ADS)

    Babaei Kia, Hojjat; Khari A'ain, Abu

    2013-01-01

    This paper presents the design of a single-ended input, differential output low noise amplifier for GPS applications in 0.18 µm CMOS technology. This Low Noise Amplifier (LNA) is composed of a common source (CS) amplifier adopted with a common gate, common source (CGCS) balun load. Instead of spiral on-chip inductor, a differential active inductor circuit (DAI) is used as an active load of balun and also gm-boosting technique is used to decrease the noise figure (NF) of Single-to-Differential Low Noise Amplifier (S-to-D LNA). The LNA is simulated with Cadence Spectra which shows the S21 of 22.94 dB at 1.57 GHz, S11 of -13.1 dB, NFmin of 2.66 dB and power dissipation of 2.5 mw from 1.8 V dc power supply. By adding an external capacitor to the DAI circuit, it is possible to reconfigure the LNA to use in multi frequency range. For using the LNA in frequency range of 1.22 GHz, the external capacitor is set to 121 fF. The result shows the S21 of 20.4 dB, S11 of -12.4 dB and NFmin of 2.4 dB.

  14. A Wideband Noise-canceling CMOS LNA Using Cross-coupled Feedback and Bulk Effect

    NASA Astrophysics Data System (ADS)

    Guo, Benqing; Yang, Guoning; An, Shiquan

    2014-05-01

    An improved wideband common-gate (CG) and common-source (CS) CMOS LNA with noise cancellation is proposed. The cross-coupled feedback between the CG input transistor and the cascode transistor of CS input stage is used to increase the input transconductance of the LNA. And the bulk effect of CS input transistors is utilized to enhance gm-boosting coefficient. Thus, comparable gain and noise are achieved by reduced bias currents of the LNA while the resulted additional NF degradation is negligible. Fabricated in a 0.13 μm RF CMOS process, the LNA achieves a flat voltage gain of 18 dB, an NF of 2.7~3.2 dB, and an IIP3 of -4.5~-7.4 dBm over a 3 dB bandwidth of 0.1~4.4 GHz. It consumes only 4.1 mA from a 1 V supply and occupies an area of 520 × 490 um2. In contrast to those of reported wideband LNAs, the proposed LNA has the merit of lower power consumption and lower supply voltage.

  15. Design and optimization of CMOS LNA with ESD protection for 2.4 GHz WSN application

    NASA Astrophysics Data System (ADS)

    Zhiqun, Li; Liang, Chen; Hao, Zhang

    2011-10-01

    A new optimization method of a source inductive degenerated low noise amplifier (LNA) with electrostatic discharge protection is proposed. It can achieve power-constrained simultaneous noise and input matching. An analysis of the input impedance and the noise parameters is also given. Based on the developed method, a 2.4 GHz LNA for wireless sensor network application is designed and optimized using 0.18-μm RF CMOS technology. The measured results show that the LNA achieves a noise figure of 1.59 dB, a power gain of 14.12 dB, an input 1 dB compression point of -8 dBm and an input third-order intercept point of 1 dBm. The DC current is 4 mA under a supply of 1.8 V.

  16. Synthesis, hybridization characteristics, and fluorescence properties of oligonucleotides modified with nucleobase-functionalized locked nucleic acid adenosine and cytidine monomers.

    PubMed

    Kaura, Mamta; Kumar, Pawan; Hrdlicka, Patrick J

    2014-07-03

    Conformationally restricted nucleotides such as locked nucleic acid (LNA) are very popular as affinity-, specificity-, and stability-enhancing modifications in oligonucleotide chemistry to produce probes for nucleic acid targeting applications in molecular biology, biotechnology, and medicinal chemistry. Considerable efforts have been devoted in recent years to optimize the biophysical properties of LNA through additional modification of the sugar skeleton. We recently introduced C5-functionalization of LNA uridines as an alternative and synthetically more straightforward approach to improve the biophysical properties of LNA. In the present work, we set out to test the generality of this concept by studying the characteristics of oligonucleotides modified with four different C5-functionalized LNA cytidine and C8-functionalized LNA adenosine monomers. The results strongly suggest that C5-functionalization of LNA pyrimidines is indeed a viable approach for improving the binding affinity, target specificity, and/or enzymatic stability of LNA-modified ONs, whereas C8-functionalization of LNA adenosines is detrimental to binding affinity and specificity. These insights will impact the future design of conformationally restricted nucleotides for nucleic acid targeting applications.

  17. Thermal stability and conformation of antiparallel duplexes formed by P-stereodefined phosphorothioate DNA/LNA chimeric oligomers with DNA and RNA matrices.

    PubMed

    Jastrzębska, Katarzyna; Maciaszek, Anna; Dolot, Rafał; Bujacz, Grzegorz; Guga, Piotr

    2015-10-21

    3'-O-(2-Thio-4,4-pentamethylene-1,3,2-oxathiaphospholane) derivatives of LNA-type nucleosides (LNA-OTPs, 2a-d; B' = Thy, Ade(Bz), Cyt(Bz), Gua(dmf), respectively) were synthesized and separated into pure P-diastereomers. X-ray analysis allowed for assignment of the absolute configuration of the phosphorus atom in the detritylated, fast-eluting diastereomer 2a. The diastereomerically pure LNA-OTP monomers were used in solid phase synthesis of P-stereodefined chimeric PS-(DNA/LNA) 11-mers containing 2-3 LNA units. Formally, among the phosphorothioate oligomers the biggest enhancement in thermal stability of Watson-Crick paired duplexes was found for [SP-PS]-(DNA/LNA)/RNA duplexes (on average 8.2 °C per LNA nucleotide), followed by [RP-PS]-(DNA/LNA)/RNA (6.3 °C), [RP-PS]-(DNA/LNA)/DNA (3.8 °C) and [SP-PS]-(DNA/LNA)/DNA (2.4 °C per LNA nucleotide). However, detailed analysis of the thermal dissociation data showed that the thermal stability of (PS-LNA)-containing duplexes does not depend on the spatial orientation of the sulfur atoms. This conclusion received support from CD measurements.

  18. Limitations of TaqMan PCR for Detecting Divergent Viral Pathogens Illustrated by Hepatitis A, B, C, and E Viruses and Human Immunodeficiency Virus

    PubMed Central

    Gardner, Shea N.; Kuczmarski, Thomas A.; Vitalis, Elizabeth A.; Slezak, Tom R.

    2003-01-01

    Recent events illustrate the imperative to rapidly and accurately detect and identify pathogens during disease outbreaks, whether they are natural or engineered. Particularly for our primary goal of detecting bioterrorist releases, detection techniques must be both species-wide (capable of detecting all known strains of a given species) and species specific. Due to classification restrictions on the publication of data for species that may pose a bioterror threat, we illustrate the challenges of finding such assays using five nonthreat organisms that are nevertheless of public health concern: human immunodeficiency virus (HIV) and four species of hepatitis viruses. Fluorogenic probe-based PCR assays (TaqMan; Perkin-Elmer Corp., Applied Biosystems, Foster City, Calif.) may be sensitive, fast methods for the identification of species in which the genome is conserved among strains, such as hepatitis A virus. For species such as HIV, however, the strains are highly divergent. We use computational methods to show that nine TaqMan primer and probe sequences, or signatures, are needed to ensure that all strains will be detected, but this is an unfeasible number, considering the cost of TaqMan probes. Strains of hepatitis B, C, and E viruses show intermediate divergence, so that two to three TaqMan signatures are required to detect all strains of each virus. We conclude that for species such as hepatitis A virus with high levels of sequence conservation among strains, signatures can be found computationally for detection by the TaqMan assay, which is a sensitive, rapid, and cost-effective method. However, for species such as HIV with substantial genetic divergence among strains, the TaqMan assay becomes unfeasible and alternative detection methods may be required. We compare the TaqMan assay with some of the alternative nucleic acid-based detection techniques of microarray, chip, and bead technologies in terms of sensitivity, speed, and cost. PMID:12791858

  19. Development of a 2′,4′-BNA/LNA-based siRNA for Dyslipidemia and Assessment of the Effects of Its Chemical Modifications In Vivo

    PubMed Central

    Wada, Shunsuke; Obika, Satoshi; Shibata, Masa-Aki; Yamamoto, Tsuyoshi; Nakatani, Moeka; Yamaoka, Tetsuji; Torigoe, Hidetaka; Harada-Shiba, Mariko

    2012-01-01

    Recent advances in RNA interference (RNAi)-based drug development have partially allowed systemic administration of these agents in vivo with promising therapeutic effects. However, before chemically modified small-interfering RNAs (siRNAs) can be applied clinically, their in vivo effects should be thoroughly assessed. And while many studies have assessed the effects of chemically modified siRNAs in vitro, there has been no comprehensive assessment of their effects in vivo. Here, we aimed to elucidate the effects of administering chemically modified siRNAs in vivo and to propose a 2′,4′-bridged nucleic acid (BNA)/locked nucleic acid (LNA)-based siRNA candidate for dyslipidemia. A potentially therapeutic siRNA, siL2PT-1M, was modified with phosphorothioate (PS) and 2′,4′-BNA/LNA in its sense strand and with 2′-methoxy (2′-OMe) nucleotides in its immunostimulatory motif; administration of siL2PT-1M resulted in sustained reductions in serum total cholesterol (TC) (24 days) and a concomitant apolipoprotein B (apoB) mRNA reduction in liver without adverse effects. The 2′,4′-BNA/LNA modification in the sense strand was greatly augmented the duration of the RNAi effect, whereas cholesterol conjugation shortened the duration. Cholesterol-conjugated immunostimulatory siRNA (isRNA) induced higher serum interferon-α (IFN-α) levels than did nonmodified isRNA, indicating that the immune reaction was facilitated by cholesterol conjugation. Our results indicated that modification of the adenosine residues complementary to the immunostimulatory motif and of central 5′-UG-3′ in the sense strand would ameliorate the negative immune response. PMID:23344237

  20. Improved case confirmation in meningococcal disease with whole blood Taqman PCR

    PubMed Central

    Hackett, S; Carrol, E; Guiver, M; Marsh, J; Sills, J; Thomson, A; Kaczmarski, E; Hart, C

    2002-01-01

    Background: The clinical diagnosis of meningococcal disease (MCD) can be difficult. Non-culture methods like the previous ELISA meningococcal PCR improved case confirmation rates, but were not ideal. A Taqman meningococcal PCR, using DNA extracted from serum (S-Taqman), which has an improved sensitivity compared to the ELISA method in vitro, was introduced into clinical practice in July 1997. A new whole blood DNA extraction method for Taqman (WB-Taqman) was introduced in September 1999. Aims: To determine the degree of improvement in the confirmation rate in clinically diagnosed MCD, following the introduction of WB-Taqman. Methods: A total of 192 patients (WB-Taqman) with possible or probable MCD, including those admitted to our paediatric intensive care unit, were studied. Admission EDTA samples obtained were sent for bacterial DNA detection at the Meningococcal Reference Unit (MRU), Manchester. These patients were compared to 319 patients with possible and probable MCD, seen at the same hospital prior to the introduction of WB-Taqman. Results: Following the introduction of WB-Taqman, 82 of the 95 probable cases (88%) had a positive meningococcal PCR result. This gives a diagnostic sensitivity and specificity for WB-Taqman of 87% and 100% respectively. Following WB-Taqman all blood culture positive patients were also PCR positive. Confirmation of cases by PCR rose from 47% (S-Taqman, n = 166) to 88% (WB-Taqman). When all confirmatory tests were included, case confirmation increased from 72% (S-Taqman) to 94% (WB-Taqman). Conclusion: The sensitivity of PCR in confirming clinical MCD has improved significantly with this new method. The gold standard for confirming cases of MCD is now the WB-Taqman PCR. PMID:12023187

  1. Evaluation of Cobas TaqMan MTB for direct detection of the Mycobacterium tuberculosis complex in comparison with Cobas Amplicor MTB.

    PubMed

    Bloemberg, Guido V; Voit, Antje; Ritter, Claudia; Deggim, Vanessa; Böttger, Erik C

    2013-07-01

    The Roche Cobas Amplicor MTB assay, recently replaced by the Roche Cobas TaqMan MTB assay, was one of the first commercially available assays for detection of the Mycobacterium tuberculosis complex based on nucleic acid amplification. We reported previously on the limited specificity of the Cobas Amplicor MTB assay, in particular for positive samples with an optical density at 660 nm (OD660) of <2.0. Using a selected set of respiratory samples, which were scored as false positive by the Cobas Amplicor test, we demonstrate here that the specificity of the Cobas TaqMan assay is significantly improved. In addition, our study of a set of 133 clinical samples revealed that the Cobas TaqMan MTB assay showed significantly less PCR inhibition than the Cobas Amplicor test. An overall concordance of 98.2% was observed between the two assays. In a subsequent prospective study, we evaluated the performance of the Roche Cobas TaqMan MTB assay on 1,143 clinical specimens, including respiratory (n = 838) and nonrespiratory (n = 305) specimens. Using culture as the gold standard, we found a sensitivity of 88.4% and a specificity of 98.8% for the 838 respiratory specimens, compared to a sensitivity of 63.6% and a specificity of 94.6% for the 305 nonrespiratory specimens. We conclude that the Cobas TaqMan MTB assay is a significantly improved tool for the direct detection of M. tuberculosis DNA in clinical specimens.

  2. Development of TaqMan real-time reverse transcription-polymerase chain reaction for the detection and quantitation of porcine kobuvirus.

    PubMed

    Zhu, Xiangdong; Wang, Yufei; Chen, Jianfei; Zhang, Xin; Shi, Hongyan; Shi, Da; Gao, Jing; Feng, Li

    2016-08-01

    Porcine kobuvirus (PKV) is a newly emerging virus that has been detected in diarrheic pigs. Presently, reverse transcription-polymerase chain reaction (RT-PCR) and RT-loop-mediated amplification are the only methods that can be used to detect PKV. To develop a TaqMan real-time RT-PCR for the rapid detection and quantitation of PKV nucleic acid in fecal samples, a pair of primers and a probe were designed to amplify the conserved 3D region of the PKV genome. After optimization, the TaqMan real-time RT-PCR was highly specific and ∼1000 times more sensitive than conventional RT-PCR, and the detection limit was as low as 30 DNA copies. Among the 148 intestinal samples from piglets with diarrhea, 136 and 118 were positive based on the TaqMan and conventional RT-PCR methods, respectively, indicating that the TaqMan RT-PCR was more sensitive than conventional RT-PCR, and the total concordance of the two methods was approximately 87.84%. Thus, the TaqMan real-time RT-PCR should be a useful tool for the early detection and quantitation of PKV.

  3. Tiny Molecular Beacons: LNA/2'-O-methyl RNA Chimeric Probes for Imaging Dynamic mRNA Processes in Living Cells

    PubMed Central

    Catrina, Irina E.; Marras, Salvatore A.E.; Bratu, Diana P.

    2012-01-01

    New approaches for imaging dynamic processes involving RNAs in living cells are continuously being developed and optimized. The use of molecular beacons synthesized from 2'-O-methylribonucleotides (which are resistant to cellular nucleases) is an established approach for visualizing native mRNAs in real time. In order to spatially and temporally resolve dynamic steps involving RNA in cells, molecular beacons need to efficiently hybridize to their RNA targets. To expand the repertoire of target sites accessible to molecular beacons, we decreased the length of their probe sequences and altered their backbone by the inclusion of LNA (locked nucleic acid) nucleotides. We named these new LNA/2'-O-methyl RNA chimera oligonucleotides, “tiny molecular beacons”. We analyzed these tiny molecular beacons and found that the incorporation of just a few LNA nucleotides enables these shorter probes to stably anneal to more structured regions of the RNA than is possible with conventional molecular beacons. The ease of synthesis of tiny molecular beacons, and the flexibility to couple them to a large variety of fluorophores and quenchers, renders them optimal for the detection of less abundant and/or highly structured RNAs. To determine their efficiency to detect endogenous mRNAs in live specimens, we designed tiny molecular beacons that were specific for oskar mRNA and microinjected them into living Drosophila melanogaster oocytes. We then imaged the live oocytes via spinning disc confocal microscopy. The results demonstrate that tiny molecular beacons hybridize to target mRNA at faster rates than classically designed molecular beacons, and are able to access previously inaccessible target regions. PMID:22738327

  4. The impact of process variations on input impedance and mitigation using a circuit technique in FinFET-based LNA

    NASA Astrophysics Data System (ADS)

    Suresh, D.; Nagarajan, K. K.; Srinivasan, R.

    2015-04-01

    The effect of process variations of a FinFET-based low noise amplifier (LNA) are mitigated by using the device in an independently driven mode, i.e. an independently driven double gate (IDDG) FinFET. A 45 nm gate length IDDG FinFET-based cascoded LNA, operating at 5 GHz, is designed and studied to assess the impact of process variation on the LNA performance metrics such as input impedance, gain and noise figure. Four geometrical parameters, gate length, channel width, gate oxide thickness and fin width, and one non-geometrical parameter, channel doping concentration, are considered in the study. The effect of these variations on the input impedance (the desired value is 50 Ω purely real) of the LNA is compensated by the second gate bias of the IDDG FinFET. Project supported by the Defense Research Development Organization (DRDO), Government of India.

  5. Spatio-Temporal Variations of High and Low Nucleic Acid Content Bacteria in an Exorheic River

    PubMed Central

    Ma, Lili; Ji, Yurui; Bartlam, Mark; Wang, Yingying

    2016-01-01

    Bacteria with high nucleic acid (HNA) and low nucleic acid (LNA) content are commonly observed in aquatic environments. To date, limited knowledge is available on their temporal and spatial variations in freshwater environments. Here an investigation of HNA and LNA bacterial abundance and their flow cytometric characteristics was conducted in an exorheic river (Haihe River, Northern China) over a one year period covering September (autumn) 2011, December (winter) 2011, April (spring) 2012, and July (summer) 2012. The results showed that LNA and HNA bacteria contributed similarly to the total bacterial abundance on both the spatial and temporal scale. The variability of HNA on abundance, fluorescence intensity (FL1) and side scatter (SSC) were more sensitive to environmental factors than that of LNA bacteria. Meanwhile, the relative distance of SSC between HNA and LNA was more variable than that of FL1. Multivariate analysis further demonstrated that the influence of geographical distance (reflected by the salinity gradient along river to ocean) and temporal changes (as temperature variation due to seasonal succession) on the patterns of LNA and HNA were stronger than the effects of nutrient conditions. Furthermore, the results demonstrated that the distribution of LNA and HNA bacteria, including the abundance, FL1 and SSC, was controlled by different variables. The results suggested that LNA and HNA bacteria might play different ecological roles in the exorheic river. PMID:27082986

  6. Spatio-Temporal Variations of High and Low Nucleic Acid Content Bacteria in an Exorheic River.

    PubMed

    Liu, Jie; Hao, Zhenyu; Ma, Lili; Ji, Yurui; Bartlam, Mark; Wang, Yingying

    2016-01-01

    Bacteria with high nucleic acid (HNA) and low nucleic acid (LNA) content are commonly observed in aquatic environments. To date, limited knowledge is available on their temporal and spatial variations in freshwater environments. Here an investigation of HNA and LNA bacterial abundance and their flow cytometric characteristics was conducted in an exorheic river (Haihe River, Northern China) over a one year period covering September (autumn) 2011, December (winter) 2011, April (spring) 2012, and July (summer) 2012. The results showed that LNA and HNA bacteria contributed similarly to the total bacterial abundance on both the spatial and temporal scale. The variability of HNA on abundance, fluorescence intensity (FL1) and side scatter (SSC) were more sensitive to environmental factors than that of LNA bacteria. Meanwhile, the relative distance of SSC between HNA and LNA was more variable than that of FL1. Multivariate analysis further demonstrated that the influence of geographical distance (reflected by the salinity gradient along river to ocean) and temporal changes (as temperature variation due to seasonal succession) on the patterns of LNA and HNA were stronger than the effects of nutrient conditions. Furthermore, the results demonstrated that the distribution of LNA and HNA bacteria, including the abundance, FL1 and SSC, was controlled by different variables. The results suggested that LNA and HNA bacteria might play different ecological roles in the exorheic river.

  7. Development of TaqMan probe-based insulated isothermal PCR (iiPCR) for sensitive and specific on-site pathogen detection.

    PubMed

    Tsai, Yun-Long; Wang, Hwa-Tang Thomas; Chang, Hsiao-Fen Grace; Tsai, Chuan-Fu; Lin, Ching-Ko; Teng, Ping-Hua; Su, Chen; Jeng, Chien-Chung; Lee, Pei-Yu

    2012-01-01

    Insulated isothermal PCR (iiPCR), established on the basis of Ralyeigh-Bénard convection, is a rapid and low-cost platform for nucleic acid amplification. However, the method used for signal detection, namely gel electrophoresis, has limited the application of iiPCR. In this study, TaqMan probe-based iiPCR system was developed to obviate the need of post-amplification processing. This system includes an optical detection module, which was designed and integrated into the iiPCR device to detect fluorescent signals generated by the probe. TaqMan probe-iiPCR assays targeting white spot syndrome virus (WSSV) and infectious myonecrosis virus were developed for preliminary evaluation of this system. Significant elevation of fluorescent signals was detected consistently among positive iiPCR reactions in both assays, correlating with amplicon detection by gel electrophoresis analysis. After condition optimization, a threshold value of S/N (fluorescent intensity(after)/fluorescent intensity(before)) for positive reactions was defined for WSSV TaqMan probe-iiPCR on the basis of 20 blank reactions. WSSV TaqMan probe-iiPCR generated positive S/Ns from as low as 10(1) copies of standard DNA and lightly infected Litopenaeus vannamei. Compared with an OIE-certified nested PCR, WSSV TaqMan probe-iiPCR showed a sensitivity of 100% and a specificity of 96.67% in 120 WSSV-free or lightly infected shrimp samples. Generating positive signals specifically and sensitively, TaqMan probe-iiPCR system has a potential as a low-cost and rapid on-site diagnostics method.

  8. Comparison of AdvanSure TB/NTM PCR and COBAS TaqMan MTB PCR for Detection of Mycobacterium tuberculosis Complex in Routine Clinical Practice.

    PubMed

    Cho, Won-Hyung; Won, Eun-Jeong; Choi, Hyun-Jung; Kee, Seung-Jung; Shin, Jong-Hee; Ryang, Dong-Wook; Suh, Soon-Pal

    2015-05-01

    The AdvanSure tuberculosis/non-tuberculous mycobacterium (TB/NTM) PCR (LG Life Science, Korea) and COBAS TaqMan Mycobacterium tuberculosis (MTB) PCR (Roche Diagnostics, USA) are commonly used in clinical microbiology laboratories. We aimed to evaluate these two commercial real-time PCR assays for detection of MTB in a large set of clinical samples over a two-year period. AdvanSure TB/NTM PCR and COBAS TaqMan MTB PCR were performed on 9,119 (75.2%) and 3,010 (24.8%) of 12,129 (9,728 respiratory and 2,401 non-respiratory) MTB specimens, with 361 (4.0%) and 102 (3.4%) acid-fast bacilli (AFB)-positive results, respectively. In MTB culture, 788 (6.5%) MTB and 514 (4.2%) NTM were identified. The total sensitivity and specificity of the AdvanSure assay were 67.8% (95% confidence interval [CI], 63.9-71.6) and 98.3% (95% CI, 98.0-98.6), while those of the COBAS TaqMan assay were 67.2% (95% CI, 60.0-73.8) and 98.4% (95% CI, 97.9-98.9), respectively. The sensitivities and specificities of the AdvanSure and COBAS TaqMan assays for AFB-positive and AFB-negative samples were comparable. Furthermore, the AdvanSure assay showed fewer invalid results compared with the COBAS TaqMan assay (5.0 vs. 20.4 invalid results/1,000 tests, P<0.001). AdvanSure assay represents a comparable yet more reliable method than COBAS TaqMan for the identification of mycobacteria in routine clinical microbiology.

  9. Detection of BRAF, NRAS, KIT, GNAQ, GNA11 and MAP2K1/2 mutations in Russian melanoma patients using LNA PCR clamp and biochip analysis

    PubMed Central

    Emelyanova, Marina; Ghukasyan, Lilit; Abramov, Ivan; Ryabaya, Oxana; Stepanova, Evgenia; Kudryavtseva, Anna; Sadritdinova, Asiya; Dzhumakova, Cholpon; Belysheva, Tatiana; Surzhikov, Sergey; Lyubchenko, Lyudmila; Zasedatelev, Alexander; Nasedkina, Tatiana

    2017-01-01

    Target inhibitors are used for melanoma treatment, and their effectiveness depends on the tumor genotype. We developed a diagnostic biochip for the detection of 39 clinically relevant somatic mutations in the BRAF, NRAS, KIT, GNAQ, GNA11, MAP2K1 and MAP2K2 genes. We used multiplex locked nucleic acid (LNA) PCR clamp for the preferable amplification of mutated over wild type DNA. The amplified fragments were labeled via the incorporation of fluorescently labeled dUTP during PCR and were hybridized with specific oligonucleotides immobilized on a biochip. This approach could detect 0.5% of mutated DNA in the sample analyzed. The method was validated on 253 clinical samples and six melanoma cell lines. Among 253 melanomas, 129 (51.0%) BRAF, 45 (17.8%) NRAS, 6 (2.4%) KIT, 4 (1.6%) GNAQ, 2 (0.8%) GNA11, 2 (0.8%) MAP2K1 and no MAP2K2 gene mutations were detected by the biochip assay. The results were compared with Sanger sequencing, next generation sequencing and ARMS/Scorpion real-time PCR. The specimens with discordant results were subjected to LNA PCR clamp followed by sequencing. The results of this analysis were predominantly identical to the results obtained by the biochip assay. Infrequently, we identified rare somatic mutations. In the present study we demonstrate that the biochip-based assay can effectively detect somatic mutations in approximately 70% of melanoma patients, who may require specific targeted therapy. PMID:28881731

  10. Detection of BRAF, NRAS, KIT, GNAQ, GNA11 and MAP2K1/2 mutations in Russian melanoma patients using LNA PCR clamp and biochip analysis.

    PubMed

    Emelyanova, Marina; Ghukasyan, Lilit; Abramov, Ivan; Ryabaya, Oxana; Stepanova, Evgenia; Kudryavtseva, Anna; Sadritdinova, Asiya; Dzhumakova, Cholpon; Belysheva, Tatiana; Surzhikov, Sergey; Lyubchenko, Lyudmila; Zasedatelev, Alexander; Nasedkina, Tatiana

    2017-08-08

    Target inhibitors are used for melanoma treatment, and their effectiveness depends on the tumor genotype. We developed a diagnostic biochip for the detection of 39 clinically relevant somatic mutations in the BRAF, NRAS, KIT, GNAQ, GNA11, MAP2K1 and MAP2K2 genes. We used multiplex locked nucleic acid (LNA) PCR clamp for the preferable amplification of mutated over wild type DNA. The amplified fragments were labeled via the incorporation of fluorescently labeled dUTP during PCR and were hybridized with specific oligonucleotides immobilized on a biochip. This approach could detect 0.5% of mutated DNA in the sample analyzed. The method was validated on 253 clinical samples and six melanoma cell lines. Among 253 melanomas, 129 (51.0%) BRAF, 45 (17.8%) NRAS, 6 (2.4%) KIT, 4 (1.6%) GNAQ, 2 (0.8%) GNA11, 2 (0.8%) MAP2K1 and no MAP2K2 gene mutations were detected by the biochip assay. The results were compared with Sanger sequencing, next generation sequencing and ARMS/Scorpion real-time PCR. The specimens with discordant results were subjected to LNA PCR clamp followed by sequencing. The results of this analysis were predominantly identical to the results obtained by the biochip assay. Infrequently, we identified rare somatic mutations. In the present study we demonstrate that the biochip-based assay can effectively detect somatic mutations in approximately 70% of melanoma patients, who may require specific targeted therapy.

  11. Nd:LNA laser optical pumping of He-4 - Application to space magnetometers

    NASA Technical Reports Server (NTRS)

    Slocum, R. E.; Schearer, L. D.; Tin, P.; Marquedant, R.

    1988-01-01

    Results obtained from laser pumping in a helium magnetometer sensor, using a tunable Nd:LNA laser pumped with a high-power diode laser, are reported. It is shown that it was possible to observe both the Hanle signals and the n = 0, p = 1 parametric resonance by monitoring the pumping radiation passing through the cell. As the diode laser-pumped Nd:LNA laser was tuned through the D0, D1, and D2 transitions, three distinct resonance signals were produced. A comparison of the slope of lamp-pumped signals and laser-pumped D1 signals showed that, under otherwise identical conditions, the slope of the D1 laser signal was 45 times greater than the lamp-pumped signal.

  12. Nd:LNA laser optical pumping of He-4 - Application to space magnetometers

    NASA Astrophysics Data System (ADS)

    Slocum, R. E.; Schearer, L. D.; Tin, P.; Marquedant, R.

    1988-12-01

    Results obtained from laser pumping in a helium magnetometer sensor, using a tunable Nd:LNA laser pumped with a high-power diode laser, are reported. It is shown that it was possible to observe both the Hanle signals and the n = 0, p = 1 parametric resonance by monitoring the pumping radiation passing through the cell. As the diode laser-pumped Nd:LNA laser was tuned through the D0, D1, and D2 transitions, three distinct resonance signals were produced. A comparison of the slope of lamp-pumped signals and laser-pumped D1 signals showed that, under otherwise identical conditions, the slope of the D1 laser signal was 45 times greater than the lamp-pumped signal.

  13. A Novel Current-Mode Differential Transconductance LNA for IEEE 802.11a Application

    NASA Astrophysics Data System (ADS)

    Tu, Yuxiang; Wang, Chunhua; Chen, Lei; Wu, Zhangbin; Zhang, Guangxiang

    2012-03-01

    In this paper, a novel current-mode differential transconductance low noise amplifier (LNA) designed in the chartered 0.18 μm CMOS technology is proposed to realize the receiver front-end. The proposed LNA frequency ranges from 4.7 GHz to 6 GHz,mainly targeting at IEEE802.11a application. It utilizes two stage structure, with a PMOS current-mirrorr using inductive series peaking technique to achieve optimized current output. The noise performance is improved through the use of a gm-boosting technique, while the gain performance is improved and power consumption is saved by using current-reused technique. Measured results demonstrate that the circuit provides flat transconductance gain higher than 17.0 dB, noise figure lower than 2.31 dB and low operating voltage of 0.85 V in frequency band. A comparison with other LNAs in similar and nearby frequency bands shows the proposed LNA has advantages of higher gain, lower noise figure and better other performances.

  14. Electronic coupling between base pair dimers of LNA:DNA oligomers.

    PubMed

    Ivanova, Anela; Jezierski, Grzegorz; Rösch, Notker

    2008-01-21

    We calculated ab initio electronic coupling elements between neighboring base-pair dimers in a set of LNA:DNA oligomers with different numbers of locked nucleotides and compared them by averaging the values over ensembles of snapshots from molecular dynamics trajectories. Averaging was based on coupling elements for various ensembles comprising of 33,000 structures. The known pronounced variations of coupling elements on the nanosecond timescale due to thermal fluctuations of the DNA structure were confirmed. We found significant differences in electronic coupling at the dimer level between a non-modified DNA:DNA duplex and the corresponding duplex containing one fully LNA-substituted strand. We rationalized these differences by very dissimilar overlap in the pi-stack as a consequence of the LNA-modified system approximating an A-DNA-type helix. The calculated coupling elements for the non-modified reference duplex were similar to those of standard B-DNA and those for the fully modified oligomer resembled the matrix elements estimated for standard A-DNA.

  15. Selection of LNA-containing DNA aptamers against recombinant human CD73.

    PubMed

    Elle, Ida C; Karlsen, Kasper K; Terp, Mikkel G; Larsen, Niels; Nielsen, Ronni; Derbyshire, Nicola; Mandrup, Susanne; Ditzel, Henrik J; Wengel, Jesper

    2015-05-01

    LNA-containing DNA aptamers against CD73 (human ecto-5'-nucleotidase), a protein frequently overexpressed in solid tumours, were isolated by SELEX. A pre-defined stem-loop library, containing LNA in the forward primer region, was enriched with CD73 binding sequences through six rounds of SELEX with recombinant his-tagged CD73 immobilised on anti-his plates. Enriched pools isolated from rounds one, three and six were subjected to next-generation sequencing and analysed for enrichment using custom bioinformatics software. The software identified aptamer sequences via the primers and then performed several steps including sequence unification, clustering and alignment to identify enriched sequences. Three enriched sequences were synthesised for further analysis, two of which showed sequence similarities. These sequences exhibited binding to the recombinant CD73 with KD values of 10 nM and 3.5 nM when tested by surface plasmon resonance. Truncated variants of these aptamers and variants where the LNA nucleotides were substituted for the DNA equivalent also exhibited affinity for the recombinant CD73 in the low nanomolar range. In enzyme inhibition assays with recombinant CD73 the aptamer sequences were able to decrease the activity of the protein. However, the aptamers exhibited no binding to cellular CD73 by flow cytometry analysis likely since the epitope recognised by the aptamer was not available for binding on the cellular protein.

  16. Towards Fluorescence In Vivo Hybridization (FIVH) Detection of H. pylori in Gastric Mucosa Using Advanced LNA Probes

    PubMed Central

    Fontenete, Sílvia; Leite, Marina; Guimarães, Nuno; Madureira, Pedro; Ferreira, Rui Manuel; Figueiredo, Céu; Wengel, Jesper; Azevedo, Nuno Filipe

    2015-01-01

    In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH) is a commonly used technique to detect H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo FISH-based method (FIVH) that directly detects and allows the visualization of the bacterium within the human body would significantly reduce the time of analysis, allowing the diagnosis to be performed during endoscopy. In a previous study we designed and synthesized a phosphorothioate locked nucleic acid (LNA)/ 2’ O-methyl RNA (2’OMe) probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization step now takes only 30 minutes and, in addition to the buffer, uses only urea and probe at non-toxic concentrations. Importantly, the sensitivity and specificity of the FISH method was maintained in the range of conditions tested, even at low stringency conditions (e.g., low pH). In conclusion, this methodology is a promising approach that might be used in vivo in the future in combination with a confocal laser endomicroscope for H. pylori visualization. PMID:25915865

  17. 0.18 um 3.1-10.6 GHz CMOS UWB LNA with 25±1dB gain

    NASA Astrophysics Data System (ADS)

    Shamsadini, Shila; Kashani, Farokh Hojat; Bathaei, Neda

    2011-05-01

    A 3.1-10.6 GHz ultra-wideband low noise-amplifier (UWB LNA) using standard 0.18 um CMOS technology which is applicable in telecommunication has been reported. A two-stage, common-gate in cascade with cascode, UWB LNA has been proposed to achieve more than 10 dB input and output return loss, maximum gain of 26 dB, and NF of 2.9 dB over the full frequency band. The proposed LNA consumes 10 mW from 1.8 V supply. The designed LNA has an excellent high gain in comparison with previous works.

  18. Polymorphism of linoleic acid (cis-9, cis-12-octadecadienoic acid) and alpha-linolenic acid (cis-9, cis-12, cis-15-octadecatrienoic acid).

    PubMed

    Ueno, S; Miyazaki, A; Yano, J; Furukawa, Y; Suzuki, M; Sato, K

    2000-10-01

    Crystallization and polymorphic properties of linoleic acid (cis-9, cis-12-Octadecadienoic acid) (LA) and alpha-linolenic acid (cis-9, cis-12, cis-15-Octadecatrienoic acid) (alpha-LNA) have been studied by optical microscopy, differential scanning calorimetry (DSC) and X-ray diffraction (XRD). The DSC analyses presented three polymorphs in LA, and two polymorphs in alpha-LNA. The XRD patterns of the higher- and lower-temperature forms in LA and alpha-LNA showed orthorhombic O'(//)+O-like and O'(//) subcell, which were similar to those of alpha- and gamma-forms of mono-unsaturated fatty acids, respectively. From the solvent crystallization of LA and alpha-LNA in acetonitrile, single crystals of the higher temperature polymorphs have been obtained. The crystal habits of truncated rhombic shape were also similar to those of alpha-forms of the mono-unsaturated fatty acids. The enthalpy and entropy values of fusion and dissolution of the alpha-forms of LA, alpha-LNA and oleic acid showed that the two values decreased with increasing number of the cis-double bond.

  19. Scaffolding along nucleic acid duplexes using 2'-amino-locked nucleic acids.

    PubMed

    Astakhova, I Kira; Wengel, Jesper

    2014-06-17

    CONSPECTUS: Incorporation of chemically modified nucleotide scaffolds into nucleic acids to form assemblies rich in function is an innovative area with great promise for nanotechnology and biomedical and material science applications. The intrinsic biorecognition potential of nucleic acids combined with advanced properties of the locked nucleic acids (LNAs) provide opportunities to develop new nanomaterials and devices like sensors, aptamers, and machines. In this Account, we describe recent research on preparation and investigation of the properties of LNA/DNA hybrids containing functionalized 2'-amino-LNA nucleotides. By application of different chemical reactions, modification of 2'-amino-LNA scaffolds can be efficiently performed in high yields and with various tags, postsynthetically or during the automated oligonucleotide synthesis. The choice of a synthetic method for scaffolding along 2'-amino-LNA mainly depends on the chemical nature of the modification, its price, its availability, and applications of the product. One of the most useful applications of the product LNA/DNA scaffolds containing 2'-amino-LNA is to detect complementary DNA and RNA targets. Examples of these applications include sensing of clinically important single-nucleotide polymorphisms (SNPs) and imaging of nucleic acids in vitro, in cell culture, and in vivo. According to our studies, 2'-amino-LNA scaffolds are efficient within diagnostic probes for DNA and RNA targets and as therapeutics, whereas both 2'-amino- and isomeric 2'-α-l-amino-LNA scaffolds have promising properties for stabilization and detection of DNA nanostructures. Attachment of fluorescent groups to the 2'-amino group results in very high fluorescent quantum yields of the duplexes and remarkable sensitivity of the fluorescence signal to target binding. Notably, fluorescent LNA/DNA probes bind nucleic acid targets with advantages of high affinity and specificity. Thus, molecular motion of nanodevices and programmable

  20. Effect of Locked-Nucleic Acid on a Biologically Active G-Quadruplex. A Structure-Activity Relationship of the Thrombin Aptamer

    PubMed Central

    Bonifacio, Laura; Church, Frank C.; Jarstfer, Michael B.

    2008-01-01

    Here we tested the ability to augment the biological activity of the thrombin aptamer, d(GGTTGGTGTGGTTGG), by using locked nucleic acid (LNA) to influence its G-quadruplex structure. Compared to un-substituted control aptamer, LNA-containing aptamers displayed varying degrees of thrombin inhibition. Aptamers with LNA substituted in either positions G5, T7, or G8 showed decreased thrombin inhibition, whereas LNA at position G2 displayed activity comparable to un-substituted control aptamer. Interestingly, the thermal stability of the substituted aptamers does not correlate to activity – the more stable aptamers with LNA in position G5, T7, or G8 showed the least thrombin inhibition, while a less stable aptamer with LNA at G2 was as active as the un-substituted aptamer. These results suggest that LNA substitution at sites G5, T7, and G8 directly perturbs aptamer-thrombin affinity. This further implies that for the thrombin aptamer, activity is not dictated solely by the stability of the G-quadruplex structure, but by specific interactions between the central TGT loop and thrombin and that LNA can be tolerated in a biologically active nucleic acid structure albeit in a position dependent fashion. PMID:19325759

  1. A broadband 47-67 GHz LNA with 17.3 dB gain in 65-nm CMOS

    NASA Astrophysics Data System (ADS)

    Chong, Wang; Zhiqun, Li; Qin, Li; Yang, Liu; Zhigong, Wang

    2015-10-01

    A broadband 47-67 GHz low noise amplifier (LNA) with 17.3 dB gain in 65-nm CMOS technology is proposed. The features of millimeter wave circuits are illustrated first and design methodologies are discussed. The wideband input matching of the LNA was achieved by source inductive degeneration, which is narrowband in the low-GHz range but wideband at millimeter-wave frequencies due to the existence of gate-drain capacitance, Cgd. In order to minimize the noise figure (NF), the LNA used a common-source (CS) structure rather than cascode in the first stage, and the noise matching principle is explored. The last two stages of the LNA used a cascode structure to increase the power gain. Analysis of the gain boost effect of the gate inductor at the common-gate (CG) transistor is also performed. T-shape matching networks between stages are intended to enlarge the bandwidth. All on-chip inductors and transmission lines are modeled and simulated with a 3-dimensional electromagnetic (EM) simulation tool to guarantee the success of the design. Measurement results show that the LNA achieves a maximum gain of 17.3 dB at 60 GHz, while the 3-dB bandwidth is 20 GHz (47-67 GHz), including the interested band of 59-64 GHz, and the minimum noise figure is 4.9 dB at 62 GHz. The LNA absorbs a current of 19 mA from a 1.2 V supply and the chip occupies an area of 900 × 550 μm2 including pads. Project supported by the National High Technology Research and Development Program of China (No. 2011AA010202).

  2. A Differential CMOS Common-Gate LNA Linearized by Cross-Coupled Post Distortion Technique

    NASA Astrophysics Data System (ADS)

    Guo, Benqing; Yang, Guomin; Bin, Xiexian

    2014-05-01

    A linearized differential common-gate CMOS low noise amplifier is proposed. The linearity is improved by a cross-coupled post distortion technique, employing auxiliary PMOS transistors in weak inversion region to cancel the third-order nonlinear currents of common-gate LNA and impair the second-order nonlinear currents of that. The negative conductance characteristic of cross-coupled auxiliary PMOS transistors improves the gain while the resulted NF is little affected. Furthermore, noise contribution and linearity deterioration from the cascode stage is eliminated by an inductor resonating with the parasitic capacitance observed at the source net of the cascode transistor. The LNA implemented in a 0.18 μm CMOS technology demonstrates that IIP3 and gain have about 8.2 dB and 1.4 dB improvements in the designed frequency band, respectively. The noise figure of 3.4 dB is obtained with a power dissipation of 6.8 mW under a 1.8 V power supply.

  3. Adsorption and surface dynamics of short DNA and LNA oligonucleotides on single-crystal Au(1 1 1) electrode surfaces

    NASA Astrophysics Data System (ADS)

    Wackerbarth, Hainer; Grubb, Mikala; Wengel, Jesper; Chorkendorff, Ib; Ulstrup, Jens

    2006-05-01

    We have studied the surface dynamics of a double-strand decanucleotide (HS-10AT L) with 10 adenine-thymine base pairs linked to a Au(1 1 1)-electrode surface via a hexamethylene thiol linker. The study is based on a combination of voltammetry, interfacial capacitance data, electrochemical in situ scanning tunnelling microscopy, and X-ray photoelectron spectroscopy. The thymine bases of the oligonucleotide are connected to furanoses locked in a C3'- endo configuration called LNA (locked nucleic acid). Hybridization in solution is effected prior to linking to the Au(1 1 1)-surface. The melting point of the linker-free locked decanucleotide, 10AT L is >63 °C. However, voltammetric reductive desorption of the adsorbed thiol-modified double-strand decanucleotide, HS-10AT L, gives almost the same charge as single-strand HS-10A, 29 ± 3 and 27 ± 5 μC cm -2, respectively. In situ STM after HS-10AT L-immobilization also gives images showing highly ordered domains, virtually indistinguishable from those of immobilized HS-10A. X-ray photoelectron spectroscopy gives an N/P ration of 5.0 for HS-10AT L in line with the expected value for single-strand HS-10A (5.0). All three sets of data suggest that HS-10AT L hybridized in solution is significantly dissociated on binding to the Au(1 1 1)-electrode surface. This points to an adsorption mechanism in which a stable high density of Au-S bonds is achieved but at the expense of significant unzipping of the more voluminous duplex form.

  4. Inhibition of Gastric Tumor Cell Growth Using Seed-targeting LNA as Specific, Long-lasting MicroRNA Inhibitors

    PubMed Central

    Staedel, Cathy; Varon, Christine; Nguyen, Phu Hung; Vialet, Brune; Chambonnier, Lucie; Rousseau, Benoît; Soubeyran, Isabelle; Evrard, Serge; Couillaud, Franck; Darfeuille, Fabien

    2015-01-01

    MicroRNAs regulate eukaryotic gene expression upon pairing onto target mRNAs. This targeting is influenced by the complementarity between the microRNA “seed” sequence at its 5′ end and the seed-matching sequences in the mRNA. Here, we assess the efficiency and specificity of 8-mer locked nucleic acid (LNA)-modified oligonucleotides raised against the seeds of miR-372 and miR-373, two embryonic stem cell-specific microRNAs prominently expressed in the human gastric adenocarcinoma AGS cell line. Provided that the pairing is perfect over all the eight nucleotides of the seed and starts at nucleotide 2 or 1 at the microRNA 5′ end, these short LNAs inhibit miR-372/373 functions and derepress their common target, the cell cycle regulator LATS2. They decrease cell proliferation in vitro upon either transfection at nanomolar concentrations or unassisted delivery at micromolar concentrations. Subcutaneously delivered LNAs reduce tumor growth of AGS xenografts in mice, upon formation of a stable, specific heteroduplex with the targeted miR-372 and -373 and LATS2 upregulation. Their therapeutic potential is confirmed in fast-growing, miR-372-positive, primary human gastric adenocarcinoma xenografts in mice. Thus, microRNA silencing by 8-mer seed-targeting LNAs appears a valuable approach for both loss-of-function studies aimed at elucidating microRNA functions and for microRNA-based therapeutic strategies. PMID:26151747

  5. Enzyme-amplified electrochemical hybridization assay based on PNA, LNA and DNA probe-modified micro-magnetic beads.

    PubMed

    Laschi, Serena; Palchetti, Ilaria; Marrazza, Giovanna; Mascini, Marco

    2009-09-01

    In the present study, we investigated the properties of PNA and LNA capture probes in the development of an electrochemical hybridization assay. Streptavidin-coated paramagnetic micro-beads were used as a solid phase to immobilize biotinylated DNA, PNA and LNA capture probes, respectively. The target sequence was then recognized via hybridization with the capture probe. After labeling the biotinylated hybrid with a streptavidin-enzyme conjugate, the electrochemical detection of the enzymatic product was performed onto the surface of a disposable electrode. The assay was applied to the analytical detection of biotinylated DNA as well as RNA sequences. Detection limits, calculated considering the slope of the linear portion of the calibration curve in the range 0-2 nM were found to be 152, 118 and 91 pM, coupled with a reproducibility of the analysis equal to 5, 9 and 6%, calculated as RSD%, for DNA, PNA and LNA probes respectively, using the DNA target. In the case of the RNA target, the detection limits were found to be 51, 60 and 78 pM for DNA, PNA and LNA probes respectively.

  6. Comparison of TaqMan and Epoch Dark Quenchers during real-time reverse transcription PCR.

    PubMed

    Daum, Luke T; Ye, Keying; Chambers, James P; Santiago, Jose; Hickman, John R; Barnes, William J; Kruzelock, Russell P; Atchley, Daniel H

    2004-06-01

    Several biotechnology companies have recently introduced novel quencher fluors for use with dual-labeled fluorogenic hydrolysis probes. The Epoch Dark Quencher trade mark fluorochrome consists of a non-fluorescent moiety capable of absorption at higher wavelengths (400-650 nm). The aim of this study was to: (1) evaluate the feasibility of using Epoch Dark Quencher fluorochromes in real-time PCR pathogen detection assays that were previously optimized with TaqMan (TAMRA) quenching fluors, and (2) compare the sensitivity based on cycle threshold (CT) between probes containing either TaqMan or Epoch Dark Quencher fluors. Our data indicate Epoch Dark Quencher probes can be used in place of TaqMan probes and their performance was not better than traditional TaqMan (TAMRA) quenchers. Marginal differences observed between quenching fluorochromes may arise from concentration differences during probe synthesis.

  7. Development and evaluation of TaqMan real-time PCR assay for detection of beak and feather disease virus.

    PubMed

    Černíková, Lenka; Vitásková, Eliška; Nagy, Alexander

    2017-03-02

    Psittacine beak and feather disease (PBFD) is one of the most significant viral diseases in psittacine birds. The aim of the presented study was to develop a highly specific and sensitive TaqMan real-time PCR assay for universal detection of beak and feather disease virus (BFDV). Primers and a hydrolysis probe were selected on the highly conserved regions belonging to the ORF1 of the BFDV genome which were identified by aligning 814 genomic sequences downloaded from the GenBank database. The evaluation of the reaction parameters suggested a reaction efficiency of 97.1%, with consistent detection of 10(1) virus copies/μl of nucleic acid extract. The low values of standard deviation and coefficient of variation indicate a high degree of reproducibility and repeatability. The diagnostic applicability of the assay was proven on 36 BFDV positive and 107 negative specimens of psittacine origin representing 28 species. The assay showed a 100% ability to detect distinct genetic variants of the virus. Our data suggest that the presented TaqMan real-time PCR represents a specific, sensitive and reliable assay facilitating the molecular detection of BFDV.

  8. Design of a Low Voltage CMOS LNA at 2 GHz with Substrate-Bias

    NASA Astrophysics Data System (ADS)

    Wan Muhamad Hatta, S. F.; Soin, N.

    2008-11-01

    A low-voltage (1.5V), 2 GHz cascode CMOS low noise amplifier (LNA) has been designed and simulated using Silvaco's SMARTSPICE RF. The proposed design employs substrate bias of 0.5V and utilizes inductive source degeneration. This paper further presents an analysis on the effect of substrate bias on the MOSFET's threshold voltage as well as the transconductance. The simulated power gain (S21) is of 5.2 dB and a noise figure (NF) of 2.2975 dB is achieved at the operating frequency of 2 GHz. The Input Referred 1dB Compression Point (P1dB) and the third-order intercept point (IP3) are -12.891 dB and -1.6844 dB respectively.

  9. Synthesis of 6'-branched locked nucleic acid by a radical TEMPO-scavanged stereoselective mercury cyclization.

    PubMed

    Enderlin, Gerald; Nielsen, Poul

    2008-09-05

    A 6'(R)-hydroxymethyl derivative of the locked nucleic acid (LNA)-thymidine monomer has been synthesized by a stereoselective mercury cyclization and subsequent use of TEMPO as a radical scavenger. This compound was converted to an azide derivative, which in a Huisgen-type [3 + 2] cycloaddition afforded a double-headed nucleoside with a triazole linking an additional thymine to the 6'-position of the LNA-nucleoside monomer.

  10. H5N1 Oseltamivir-resistance detection by real-time PCR using two high sensitivity labeled TaqMan probes.

    PubMed

    Chutinimitkul, Salin; Suwannakarn, Kamol; Chieochansin, Thaweesak; Mai, Le Quynh; Damrongwatanapokin, Sudarat; Chaisingh, Arunee; Amonsin, Alongkorn; Landt, Olfert; Songserm, Thaweesak; Theamboonlers, Apiradee; Poovorawan, Yong

    2007-01-01

    A single amino acid substitution, from histidine to tyrosine at position 274 of the neuraminidase gene has converted Oseltamivir sensitive H5N1 influenza A virus into a resistant strain. Currently, Oseltamivir is being stockpiled in many countries potentially affected by the influenza A virus subtype H5N1 epidemic. To identify this change in Oseltamivir-treated patients, a method based on real-time PCR using two labeled TaqMan probes was developed for its rapid detection. In order to validate the method, Oseltamivir specimen from treated (Oseltamivir-resistant strain from a Vietnamese patient, two Oseltamivir-treated tigers) and untreated subjects have been used for this study. The results thus obtained as well as those derived from clone selection and sequencing showed that TaqMan probes could clearly discriminate wild type H274 from the mutant 274Y variant. The sensitivity of this assay was as low as 10 copies/microl and allowed the detection of the mutation in a mixture of wild type and mutant. Overall, the assay based on real-time PCR with two labeled TaqMan probes described here should be useful for detecting Oseltamivir-resistant H274Y H5N1 influenza A virus in many species and various sources of specimens with high sensitivity and specificity. Such studies can address potential differences in the diagnostic outcomes between patients who develop detectable Oseltamivir resistance and those who retain only the wild type strain of H5N1.

  11. Current Status and Perspectives Regarding LNA-anti-miR Oligonucleotides and microRNA miR-21 Inhibitors as a Potential Therapeutic Option in Treatment of Colorectal Cancer.

    PubMed

    Nedaeinia, Reza; Avan, Amir; Ahmadian, Mehdi; Nedaee Nia, Sasan; Ranjbar, Maryam; Sharifi, Mohammadreza; Goli, Mohammad; Piroozmand, Ahmad; Nourmohammadi, Esmail; Manian, Mostafa; Ferns, Gordon A; Ghayour-Mobarhan, Majid; Salehi, Rasoul

    2017-04-12

    Colorectal cancer (CRC) is among the leading causes of cancer-related death, principally due to its metastatic spread and multifactorial chemoresistance. The therapeutic failure can also be explained by inter- or intra-tumor genetic heterogeneity and tumor stromal content. Thus, the identification of novel prognostic biomarkers and therapeutic options are warranted in the management of CRC patients. There are data showing that microRNA-21 is elevated in different types of cancer, particularly colon adenocarcinoma and that this is association with a poor prognosis. This suggests that microRNA-21 may be of value as a potential therapeutic target. Furthermore, locked nucleic acid (LNA)-modified oligonucleotides have recently emerged as a therapeutic option for targeting dysregulated miRNAs in cancer therapy, through antisense-based gene silencing. Further work is required to identify innovative anticancer drugs that improve the current therapy either through novel combinatorial approaches or with better efficacy than conventional drugs. We aimed to provide an overview of the preclinical and clinical studies targeting key dysregulated signaling pathways in CRC as well as the therapeutic application of LNA-modified oligonucleotides and miR inhibitors in the treatment of CRC patients. This article is protected by copyright. All rights reserved.

  12. Quantification of low-expressed mRNA using 5' LNA-containing real-time PCR primers

    SciTech Connect

    Malgoyre, A.; Banzet, S.; Mouret, C.; Bigard, A.X.; Peinnequin, A. . E-mail: andrepeinnequin@crssa.net

    2007-03-02

    Real-time RT-PCR is the most sensitive and accurate method for mRNA quantification. Using specific recombinant DNA as a template, real-time PCR allows accurate quantification within a 7-log range and increased sensitivity below 10 copies. However, when using RT-PCR to quantify mRNA in biological samples, a stochastic off-targeted amplification can occur. Classical adjustments of assay parameters have minimal effects on such amplification. This undesirable amplification appears mostly to be dependent on specific to non-specific target ratio rather than on the absolute quantity of the specific target. This drawback, which decreases assay reliability, mostly appears when quantifying low-expressed transcript in a whole organ. An original primer design using properties of LNA allows to block off-target amplification. 5'-LNA substitution strengthens 5'-hybridization. Consequently on-target hybridization is stabilized and the probability for the off-target to lead to amplification is decreased.

  13. A High Linoleic Acid Diet does not Induce Inflammation in Mouse Liver or Adipose Tissue.

    PubMed

    Vaughan, Roger A; Garrison, Richard L; Stamatikos, Alexis D; Kang, Minsung; Cooper, Jamie A; Paton, Chad M

    2015-11-01

    Recently, the pro-inflammatory effects of linoleic acid (LNA) have been re-examined. It is now becoming clear that relatively few studies have adequately assessed the effects of LNA, independent of obesity. The purpose of this work was to compare the effects of several fat-enriched but non-obesigenic diets on inflammation to provide a more accurate assessment of LNA's ability to induce inflammation. Specifically, 8-week-old male C57Bl/6 mice were fed either saturated (SFA), monounsaturated (MUFA), LNA, or alpha-linolenic acid enriched diets (50 % Kcal from fat, 22 % wt/wt) for 4 weeks. Chow and high-fat, hyper-caloric diets were used as negative and positive controls, respectively. Expression of pro-inflammatory and pro-coagulant markers from epididymal fat, liver, and plasma were measured along with food intake and body weights. Mice fed the high SFA, MUFA, and high-fat diets exhibited increased pro-inflammatory markers in liver and adipose tissue; however, mice fed LNA for four weeks did not display significant changes in pro-inflammatory or pro-coagulant markers in epididymal fat, liver, or plasma. The present study demonstrates that LNA alone is insufficient to induce inflammation. Instead, it is more likely that hyper-caloric diets are responsible for diet-induced inflammation possibly due to adipose tissue remodeling.

  14. DESIGN OF 2.4 GHZ CMOS DIRECT CONVERSION LNA AND MIXER COMBINATION FOR WIRLESS DATA LINK TRANSCEIVER.

    SciTech Connect

    ZHAO, D.; OCONNOR, P.

    2002-04-10

    Three LNA and mixer combinations in 0.6{micro}m and 0.4{micro}m standard CMOS processes for direct-conversion receiver of 2.4GHz ISM band short-range wireless data-link applications are described in this paper. Taking low power dissipation as first consideration, these designs, employing differential common-source LNA and double balanced mixer architectures, achieve total conversion gain as high as 42.4dB, DSB noise figure as low as 9.5dB, output-referred IP3 as high as of 21.3dBm at about 4mA DC current consumption. This proves it is possible to apply standard CMOS process to implement receiver front-end with low power dissipation for this kind of application, but gain changeable LNA is needed to combat the dominant flicker noise of the mixer in order to achieve acceptable sensitivity and dynamic range at the same time.

  15. Interim Report on Multiple Sequence Alignments and TaqMan Signature Mapping to Phylogenetic Trees

    SciTech Connect

    Gardner, S; Jaing, C

    2012-03-27

    The goal of this project is to develop forensic genotyping assays for select agent viruses, addressing a significant capability gap for the viral bioforensics and law enforcement community. We used a multipronged approach combining bioinformatics analysis, PCR-enriched samples, microarrays and TaqMan assays to develop high resolution and cost effective genotyping methods for strain level forensic discrimination of viruses. We have leveraged substantial experience and efficiency gained through year 1 on software development, SNP discovery, TaqMan signature design and phylogenetic signature mapping to scale up the development of forensics signatures in year 2. In this report, we have summarized the Taqman signature development for South American hemorrhagic fever viruses, tick-borne encephalitis viruses and henipaviruses, Old World Arenaviruses, filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus and Japanese encephalitis virus.

  16. High-Throughput Genotyping with TaqMan Allelic Discrimination and Allele-Specific Genotyping Assays.

    PubMed

    Heissl, Angelika; Arbeithuber, Barbara; Tiemann-Boege, Irene

    2017-01-01

    Real-time PCR-based genotyping methods, such as TaqMan allelic discrimination assays and allele-specific genotyping, are particularly useful when screening a handful of single nucleotide polymorphisms in hundreds of samples; either derived from different individuals, tissues, or pre-amplified DNA. Although real-time PCR-based methods such as TaqMan are well-established, alternative methods, like allele-specific genotyping, are powerful alternatives, especially for genotyping short tandem repeat (STR) length polymorphisms. Here, we describe all relevant aspects when developing an assay for a new SNP or STR using either TaqMan or allele-specific genotyping, respectively, such as primer and probe design, optimization of reaction conditions, the experimental procedure for typing hundreds of samples, and finally the data evaluation. Our goal is to provide a guideline for developing genotyping assays using these two approaches that render reliable and reproducible genotype calls involving minimal optimization.

  17. Inhibition of Hsp27 Radiosensitizes Head-and-Neck Cancer by Modulating Deoxyribonucleic Acid Repair

    SciTech Connect

    Guttmann, David M.; Hart, Lori; Du, Kevin; Seletsky, Andrew; Koumenis, Constantinos

    2013-09-01

    Purpose: To present a novel method of tumor radiosensitization through Hsp27 knockdown using locked nucleic acid (LNA) and to investigate the role of Hsp27 in DNA double strand break (DSB) repair. Methods and Materials: Clonogenic survival assays, immunoblotting, the proximity ligation assay, and γH2AX foci analysis were conducted in SQ20B and FaDu human head-and-neck cancer cell lines treated with Hsp27 LNA and Hsp27 short hairpin RNA (shRNA). Additionally, nude mice with FaDu flank tumors were treated with fractionated radiation therapy after pretreatment with Hsp27 LNA and monitored for tumor growth. Results: Hsp27 LNA and Hsp27 shRNA radiosensitized head-and-neck cancer cell lines in an Hsp27-dependent manner. Ataxia-Telangectasia Mutated-mediated DNA repair signaling was impaired in irradiated cells with Hsp27 knockdown. ATM kinase inhibition abrogated the radiosensitizing effect of Hsp27. Furthermore, Hsp27 LNA and shRNA both attenuated DNA repair kinetics after radiation, and Hsp27 was found to colocalize with ATM in both untreated and irradiated cells. Last, combined radiation and Hsp27 LNA treatment in tumor xenografts in nude mice suppressed tumor growth compared with either treatment alone. Conclusions: These results support a radiosensitizing property of Hsp27 LNA in vitro and in vivo, implicate Hsp27 in double strand break repair, and suggest that Hsp27 LNA might eventually serve as an effective clinical agent in the radiotherapy of head-and-neck cancer.

  18. TaqMan PCR for Detection of Vibrio cholerae O1, O139, Non-O1, and Non-O139 in Pure Cultures, Raw Oysters, and Synthetic Seawater†

    PubMed Central

    Lyon, W. J.

    2001-01-01

    Vibrio cholerae is recognized as a leading human waterborne pathogen. Traditional diagnostic testing for Vibrio is not always reliable, because this bacterium can enter a viable but nonculturable state. Therefore, nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing. In this article, a TaqMan PCR assay is presented for quantitative detection of V. cholerae in pure cultures, oysters, and synthetic seawater. Primers and probe were designed from the nonclassical hemolysin (hlyA) sequence of V. cholerae strains. This probe was applied to DNA from 60 bacterial strains comprising 21 genera. The TaqMan PCR assay was positive for all of the strains of V. cholerae tested and negative for all other species of Vibrio tested. In addition, none of the other genera tested was amplified with the TaqMan primers and probe used in this study. The results of the TaqMan PCR with raw oysters and spiked with V. cholerae serotypes O1 and O139 were comparable to those of pure cultures. The sensitivity of the assay was in the range of 6 to 8 CFU g−1 and 10 CFU ml−1 in spiked raw oyster and synthetic seawater samples, respectively. The total assay could be completed in 3 h. Quantification of the Vibrio cells was linear over at least 6 log units. The TaqMan probe and primer set developed in this study can be used as a rapid screening tool for the presence of V. cholerae in oysters and seawater without prior isolation and characterization of the bacteria by traditional microbiological methods. PMID:11571173

  19. A high-fat, high-oleic diet, but not a high-fat, saturated diet, reduces hepatic alpha-linolenic acid and eicosapentaenoic acid content in mice

    USDA-ARS?s Scientific Manuscript database

    Considerable research centers upon the role of linoleic acid (LNA; 18:2n6) as a competitive inhibitor of a-linolenic (ALA; 18:3n3) metabolism; however, little data exist as to the impact of saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) on ALA metabolism. We tested the hypothesi...

  20. Rapid Detection of Rifampicin- and Isoniazid-Resistant Mycobacterium tuberculosis using TaqMan Allelic Discrimination.

    PubMed

    Darban-Sarokhalil, Davood; Nasiri, Mohammad J; Fooladi, Abbas A I; Heidarieh, Parvin; Feizabadi, Mohammad M

    2016-04-01

    Multidrug-resistant tuberculosis (MDR-TB) is a global problem that many countries are challenged with. Rapid and accurate detection of MDR-TB is critical for appropriate treatment and controlling of TB. The aim of the present study was to evaluate the TaqMan allelic discrimination without minor groove binder (MGB) as a rapid, efficient, and low-cost method for detection of drug resistant strains of Mycobacterium tuberculosis. A total of 112 M. tuberculosis isolates from cases with diagnosed TB were subjected to drug susceptibility testing (DST), using the proportion method. Resistant isolates were tested for characterization of mutations in the rpoB and KatG genes by TaqMan genotyping. Of 112 M. tuberculosis isolates for which DST was performed, three, one, and two isolates were MDR, rifampin (RIF) resistant, and isoniazid (INH) resistant, respectively. According to the threshold cycle (Ct) and curve pattern of mutants, TaqMan probes detect all of the mutations in the analyzed genes (katG 315, AGC→ACC, rpoB 531, TCG→TTG, and rpoB 531, TCG→TGG). The present study suggests that drug-resistant strains of M. tuberculosis can be detected by pattern's curve or Ct with TaqMan probes without MGB in real-time polymerase chain reaction (PCR).

  1. Utility of real-time Taqman PCR for antemortem and postmortem diagnosis of human rabies.

    PubMed

    Mani, Reeta Subramaniam; Madhusudana, Shampur Narayan; Mahadevan, Anita; Reddy, Vijayalakshmi; Belludi, Ashwin Yajaman; Shankar, Susarla Krishna

    2014-10-01

    Rabies, a fatal zoonotic viral encephalitis remains a neglected disease in India despite a high disease burden. Laboratory confirmation is essential, especially in patients with paralytic rabies who pose a diagnostic dilemma. However, conventional tests for diagnosis of rabies have several limitations. In the present study the utility of a real-time TaqMan PCR assay was evaluated for antemortem/postmortem diagnosis of rabies. Human clinical samples received for antemortem rabies diagnosis (CSF, saliva, nuchal skin biopsy, serum), and samples obtained postmortem from laboratory confirmed rabies in humans (brain tissue, CSF, serum) and animals (brain tissue) were included in the study. All CSF and sera were tested for rabies viral neutralizing antibodies (RVNA) by rapid fluorescent focus inhibition test (RFFIT) and all samples (except sera) were processed for detection of rabies viral RNA by real-time TaqMan PCR. All the 29 (100%) brain tissues from confirmed cases of human and animal rabies, and 11/14 (78.5%) CSF samples obtained postmortem from confirmed human rabies cases were positive by real-time TaqMan PCR. Rabies viral RNA was detected in 5/11 (45.4%) CSF samples, 6/10 (60%) nuchal skin biopsies, and 6/7 (85.7%) saliva samples received for antemortem diagnosis. Real-time TaqMan PCR alone could achieve antemortem rabies diagnosis in 11/13 (84.6%) cases; combined with RVNA detection in CSF antemortem rabies diagnosis could be achieved in all 13 (100%) cases. Real-time TaqMan PCR should be made available widely as an adjunctive test for diagnosis of human rabies in high disease burden countries like India.

  2. Taqman real-time PCR assays for rapid detection of avian pathogenic Escherichia coli isolates.

    PubMed

    Ikuta, Nilo; De Oliveira Solla Sobral, Fabiana; Lehmann, Fernanda Kieling Moreira; da Silveira, Proença Vinicius; de Carli, Silvia; Casanova, Yara Silva; Celmer, Álvaro José; Fonseca, André Salvador Kazantzi; Lunge, Vagner Ricardo

    2014-12-01

    Avian pathogenic Escherichia coli (APEC) isolates are currently differentiated from nonpathogenic strains by classical PCR of virulence genes. This study improves the detection of the five main virulence genes used for APEC detection with the development of duplex and single Taqman real-time PCR to these targets. Primers and probes targeted to ompT, hlyF, iroN, iutA, and iss genes were designed and used in the implementation of single (iss) and duplex (hlyF/ompT and iroN/iutA) Taqman PCR assays. All five virulence genes of E coli strains were successfully detected by classical and Taqman real-time (single and duplex) PCR. A panel of 111 E coli isolates, obtained from avian samples collected in different Brazilian regions between 2010 and 2011, were further tested by both assays. Complete agreement was observed in the detection of four genes, ompT, hlyF, iron, iutA, but not for iss. This issue was addressed by combining the forward primer of the classical PCR to the new iss reverse primer and probe, resulting in complete agreement for all five genes. In total, 61 (55%) Brazilian E. coli isolates were detected as APEC, and the remaining 50 (45%) as avian fecal E. coli (AFEC). In conclusion, classical and Taqman real-time PCR presented exactly the same analytical performance for the differentiation of APEC and AFEC isolates. The developed real-time Taqman PCR assays could be used for the detection and differentiation of APEC isolates.

  3. Vertical and Seasonal Variations of Bacterioplankton Subgroups with Different Nucleic Acid Contents: Possible Regulation by Phosphorus†

    PubMed Central

    Nishimura, Yoko; Kim, Chulgoo; Nagata, Toshi

    2005-01-01

    We used flow cytometry to examine seasonal variations in basin-scale distributions of bacterioplankton in Lake Biwa, Japan, a large mesotrophic freshwater lake with an oxygenated hypolimnion. The bacterial communities were divided into three subgroups: bacteria with very high nucleic acid contents (VHNA bacteria), bacteria with high nucleic acid contents (HNA bacteria), and bacteria with low nucleic acid contents (LNA bacteria). During the thermal stratification period, the relative abundance of VHNA bacteria (%VHNA) increased with depth, while the reverse trend was evident for LNA bacteria. Seasonally, the %VHNA was strongly positively correlated (r = 0.87; P < 0.001) with the concentration of dissolved inorganic phosphorus, but not with the concentration of chlorophyll a. The growth of VHNA bacteria was significantly enhanced by addition of phosphate or phosphate plus glucose but not by addition of glucose alone. Although the growth of VHNA and HNA bacteria generally exceeded that of LNA bacteria, our data also revealed that LNA bacteria grew faster than and were grazed as fast as VHNA bacteria in late August, when nutrient limitation was presumably severe. Based on these results, we hypothesize that in severely P-limited environments such as Lake Biwa, P limitation exerts more severe constraints on the growth of bacterial groups with higher nucleic acid contents, which allows LNA bacteria to be competitive and become an important component of the microbial loop. PMID:16204494

  4. [Evaluation of COBAS TaqMan: a real-time PCR-based diagnostic kit for mycobacteria].

    PubMed

    Yonemaru, Makoto; Horiba, Masahide; Tada, Atsuhiko; Nagai, Takayuki

    2009-12-01

    The real-time PCR-based diagnostic kits, COBAS TaqMan MTB and COBAS TaqMan MAI (Roche Diagnostics, Tokyo, Japan), were developed to detect Mycobacterium tuberculosis (MTB) and M. avium (MAV)/M. intracellulare (MIN), respectively. The TaqMan kits simultaneously perform amplification and detection of mycobacterial DNA to reduce assay time. We evaluated the diagnostic accuracy of both TaqMan kits in 781 clinical specimens, and compared the results with those obtained from the AMPLICOR MTB and MAI kits. With smear-positive specimens, the TaqMan kits showed 100% concordance with AMPLICOR in MTB, MAV and MIN. With all specimens, the concordances of TaqMan with AMPLICOR were 99.1%, 99.0%, and 99.7% in MTB, MAV and MIN, respectively. Four specimens for MTB and one for MAV were AMPLICOR positive/TaqMan negative. Among them, two specimens were culture-positive for MTB and one for MAV. Three specimens for MTB, seven for MAV, and two for MIN were AMPLICOR negative/TaqMan positive. Among them, two specimens were culture-positive for MTB, seven for MAV, and one for MIN. In twelve out of 21 specimens in which AMPLICOR failed to activate PCR, TaqMan successfully determined the results which were in concordance with those of mycobacterial culture. Thus, our data suggest that the accuracy of TaqMan in detecting mycobacterial DNA is superior to that of AMPLICOR. We conclude that TaqMan, which is an easy and rapid DNA amplification test, is useful for detecting MTB, MAV and MIN.

  5. Assessment of configurations and chemistries of bridged nucleic acids-containing oligomers as external guide sequences: a methodology for inhibition of expression of antibiotic resistance genes

    PubMed Central

    Jackson, Alexis; Jani, Saumya; Sala, Carol Davies; Zorreguieta, Angeles; Tolmasky, Marcelo E.

    2016-01-01

    EGSs (external guide sequences) are short antisense oligoribonucleotides that elicit RNase P-mediated cleavage of a target mRNA, which results in inhibition of gene expression. EGS technology is used to inhibit expression of a wide variety of genes, a strategy that may lead to development of novel treatments of numerous diseases, including multidrug-resistant bacterial and viral infections. Successful development of EGS technology depends on finding nucleotide analogs that resist degradation by nucleases present in biological fluids and the environment but still elicit RNase P-mediated degradation when forming a duplex with a target mRNA. Previous results suggested that locked nucleic acids (LNA)/DNA chimeric oligomers have these properties. LNA are now considered the first generation of compounds collectively known as bridged nucleic acids (BNA), modified ribonucleotides that contain a bridge at the 2′,4′-position of the ribose. LNA and the second generation BNA, known as BNANC, differ in the chemical nature of the bridge. Chimeric oligomers containing LNA or BNANC and deoxynucleotide monomers in different configurations are nuclease resistant and could be excellent EGS compounds. However, not all configurations may be equally active as EGSs. RNase P cleavage assays comparing LNA/DNA and BNANC/DNA chimeric oligonucleotides that share identical nucleotide sequence but with different configurations were carried out using as target the amikacin resistance aac(6′)-Ib mRNA. LNA/DNA gapmers with 5 and 3/4 LNA residues at the 5′- and 3′-ends, respectively, were the most efficient EGSs while all BNANC/DNA gapmers showed very poor activity. When the most efficient LNA/DNA gapmer was covalently bound to a cell penetrating peptide (CPP), the hybrid compound conserved the EGS activity as determined by RNase P cleavage assays and reduced the levels of resistance to amikacin when added to Acinetobacter baumannii cells in culture, an indication of cellular uptake and

  6. Dietary linoleic acid elevates the endocannabinoids 2-AG and anandamide and promotes weight gain in mice fed a low fat diet.

    PubMed

    Alvheim, Anita Røyneberg; Torstensen, Bente E; Lin, Yu Hong; Lillefosse, Haldis Haukås; Lock, Erik-Jan; Madsen, Lise; Frøyland, Livar; Hibbeln, Joseph R; Malde, Marian Kjellevold

    2014-01-01

    Dietary intake of linoleic acid (LNA, 18:2n-6) has increased dramatically during the 20th century and is associated with greater prevalence of obesity. The endocannabinoid system is involved in regulation of energy balance and a sustained hyperactivity of the endocannabinoid system may contribute to obesity. Arachidonic acid (ARA, 20:4n-6) is the precursor for 2-AG and anandamide (AEA), and we sought to determine if low fat diets (LFD) could be made obesogenic by increasing the endocannabinoid precursor pool of ARA, causing excessive endocannabinoid signaling leading to weight gain and a metabolic profile associated with obesity. Mice (C57BL/6j, 6 weeks of age) were fed 1 en% LNA and 8 en% LNA in low fat (12.5 en%) and medium fat diets (MFD, 35 en%) for 16 weeks. We found that increasing dietary LNA from 1 to 8 en% in LFD and MFD significantly increased ARA in phospholipids (ARA-PL), elevated 2-AG and AEA in liver, elevated plasma leptin, and resulted in larger adipocytes and more macrophage infiltration in adipose tissue. In LFD, dietary LNA of 8 en% increased feed efficiency and caused greater weight gain than in an isocaloric reduction to 1 en% LNA. Increasing dietary LNA from 1 to 8 en% elevates liver endocannabinoid levels and increases the risk of developing obesity. Thus a high dietary content of LNA (8 en%) increases the adipogenic properties of a low fat diet.

  7. Application of locked nucleic acid-based probes in fluorescence in situ hybridization.

    PubMed

    Fontenete, Sílvia; Carvalho, Daniel; Guimarães, Nuno; Madureira, Pedro; Figueiredo, Céu; Wengel, Jesper; Azevedo, Nuno Filipe

    2016-07-01

    Fluorescence in situ hybridization (FISH) employing nucleic acid mimics as probes is becoming an emerging molecular tool in the microbiology area for the detection and visualization of microorganisms. However, the impact that locked nucleic acid (LNA) and 2'-O-methyl (2'-OMe) RNA modifications have on the probe that is targeting microorganisms is unknown. In this study, the melting and hybridization efficiency properties of 18 different probes in regards to their use in FISH for the detection of the 16S rRNA of Helicobacter pylori were compared. For the same sequence and target, probe length and the type of nucleic acid mimics used as mixmers in LNA-based probes strongly influence the efficiency of detection. LNA probes with 10 to 15 mers showed the highest efficiency. Additionally, the combination of 2'-OMe RNA with LNA allowed an increase on the fluorescence intensities of the probes. Overall, these results have significant implications for the design and applications of LNA probes for the detection of microorganisms.

  8. Detection of EGFR mutations by TaqMan mutation detection assays powered by competitive allele-specific TaqMan PCR technology.

    PubMed

    Roma, Cristin; Esposito, Claudia; Rachiglio, Anna Maria; Pasquale, Raffaella; Iannaccone, Alessia; Chicchinelli, Nicoletta; Franco, Renato; Mancini, Rita; Pisconti, Salvatore; De Luca, Antonella; Botti, Gerardo; Morabito, Alessandro; Normanno, Nicola

    2013-01-01

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

  9. Development, Evaluation, and Standardization of a Real-Time TaqMan Reverse Transcription-PCR Assay for Quantification of Hepatitis A Virus in Clinical and Shellfish Samples

    PubMed Central

    Costafreda, M. Isabel; Bosch, Albert; Pintó, Rosa M.

    2006-01-01

    A standardized real-time reverse transcription-PCR (RT-PCR) assay has been developed for an accurate estimation of the number of genome copies of hepatitis A virus (HAV) in clinical and shellfish samples. Real-time procedures were based on the amplification of a fragment of the highly conserved 5′ noncoding region and detection through an internal fluorescent probe, including TaqMan and beacon chemistries, in one- and two-step RT-PCR formats. The best performance in terms of sensitivity and reproducibility was achieved by a one-step TaqMan RT-PCR, with a sensitivity enabling the detection of 0.05 infectious unit and 10 copies of a single-stranded RNA (ssRNA) synthetic transcript. Standard reagents, such as a mengovirus strain and an ssRNA transcript, were employed as controls of nucleic acid extraction and RT-PCR, respectively. The test proved to be highly specific after a broad panel of enteric viruses was tested. Sequence alignment of target regions of the primers and probe proved them to be adequate for the quantification of all HAV genotypes. In addition, a quasispecies analysis of the mutant spectrum indicated that these regions are not prone to variability, thus confirming their robustness. PMID:16751488

  10. [Development and application of TaqMan MGB probe fluorescence quantitative PCR method for rapid detection of Clostridium piliforme].

    PubMed

    Gao, Zheng-qin; Yue, Bing-fei; He, Zheng-ming

    2012-02-01

    To develop a TaqMan MGB probe-based, sensitive and specific fluorescence quantitative PCR assay method for rapid detection of Clostridium piliforme. Primers and probes specific to 16S rRNA gene of Clostridium piliforme were designed. A TaqMan MGB probe-based, fluorescence quantitative PCR method was established. Specificity, sensitivity and stability of the method were assessed, followed by real-time quantitative PCR assay to detect Clostridium piliforme on 1156 clinical specimens during 2008-2011 and compared with conventional PCR assay. The specificity of TaqMan MGB probe-based fluorescence quantitative PCR was high and did not show cross-reactivity with Helicobacter hepaticus, Helicobacter pylori, Campylobacter jejuni, Pasteurella pneumotropica, Escherichia coli or Pseudomonas aeruginosa. The detection limit was 2.2 copies/µl. The correlation coefficient and slope value of standard curve were 0.999 and -3.204, respectively and the efficiency of TaqMan MGB-based probe fluorescence quantitative PCR assay was 100%. When the TaqMan MGB-based probe fluorescence quantitative PCR assay was preformed to detect Clostridium piliforme on 1156 clinical specimens, a total of 101 specimens showed positive on Clostridium piliforme. However, only 44 specimens showed positive when conventional PCR was used. The real-time quantitative PCR for Clostridium piliforme could be completed within 2 hours. The TaqMan MGB-based probe fluorescence quantitative PCR assay method was a reliable, specific, sensitive and useful tool for rapid detection of Clostridium piliforme.

  11. Evaluation of locked nucleic acid-modified small interfering RNA in vitro and in vivo.

    PubMed

    Mook, Olaf R; Baas, Frank; de Wissel, Marit B; Fluiter, Kees

    2007-03-01

    RNA interference has become widely used as an experimental tool to study gene function. In addition, small interfering RNA (siRNA) may have great potential for the treatment of diseases. Recently, it was shown that siRNA can be used to mediate gene silencing in mouse models. Locally administered siRNAs entered the first clinical trials, but strategies for successful systemic delivery of siRNA are still under development. Challenges still exist about the stability, delivery, and therapeutic efficacy of siRNA. In the present study, we compare the efficacy of two methods of systemic siRNA delivery and the effects of siRNA modifications using locked nucleic acids (LNA) in a xenograft cancer model. Low volume tail vein bolus injections and continuous s.c. delivery using osmotic minipumps yielded similar uptake levels of unmodified siRNA by tumor xenografts. Both routes of administration mediated sequence-specific inhibition of two unrelated targets inside tumor xenografts. Previous studies have shown that LNA can be incorporated into the sense strand of siRNA while the efficacy is retained. Modification of siRNA targeting green fluorescent protein with LNA results in a significant increase in serum stability and thus may be beneficial for clinical applications. We show that minimal 3' end LNA modifications of siRNA are effective in stabilization of siRNA. Multiple LNA modifications in the accompanying strand further increase the stability but negate the efficacy in vitro and in vivo. In vivo, LNA-modified siRNA reduced off-target gene regulation compared with nonmodified siRNA. End-modified siRNA targeting green fluorescent protein provides a good trade-off between stability and efficacy in vivo using the two methods of systemic delivery in the nude mouse model. Therefore, LNA-modified siRNA should be preferred over unmodified siRNA.

  12. Ability of two commercially available assays (Abbott RealTime HIV-1 and Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 Version 2.0) to quantify low HIV-1 RNA Levels (<1,000 copies/milliliter): comparison with clinical samples and NIBSC working reagent for nucleic acid testing assays.

    PubMed

    Amendola, Alessandra; Marsella, Patrizia; Bloisi, Maria; Forbici, Federica; Angeletti, Claudio; Capobianchi, Maria R

    2014-06-01

    Concordance between molecular assays may be suboptimal at low HIV-1 viremia levels (<1,000 copies/ml); therefore, it may be difficult to define and compare virologic endpoints for successful and failed therapy. We compared two commercial assays (the Abbott RealTime HIV-1 and the Roche Cobas AmpliPrep/TaqMan HIV-1 version 2.0) for their ability to detect and quantify low viral loads. A comparison was performed using 167 residual clinical samples (with values ranging from "not detected" to 1,000 copies/ml, as measured by the Abbott assay) and the National Institute and Biological Standards and Control (NIBSC) HIV-1 RNA working reagent 1 for nucleic acid amplification techniques (NAT) assays (serially diluted to a range from 1 to 1,000 copies/ml). Quantitative results were compared using Lin's concordance correlation coefficient and a Bland-Altman plot. Concordance with the qualitative results was measured by Cohen's kappa statistic. With clinical samples, the degree of interassay concordance of the qualitative results at a 40-copies/ml HIV-1 RNA threshold was substantial (κ = 0.762); the correlation among the quantified samples was suboptimal (concordance correlation coefficient, 0.728; P < 0.0001); the mean difference of the values between the Roche and Abbott assays was 0.193 log10 copies/ml. Using the HIV-1 RNA working reagent 1 for NAT assays, the results provided by the Roche assay were, on average, 3 times higher than expected, while the Abbott assay showed high accuracy. The Roche assay was highly sensitive, being able to detect a level as low as 3.5 copies/ml HIV-1 RNA with 95% probability. The performance characteristics of each molecular assay should be taken into account when HIV-1 RNA threshold values for "virologic suppression," "virologic failure," "persistent low viral loads," etc., are defined and indicated in the support of clinical decisions. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  13. Application of locked nucleic acids to improve aptamer in vivo stability and targeting function

    PubMed Central

    Schmidt, Kathrin S.; Borkowski, Sandra; Kurreck, Jens; Stephens, Andrew W.; Bald, Rolf; Hecht, Maren; Friebe, Matthias; Dinkelborg, Ludger; Erdmann, Volker A.

    2004-01-01

    Aptamers are powerful candidates for molecular imaging applications due to a number of attractive features, including rapid blood clearance and tumor penetration. We carried out structure–activity relationship (SAR) studies with the Tenascin-C binding aptamer TTA1, which is a promising candidate for application in tumor imaging with radioisotopes. The aim was to improve its in vivo stability and target binding. We investigated the effect of thermal stabilization of the presumed non-binding double-stranded stem region on binding affinity and resistance against nucleolytic degradation. To achieve maximal thermal stem stabilization melting experiments with model hexanucleotide duplexes consisting of unmodified RNA, 2′-O-methyl RNA (2′-OMe), 2′-Fluoro RNA (2′-F) or Locked Nucleic Acids (LNAs) were initially carried out. Extremely high melting temperatures have been found for an LNA/LNA duplex. TTA1 derivatives with LNA and 2′-OMe modifications within the non-binding stem have subsequently been synthesized. Especially, the LNA-modified TTA1 derivative exhibited significant stem stabilization and markedly improved plasma stability while maintaining its binding affinity to the target. In addition, higher tumor uptake and longer blood retention was found in tumor-bearing nude mice. Thus, our strategy to introduce LNA modifications after the selection procedure is likely to be generally applicable to improve the in vivo stability of aptamers without compromising their binding properties. PMID:15509871

  14. Dietary alpha-linolenic acid/linoleic acid ratios modulate intestinal immunity, tight junctions, anti-oxidant status and mRNA levels of NF-κB p65, MLCK and Nrf2 in juvenile grass carp (Ctenopharyngodon idella).

    PubMed

    Zeng, Yun-Yun; Jiang, Wei-Dan; Liu, Yang; Wu, Pei; Zhao, Juan; Jiang, Jun; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu; Feng, Lin

    2016-04-01

    This study was conducted to investigate the effects of dietary alpha-linolenic acid/linoleic acid (ALA/LNA) ratios on the immune response, tight junctions, antioxidant status and immune-related signaling molecules mRNA levels in the intestine of juvenile grass carp (Ctenopharyngodon idella). A total of 1260 juvenile grass carp with an average initial weight of 8.78 ± 0.03 g were fed diets with different ALA/LNA ratios (0.01, 0.34, 0.68, 1.03, 1.41, 1.76 and 2.15) for 60 days. Results indicated that ALA/LNA ratio of 1.03 significantly increased acid phosphatase, lysozyme activities and complement C3 contents, promoted interleukin 10, transforming growth factor β1 and κB inhibitor α mRNA abundance, whereas suppressed pro-inflammatory cytokines (interleukin 1β, interleukin 8, tumor necrosis factor α and interferon γ2) and signal molecules (IκB kinase β, IκB kinase γ and nuclear factor κB p65) mRNA levels in the intestine (P < 0.05), suggesting that optimal dietary ALA/LNA ratio improved intestinal immune response of juvenile fish. Additionally, ALA/LNA ratio of 1.03 significantly promoted Claudin-3, Claudin-b, Claudin-c, Occludin and ZO-1 gene transcription, whereas reduced Claudin-15a and myosin light-chain kinase mRNA levels in the intestine, suggesting that appropriate dietary ALA/LNA ratio strengthened tight junctions in the intestine of juvenile fish. Meanwhile, ALA/LNA ratio of 1.03 noticeably elevated glutathione contents, copper/zinc superoxide dismutase, glutathione peroxidase, glutathione S-transferase and glutathione reductase activities and mRNA levels, as well as signaling molecule nuclear factor erythoid 2-related factor 2 gene transcriptional abundance in the intestine, suggesting that proper ratio of dietary ALA/LNA ameliorate the intestinal antioxidant status of juvenile fish. Based on the quadratic regression analysis of the complement C3 content in the distal intestine and malondialdehyde content in the whole intestine, optimal ALA/LNA

  15. Development of 20 TaqMan assays differentiating the endangered shortnose and Lost River suckers

    USGS Publications Warehouse

    Hoy, Marshal S.; Ostberg, Carl O.

    2015-01-01

    Accurate species identification is vital to conservation and management of species at risk. Species identification is challenging when taxa express similar phenotypic characters and form hybrids, for example the endangered shortnose sucker (Chasmistes brevirostris) and Lost River sucker (Deltistes luxatus). Here, we developed 20 Taqman assays that differentiate these species (19 nuclear DNA and one mitochondrial DNA). Assays were evaluated in 160 young-of-the-year identified to species using meristic counts. Alleles were not fixed between species, but species were highly differentiated (F ST = 0.753, P < 0.001). The assays developed herein will be a valuable tool for resource managers.

  16. High-affinity DNA-targeting using Readily Accessible Mimics of N2′-Functionalized 2′-Amino-α-L-LNA

    PubMed Central

    Karmakar, Saswata; Anderson, Brooke A.; Rathje, Rie L.; Andersen, Sanne; Jensen, Troels B.; Nielsen, Poul; Hrdlicka, Patrick J.

    2011-01-01

    N2′-Pyrene-functionalized 2′-amino-α-L-LNAs (Locked Nucleic Acids) display extraordinary affinity toward complementary DNA targets due to favorable preorganization of the pyrene moieties for hybridization-induced intercalation. Unfortunately, the synthesis of these monomers is challenging (~20 steps, <3% overall yield), which has precluded full characterization of DNA-targeting applications based on these materials. Access to more readily accessible functional mimics would be highly desirable. Here we describe short synthetic routes toward a series of O2′-intercalator-functionalized uridine and N2′-intercalator-functionalized 2′-N-methyl-2′-aminouridine monomers and demonstrate – via thermal denaturation, UV-visible absorption and fluorescence spectroscopy experiments – that several of them mimic the DNA-hybridization properties of N2′-pyrene-functionalized 2′-amino-α-L-LNAs. For example, oligodeoxyribonucleotides (ONs) modified with 2′-O-(coronen-1-yl)methyluridine monomer Z, 2′-O-(pyren-1-yl)methyluridine monomer Y or 2′-N-(pyren-1-ylmethyl)-2′-N-methylaminouridine monomer Q, display prominent increases in thermal affinity toward complementary DNA relative to reference strands (average ΔTm/mod up to +12 °C), pronounced DNA-selectivity, and higher target specificity than 2′-amino-β-L-LNA benchmark probes. In contrast, ONs modified with 2′-O-(2-napthyl)uridine monomer W, 2′-O-(pyren-1-yl)uridine monomer X or 2′-N-(pyren-1-ylcarbonyl)-2′-N-methylaminouridine monomer S display very low affinity toward DNA targets. This demonstrates that even conservative alterations in linker chemistry, linker length and surface area of the appended intercalators have marked impact on DNA-hybridization characteristics. Straightforward access to high-affinity building blocks such as Q/Y/Z is likely to accelerate their use in DNA-targeting applications within nucleic acid based diagnostics, therapeutics, and material science. PMID:21827174

  17. Effect of pH on conjugated linoleic acid (CLA) formation of linolenic acid biohydrogenation by ruminal microorganisms.

    PubMed

    Lee, Yongjae

    2013-08-01

    Conventional beliefs surrounding the linolenic acid (LNA; cis-9 cis-12 cis-15 C18:3) biohydrogenation (BH) pathway propose that it converts to stearic acid (SA) without the formation of conjugated linoleic acid (CLA) as intermediate isomers. However, an advanced study (Lee and Jenkins, 2011) verified that LNA BH yields multiple CLAs. This study utilized the stable isotope tracer to investigate the BH intermediates of (13)C-LNA with different pH conditions (5.5 and 6.5). The (13)C enrichment was calculated as a (13)C/(12)C ratio of labeled minus unlabeled. After 24 h, eight CLA isomers were significantly enriched on both pH treatment, this result verifies that these CLAs originated from (13)C-LNA BH which supports the results of Lee and Jenkins (2011). The enrichment of cis-cis double bond CLAs (cis-9 cis-11 and cis-10 cis-12 CLA) were significantly higher at low pH conditions. Furthermore, the concentration of cis-10 cis-12 CLA at low pH was four times higher than at high pH conditions after a 3 h incubation. These differences support the LNA BH pathways partial switch under different pH conditions, with a strong influence on the cis-cis CLA at low pH. Several mono-, di-, and tri-enoic fatty acid isomers were enriched during 24 h of incubation, but the enrichment was decreased or restricted at low pH treatment. Based on these results, it is proposed that low pH conditions may cause a changed or limited capacity of the isomerization and reduction steps in BH.

  18. Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids.

    PubMed

    Fontenete, Silvia; Silvia, Fontenete; Barros, Joana; Joana, Barros; Madureira, Pedro; Pedro, Madureira; Figueiredo, Céu; Céu, Figueiredo; Wengel, Jesper; Jesper, Wengel; Azevedo, Nuno Filipe; Filipe, Azevedo Nuno

    2015-05-01

    In the past few years, several researchers have focused their attention on nucleic acid mimics due to the increasing necessity of developing a more robust recognition of DNA or RNA sequences. Fluorescence in situ hybridization (FISH) is an example of a method where the use of these novel nucleic acid monomers might be crucial to the success of the analysis. To achieve the expected accuracy in detection, FISH probes should have high binding affinity towards their complementary strands and discriminate effectively the noncomplementary strands. In this study, we investigate the effect of different chemical modifications in fluorescent probes on their ability to successfully detect the complementary target and discriminate the mismatched base pairs by FISH. To our knowledge, this paper presents the first study where this analysis is performed with different types of FISH probes directly in biological targets, Helicobacter pylori and Helicobacter acinonychis. This is also the first study where unlocked nucleic acids (UNA) were used as chemistry modification in oligonucleotides for FISH methodologies. The effectiveness in detecting the specific target and in mismatch discrimination appears to be improved using locked nucleic acids (LNA)/2'-O-methyl RNA (2'OMe) or peptide nucleic acid (PNA) in comparison to LNA/DNA, LNA/UNA, or DNA probes. Further, the use of LNA modifications together with 2'OMe monomers allowed the use of shorter fluorescent probes and increased the range of hybridization temperatures at which FISH would work.

  19. Features of “All LNA” Duplexes Showing a New Type of Nucleic Acid Geometry

    PubMed Central

    Förster, Charlotte; Eichert, André; Oberthür, Dominik; Betzel, Christian; Geßner, Reinhard; Nitsche, Andreas; Fürste, Jens P.

    2012-01-01

    “Locked nucleic acids” (LNAs) belong to the backbone-modified nucleic acid family. The 2′-O,4′-C-methylene-β-D-ribofuranose nucleotides are used for single or multiple substitutions in RNA molecules and thereby introduce enhanced bio- and thermostability. This renders LNAs powerful tools for diagnostic and therapeutic applications. RNA molecules maintain the overall canonical A-type conformation upon substitution of single or multiple residues/nucleotides by LNA monomers. The structures of “all” LNA homoduplexes, however, exhibit significant differences in their overall geometry, in particular a decreased twist, roll and propeller twist. This results in a widening of the major groove, a decrease in helical winding, and an enlarged helical pitch. Therefore, the LNA duplex structure can no longer be described as a canonical A-type RNA geometry but can rather be brought into proximity to other backbone-modified nucleic acids, like glycol nucleic acids or peptide nucleic acids. LNA-modified nucleic acids provide thus structural and functional features that may be successfully exploited for future application in biotechnology and drug discovery. PMID:22666550

  20. Fate of orally administered radioactive fatty acids in the late-pregnant rat.

    PubMed

    López-Luna, Pilar; Ortega-Senovilla, Henar; López-Soldado, Iliana; Herrera, Emilio

    2016-03-01

    To investigate the biodisponibility of placental transfer of fatty acids, rats pregnant for 20 days were given tracer amounts of [(14)C]palmitic (PA), oleic (OA), linoleic (LA), α-linolenic (LNA), or docosahexaenoic acid (DHA) orally and euthanized at 0.5, 1.0, 2.0, or 8.0 h thereafter. Maternal plasma radioactivity in lipids initially increased only to decline at later times. Most of the label appeared first as triacylglycerols (TAG); later, the proportion in phospholipids (PhL) increased. The percentage of label in placental lipids was also always highest shortly after administration and declined later; again, PhL increased with time. Fetal plasma radioactivity increased with time, with its highest value at 8.0 h after DHA or LNA administration. DHA initially appeared primarily in the nonesterified fatty acids (NEFA) and PA, OA, LA, and LNA as TAG followed by NEFA; in all cases, there was an increase in PhL at later times. Measurement of fatty acid concentrations allowed calculation of specific (radio)activities, and the ratio (fetal/maternal) of these in the plasmas gave an index of placental transfer activity, which was LNA > LA > DHA = OA > PA. It is proposed that a considerable proportion of most fatty acids transferred through the placenta are released into the fetal circulation in the form of TAG.

  1. T.I.M.S: TaqMan Information Management System, tools to organize data flow in a genotyping laboratory

    PubMed Central

    Monnier, Stéphanie; Cox, David G; Albion, Tim; Canzian, Federico

    2005-01-01

    Background Single Nucleotide Polymorphism (SNP) genotyping is a major activity in biomedical research. The Taqman technology is one of the most commonly used approaches. It produces large amounts of data that are difficult to process by hand. Laboratories not equipped with a Laboratory Information Management System (LIMS) need tools to organize the data flow. Results We propose a package of Visual Basic programs focused on sample management and on the parsing of input and output TaqMan files. The code is written in Visual Basic, embedded in the Microsoft Office package, and it allows anyone to have access to those tools, without any programming skills and with basic computer requirements. Conclusion We have created useful tools focused on management of TaqMan genotyping data, a critical issue in genotyping laboratories whithout a more sophisticated and expensive system, such as a LIMS. PMID:16221298

  2. T.I.M.S: TaqMan Information Management System, tools to organize data flow in a genotyping laboratory.

    PubMed

    Monnier, Stéphanie; Cox, David G; Albion, Tim; Canzian, Federico

    2005-10-12

    Single Nucleotide Polymorphism (SNP) genotyping is a major activity in biomedical research. The Taqman technology is one of the most commonly used approaches. It produces large amounts of data that are difficult to process by hand. Laboratories not equipped with a Laboratory Information Management System (LIMS) need tools to organize the data flow. We propose a package of Visual Basic programs focused on sample management and on the parsing of input and output TaqMan files. The code is written in Visual Basic, embedded in the Microsoft Office package, and it allows anyone to have access to those tools, without any programming skills and with basic computer requirements. We have created useful tools focused on management of TaqMan genotyping data, a critical issue in genotyping laboratories without a more sophisticated and expensive system, such as a LIMS.

  3. Assessment of Legionella pneumophila in recreational spring water with quantitative PCR (Taqman) assay

    PubMed Central

    Shen, Shu-Min; Chou, Ming-Yuan; Ji, Wen-Tsai; Hsu, Tsui-Kang; Tsai, Hsiu-Feng; Huang, Yu-Li; Chiu, Yi-Chou; Kao, Erl-Shyh; Kao, Po-Min; Fan, Cheng-Wei

    2015-01-01

    Legionella spp. are common in various natural and man-made aquatic environments. Recreational hot spring is frequently reported as an infection hotspot because of various factors such as temperature and humidity. Although polymerase chain reaction (PCR) had been used for detecting Legionella, several inhibitors such as humic substances, calcium, and melanin in the recreational spring water may interfere with the reaction thus resulting in risk underestimation. The purpose of this study was to compare the efficiencies of conventional and Taqman quantitative PCR (qPCR) on detecting Legionella pneumophila in spring facilities and in receiving water. In the results, Taqman PCR had much better efficiency on specifying the pathogen in both river and spring samples. L. pneumophila was detected in all of the 27 river water samples and 45 of the 48 hot spring water samples. The estimated L. pneumophela concentrations ranged between 1.0 × 102 and 3.3 × 105 cells/l in river water and 72.1–5.7 × 106 cells/l in hot spring water. Total coliforms and turbidity were significantly correlated with concentrations of L. pneumophila in positive water samples. Significant difference was also found in water temperature between the presence/absence of L. pneumophila. Our results suggest that conventional PCR may be not enough for detecting L. pneumophila particularly in the aquatic environments full of reaction inhibitors. PMID:26184706

  4. [Implementation of the COBAS Taqman HIV-1 Test, v1.0 for vertical transmission diagnosis].

    PubMed

    Castro, Gonzalo M; Sosa, María P; Gallego, Sandra V; Sicilia, Paola; Marin, Ángeles L; Altamirano, Natalia; Kademian, Silvia; Barbás, María G; Cudolá, Analía

    2015-01-01

    Vertical transmission is the main route of HIV infection in childhood. Because of the persistence of maternal HIV antibodies, virologic assays that directly detect HIV are required to diagnose HIV infection in infants younger than 18 months of age. The sensitivity of HIV RNA/DNA assays increases as the child becomes older. These tests have specificity values greater than 95%. The aim of this study was to evaluate the performance of the COBAS Taqman HIV-1 Test, v1.0 assay (Roche) and its concordance with a Multiplex Nested-PCR. Of 341 samples processed, 15 were positive and 326 negative by both methods. Sensitivity and specificity overall values for the viral load assay were 88.2% and 100%, respectively. Our results indicate that the COBAS Taqman assay evaluated could be used as an alternative method to diagnose HIV congenital infection. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  5. Assessment of Legionella pneumophila in recreational spring water with quantitative PCR (Taqman) assay.

    PubMed

    Shen, Shu-Min; Chou, Ming-Yuan; Hsu, Bing-Mu; Ji, Wen-Tsai; Hsu, Tsui-Kang; Tsai, Hsiu-Feng; Huang, Yu-Li; Chiu, Yi-Chou; Kao, Erl-Shyh; Kao, Po-Min; Fan, Cheng-Wei

    2015-07-01

    Legionella spp. are common in various natural and man-made aquatic environments. Recreational hot spring is frequently reported as an infection hotspot because of various factors such as temperature and humidity. Although polymerase chain reaction (PCR) had been used for detecting Legionella, several inhibitors such as humic substances, calcium, and melanin in the recreational spring water may interfere with the reaction thus resulting in risk underestimation. The purpose of this study was to compare the efficiencies of conventional and Taqman quantitative PCR (qPCR) on detecting Legionella pneumophila in spring facilities and in receiving water. In the results, Taqman PCR had much better efficiency on specifying the pathogen in both river and spring samples. L. pneumophila was detected in all of the 27 river water samples and 45 of the 48 hot spring water samples. The estimated L. pneumophela concentrations ranged between 1.0 × 10(2) and 3.3 × 10(5) cells/l in river water and 72.1-5.7 × 10(6) cells/l in hot spring water. Total coliforms and turbidity were significantly correlated with concentrations of L. pneumophila in positive water samples. Significant difference was also found in water temperature between the presence/absence of L. pneumophila. Our results suggest that conventional PCR may be not enough for detecting L. pneumophila particularly in the aquatic environments full of reaction inhibitors.

  6. Cobas ampliprep/cobas TaqMan HIV-1 v2.0 assay: consequences at the cohort level.

    PubMed

    Taylor, Ninon; Grabmeier-Pfistershammer, Katharina; Egle, Alexander; Greil, Richard; Rieger, Armin; Ledergerber, Bruno; Oberkofler, Hannes

    2013-01-01

    High-sensitive real-time PCR assays are routinely used to monitor HIV-1 infected subjects. Inter-assay discrepancies have been described at the low viral load (VL) end, where clinical decisions regarding possible virological rebound are based. A retrospective study was performed to analyze frequencies of viral blips after transition to the COBAS Ampliprep/COBAS TaqMan v2.0 HIV-1 assay (Taqman v2.0) in patients with prior undetectable VLs as measured with the Roche Cobas Ampliprep Amplicor HIV-1 Monitor Test, v1.5 (Amplicor) and was evaluated in comparison to a group of patients monitored with the Abbott Real-time HIV-1 assay (Abbott RT) during the same period of time. 85 of 373 patients with VLs below the limit of quantification with Amplicor had VLs >50 copies/mL after transition to the TaqMan v2.0 assay. Among these 74.1% had VLs ranging from 50-499 copies/mL, 22.9% had VLs >500 copies/mL. From 22 patients with initial Taqman v2.0 based VLs exceeding 500 copies/mL, 6 patients had VLs <20 copies/mL after novel VL measurement on a next visit. In our control group with VL quantification using the Abbott RT assay, only 1 patient became detectable and showed a VL of <40 copies/mL after new measurement. Transition to the Taqman v2.0 assay was accompanied by an increase of quantifiable HIV-1 VLs in patients with long term viral suppression under antiretroviral therapy that might be attributed to technical shortcomings of the Taqman v2.0 assay. A high test variability at the low VL end but also beyond was observed, making meaningful clinical interpretation of viral blips derived from different assays difficult.

  7. Design of a LNA in the frequency band 1.8-2.2GHz in 0.13μm CMOS Technology

    NASA Astrophysics Data System (ADS)

    di Gioia, E.; Hermann, C.; Klar, H.

    2005-05-01

    The subject of this work is a low noise amplifier (LNA), operating in the frequency range 1.8-2.1GHz. The CMOS 0.13μm technology is used in respect to the low cost of the final device. Among the specifications, a variable gain and an adjustable working frequency are required. In particular, four different working modes are provided: 1.8, 1.9 and 2.1GHz high gain and 2.1GHz low gain. The amplifier is designed to be used as first stage of a receiver for mobile telephony. For this reason low power consumption is taken into consideration (low supply voltage and low drain currents). A simple digital circuit, integrated on-chip, is used to select the operating mode of the LNA by means of two input pins. A Noise figure of 1dB is obtained with a supply voltage of 0.8V.

  8. A 0.75 dB NF LNA in GaAs pHEMT utilizing gate-drain capacitance and gradual inductor

    NASA Astrophysics Data System (ADS)

    Shuo, Wang; Xinnian, Zheng; Hao, Yang; Haiying, Zhang

    2015-07-01

    A two-stage monolithic microwave integrated circuit (MMIC) low noise amplifier (LNA) fabricated in 0.5 μm GaAs pHEMT is presented. The Miller effect introduced by the parasitic gate-drain capacitance is utilized to decrease the value of the input inductor. Additionally, the input on-chip inductor is a novel high Q gradual structure. The noise figure is reduced with these two methods. With good input and output matching, the LNA achieves a noise figure of 0.75 dB and a small signal gain of 32.7 dB over 698-806 MHz. The input 1 dB compression point is -21.8 dBm and the input third order interception point is -10 dBm. Project supported by the External Cooperation Program of BIC, Chinese Academy of Sciences (No. 172511KYSB20130108).

  9. Mapping the nicking efficiencies of nickase R.BbvCI for side-specific LNA-substituted substrates using rolling circle amplification

    PubMed Central

    Wei, Hua; Zhao, Guojie; Hu, Tianyu; Tang, Suming; Jiang, Jiquan; Hu, Bo; Guan, Yifu

    2016-01-01

    We used a novel asymmetric cleavage analysis method based on rolling circle amplification (RCA) to determine the effects of LNA modification of substrate on the two subunits of R.BbvCI cleavage. We designed two sets of cleavage circular substrates by using two different ligation strategies and analyzed the single strand cleavage efficiency affected by different modification positions both from the cleaved strands and the uncleaved strands. Results showed that the effects of LNA on cleavage rates of modified strands and unmodified strands were both site-dependent. The Nb.BbvCI and Nt.BbvCI were affected by LNA modification in different way. Most of the modification positions showed strong inhibition of both of these two nickases cleavage. However, the modification in T3 position of bottom strand hardly affected both of the two nickases activities. The results suggested an intimated interaction between the two subunits of R.BbvCI, and the T3 position in bottom strand might be a less tight position which was hard to be disturbed. PMID:27582033

  10. A sensitive one-step TaqMan amplification approach for detection of rubella virus clade I and II genotypes in clinical samples.

    PubMed

    Claus, C; Bergs, S; Emmrich, N C; Hübschen, J M; Mankertz, A; Liebert, U G

    2017-02-01

    Although teratogenic rubella virus (RV) causes a vaccine-preventable disease, it is still endemic in several countries worldwide. Thus, there is a constant risk of RV importation into non-endemic areas. RV monitoring, especially during measles and Zika virus outbreaks, requires reliable diagnostic tools. For this study, a TaqMan-based one-step reverse transcription-quantitative PCR (RT-qPCR) assay, with the p90 gene as a novel and so far unexplored target for detection of clade I and II genotypes, was developed and evaluated. Automated nucleic acid extraction was carried out. Performance characteristics of the TaqMan RT-qPCR assay were determined for a RV plasmid standard and RNA extracted from virus-infected cell culture supernatants representing clade I and II genotypes. Diagnostic specificity and sensitivity were validated against other RNA and DNA viruses, relevant for RV diagnostic approaches and for RV-positive clinical samples, respectively. The assay is specific and highly sensitive with a limit of detection as low as five to one copies per reaction or 200 infectious virus particles per ml. The coefficients of variation (CV) were specified as intra- (within one run) and inter- (between different runs) assay variation, and calculated based on the standard deviations for the obtained Ct values of the respective samples. Intra- and inter-assay CV values were low, with a maximum of 3.4% and 2.4%, respectively. The assay was shown to be suitable and specific for the analysis of clinical samples. With p90 as a novel target, the highly sensitive and specific TaqMan assay outlined in this study is suitable for RV diagnosis worldwide.

  11. Utility of IgM ELISA, TaqMan real-time PCR, reverse transcription PCR, and RT-LAMP assay for the diagnosis of Chikungunya fever.

    PubMed

    Reddy, Vijayalakshmi; Ravi, Vasanthapuram; Desai, Anita; Parida, Manmohan; Powers, Ann M; Johnson, Barbara W

    2012-11-01

    Chikungunya fever a re-emerging infection with expanding geographical boundaries, can mimic symptoms of other infections like dengue, malaria which makes the definitive diagnosis of the infection important. The present study compares the utility of four laboratory diagnostic methods viz. IgM capture ELISA, an in house reverse transcription PCR for the diagnosis of Chikungunya fever, TaqMan real-time PCR, and a one step reverse transcription-loop mediated isothermal amplification assay (RT-LAMP). Out of the 70 serum samples tested, 29 (41%) were positive for Chikungunya IgM antibody by ELISA and 50 (71%) samples were positive by one of the three molecular assays. CHIKV specific nucleic acid was detected in 33/70 (47%) by reverse transcription PCR, 46/70 (66%) by TaqMan real-time PCR, and 43/70 (62%) by RT-LAMP assay. A majority of the samples (62/70; 89%) were positive by at least one of the four assays used in the study. The molecular assays were more sensitive for diagnosis in the early stages of illness (2-5 days post onset) when antibodies were not detectable. In the later stages of illness, the IgM ELISA is a more sensitive diagnostic test. In conclusion we recommend that the IgM ELISA be used as an initial screening test followed one of the molecular assays in samples that are collected in the early phase of illness and negative for CHIKV IgM antibodies. Such as approach would enable rapid confirmation of the diagnosis and implementation of public health measures especially during outbreaks.

  12. Comparison of Simplexa HSV 1 & 2 PCR with culture, immunofluorescence, and laboratory-developed TaqMan PCR for detection of herpes simplex virus in swab specimens.

    PubMed

    Gitman, Melissa R; Ferguson, David; Landry, Marie L

    2013-11-01

    The Simplexa HSV 1 & 2 direct PCR assay was compared with conventional cell culture, cytospin-enhanced direct fluorescent antibody (DFA), and a laboratory-developed real-time TaqMan PCR (LDT HSV PCR) using extracted nucleic acid for the detection of herpes simplex virus (HSV) in dermal, genital, mouth, ocular, and other swab samples. One hundred seventy-one swabs were tested prospectively, and 58 were positive for HSV (34 HSV-1 and 24 HSV-2). Cytospin-DFA detected 50 (86.2%), conventional cell culture 51 (87.9%), Simplexa direct 55 (94.8%), and LDT HSV PCR 57 (98.3%) of 58 true positives. Simplexa direct detected more positives than DFA and culture, but the differences were not significant (P = 0.0736 and P = 0.3711, respectively, by the McNemar test). Samples that were positive by all methods (n = 48) were strong positives (LDT cycle threshold [CT] value, 14.4 to 26.1). One strongly positive sample was falsely negative by LDT HSV PCR due to a failure of TaqMan probe binding. Three samples falsely negative by Simplexa direct had high CT values by LDT HSV PCR (LDT CT, 35.8 to 38.2). Omission of the DNA extraction step by Simplexa direct led to a drop in sensitivity compared to the sensitivity of LDT HSV PCR using extracted samples (94.8% versus 98.3%, respectively), but the difference was not significant (P = 0.6171). Simplexa HSV 1 & 2 direct PCR was the most expensive but required the least training of the assays used, had the lowest hands-on time and fastest assay time (75 min, versus 3 h by LDT HSV PCR), and provided the HSV type.

  13. Locked Nucleic Acid and Flow Cytometry-Fluorescence In Situ Hybridization for the Detection of Bacterial Small Noncoding RNAs

    PubMed Central

    Robertson, Kelly L.

    2012-01-01

    We describe the development and testing of a high-throughput method that enables the detection of small noncoding RNAs (ncRNAs) from single bacterial cells using locked nucleic acid probes (LNA) and flow cytometry-fluorescence in situ hybridization (flow-FISH). The LNA flow-FISH method and quantitative reverse transcription-PCR (qRT-PCR) were used to monitor the expression of three ncRNAs (6S, CsrB, and TPP-2) in Vibrio campbellii ATCC BAA-1116 cultures during lag phase, mid-log phase, and stationary phase. Both LNA flow-FISH and qRT-PCR revealed that CsrB and TPP-2 were highly expressed during lag phase but markedly reduced in mid-log phase and stationary phase, whereas 6S demonstrated no to little expression during lag phase but increased thereafter. Importantly, while LNA flow-FISH and qRT-PCR demonstrated similar overall expression trends, only LNA flow-FISH, which enabled the detection of ncRNAs in individual cells as opposed to the lysate-based ensemble measurements generated by qRT-PCR, was able to capture the cell-to-cell heterogeneity in ncRNA expression. As such, this study demonstrates a new method that simultaneously enables the in situ detection of ncRNAs and the determination of gene expression heterogeneity within an isogenic bacterial population. PMID:22057868

  14. Molecular cloning and functional characterization of arachidonate 5-lipoxygenase (Alox5), and its expression in response to the ratio of linolenic acid to linoleic acid in diets of large yellow croaker (Larmichthys crocea).

    PubMed

    Wang, Tianjiao; Zuo, Rantao; Mai, Kangsen; Xu, Wei; Ai, Qinghui

    2016-11-01

    This study was conducted to clone and functionally characterize a full-length cDNA encoding arachidonate 5-lipoxygenase (Alox5) from large yellow croaker (Larmichthys crocea) and investigate its gene expression in response to graded dietary ratio of linolenic acid (ALA) to linoleic acid (LNA) (0.03, 0.06, 0.45, 0.90 and 1.51). An isolated 2372bp cDNA clone of Alox5 contained an open reading frame spanning 2025bp encoding a protein with the ability to modify arachidonate acid (AA) to 5-hydroxyeicosatetraenoic (5-HETE). In the liver, the Alox5 mRNA expression levels significantly increased to the maximum when the dietary ALA/LNA increased from 0.03 to 0.06, and then significantly decreased with dietary ALA/LNA increased to 1.51 (P<0.05). In the kidney, the expression levels of Alox5 of fish fed diets with low dietary ALA/LNA (0.03-0.06) were significantly higher than those of fish fed diets with high dietary ALA/LNA (0.45-1.51) (P<0.05). The dual-luciferase reporter assays showed that the nuclear factor kappa B (NF-κB) could act on cognate cis-acting elements in the promoter of Alox5 and increased the transcription of Alox5. Results of the present study suggested that the expression of Alox5 is higher in croakers fed high concentrations of LNA compared to those fed high concentrations of ALA, which might be regulated by NF-κB and contribute to the inflammation process by catalyzing the dioxygenation of AA.

  15. Enhancing the intestinal absorption of low molecular weight chondroitin sulfate by conjugation with α-linolenic acid and the transport mechanism of the conjugates.

    PubMed

    Xiao, Yuliang; Li, Pingli; Cheng, Yanna; Zhang, Xinke; Sheng, Juzheng; Wang, Decai; Li, Juan; Zhang, Qian; Zhong, Chuanqing; Cao, Rui; Wang, Fengshan

    2014-04-25

    The purpose of this report was to demonstrate the effect of amphiphilic polysaccharides-based self-assembling micelles on enhancing the oral absorption of low molecular weight chondroitin sulfate (LMCS) in vitro and in vivo, and identify the transepithelial transport mechanism of LMCS micelles across the intestinal barrier. α-Linolenic acid-low molecular weight chondroitin sulfate polymers(α-LNA-LMCS) were successfully synthesized, and characterized by FTIR, (1)HNMR, TGA/DSC, TEM, laser light scattering and zeta potential. The significant oral absorption enhancement and elimination half-life (t₁/₂) extension of LNA-LMCS2 in rats were evidenced by intragastric administration in comparison with CS and LMCS. Caco-2 transport studies demonstrated that the apparent permeability coefficient (Papp) of LNA-LMCS2 was significantly higher than that of CS and LMCS (p<0.001), and no significant effects on the overall integrity of the monolayer were observed during the transport process. In addition, α-LNA-LMCS micelles accumulated around the cell membrane and intercellular space observed by confocal laser scanning microscope (CLSM). Furthermore, evident alterations in the F-actin cytoskeleton were detected by CLSM observation following the treatment of the cell monolayers with α-LNA-LMCS micelles, which further certified the capacity of α-LNA-LMCS micelles to open the intercellular tight junctions rather than disrupt the overall integrity of the monolayer. Therefore, LNA-LMCS2 with low cytotoxicity and high bioavailability might be a promising substitute for CS in clinical use, such as treating osteoarthritis, atherosclerosis, etc.

  16. Effects of metal ions on lipid peroxidation in cultured rat hepatocytes loaded with alpha-linolenic acid.

    PubMed

    Furono, K; Suetsuga, T; Sugihara, N

    1996-06-07

    We investigated the ability of various redox-active metal ions to induce lipid peroxidation in normal and alpha-linolenic acid-loaded (LNA-loaded) cultured rat hepatocytes. Lipid peroxidation was estimated by the accumulation of malondialdehyde (MDA) in the culture medium. At low concentrations induction was highest with ferrous ions (Fe), whereas at high concentrations, vanadium (V) and copper ions (Cu) had the greatest effect on both groups of hepatocytes. With any one of the three metal ions, the extent of lipid peroxidation in LNA-loaded hepatocytes was several times greater compared to normal cells. In addition, upon the addition of Fe or V, LNA-loaded hepatocytes were injured whereas normal cells were not. The addition of Cu caused substantial cell injury in normal hepatocytes, and even greater injury in LNA-loaded cells. The prevention of lipid peroxidation in LNA-loaded hepatocytes by addition of an antioxidant like N,N'-diphenyl-p-phenylene-diamine (DPPD) almost completely prevented Fe- and V-induced cell injury, and reduced Cu-induced cell injury. alpha-Tocopherol behaved in a way similar to but less effective than DPPD. .OH radical scavengers such as mannitol and dimethyl sulfoxide (DMSO) had no effect on lipid peroxidation induced by any metal ions in LNA-loaded hepatocytes. Addition of cadmium ions (Cd), which required the lowest concentration to cause cell injury, induced a slight increase in lipid peroxidation in normal hepatocytes, but did not induce lipid peroxidation to the same extent as seen in LNA-loaded cells treated with any of the three metal ions already mentioned. The inhibition of lipid peroxidation by DPPD scarcely protected LNA-loaded hepatocytes from Cd-induced cell injury. None of the other metal ions including aluminum (Al), chromium (Cr), manganese (Mn), nickel (Ni), lead (Pb), and tin (Sn) ions, effectively induced lipid peroxidation in either group of hepatocytes, except cobalt ions (Co), which had a peroxidative effect in LNA

  17. Utility of a novel multiplex TaqMan PCR assay for metallo-β-lactamase genes plus other TaqMan assays in detecting genes encoding serine carbapenemases and clinically significant extended-spectrum β-lactamases.

    PubMed

    Swayne, Rosemary; Ellington, Matthew J; Curran, Martin D; Woodford, Neil; Aliyu, Sani H

    2013-10-01

    Prompt detection of infections caused by Enterobacteriaceae that produce therapeutically important β-lactamases [metallo-β-lactamases (MBLs), serine carbapenemases, acquired AmpC and CTX-M extended-spectrum β-lactamases (ESBLs)] is crucial for infection prevention and control and surveillance purposes, and, more contentiously, also for effective patient management. A novel TaqMan PCR assay was developed to detect genes encoding IMP, VIM, NDM, SPM, SIM and GIM MBLs. Published PCR assays for acquired genes encoding CTX-M ESBLs and AmpC β-lactamases were updated and adapted to the TaqMan format, respectively. A published TaqMan assay for serine carbapenemase genes was used. Assay specificity was tested using a panel of 59 isolates with known acquired genes from the four different β-lactamase groupings. The four TaqMan assays correctly identified the most clinically relevant acquired β-lactamase genes in the panel of 59 resistant Enterobacteriaceae, which included 3 VIM-, 7 NDM- and 12 IMP-producers. Consecutive, non-duplicate isolates of Enterobacteriaceae from 965 urinary and 343 blood cultures during 2010 were then screened for β-lactamase genes using these TaqMan assays. Amongst the urinary and blood culture isolates tested, 69 CTX-M-producers and 21 acquired AmpC β-lactamase-producers were identified; the CTX-M rate amongst blood culture isolates (9.3%) broadly reflects the UK national average. During the study period, one Klebsiella pneumoniae isolate producing an NDM carbapenemase was identified from a wound sample. The assays developed and/or used will enable the future surveillance and the rapid detection and appropriate early treatment of infections caused by Gram-negative bacteria producing clinically important β-lactamases, including carbapenemases.

  18. Duplex TaqMan real-time PCR assay for quantitative detection of Pantoea stewartii subsp. stewartii and Stenocarpella maydis

    USDA-ARS?s Scientific Manuscript database

    A new TaqMan real-time PCR assay was developed for the simultaneous quantitative detection of two seedborne maize pathogens in a single assay. Pantoea stewartii subsp. stewartii (Pnss) (syn. Erwinia stewartii) is the causal agent of Stewart's bacterial wilt and leaf blight of maize. Stewart's wilt i...

  19. Comparison of TaqMan and SYBR Green qPCR methods for quantitative gene expression in tung tree tissues

    USDA-ARS?s Scientific Manuscript database

    Quantitative real-time-PCR (qPCR) is widely used for gene expression analysis due to its large dynamic range, tremendous sensitivity, high sequence-specificity, little to no post-amplification processing, and sample throughput. TaqMan and SYBR Green qPCR are two frequently used methods. However, dir...

  20. Comparison of TaqMan and SYBR Green qPCR methods for quantitative gene expression in tung tree tissues.

    PubMed

    Cao, Heping; Shockey, Jay M

    2012-12-19

    Quantitative real-time-PCR (qPCR) is widely used for gene expression analysis due to its large dynamic range, tremendous sensitivity, high sequence specificity, little to no postamplification processing, and sample throughput. TaqMan and SYBR Green qPCR are two frequently used methods. However, direct comparison of both methods using the same primers and biological samples is still limited. We compared both assays using seven RNAs from the seeds, leaves, and flowers of tung tree (Vernicia fordii), which produces high-value industrial oil. High-quality RNA were isolated from tung tissues, as indicated by a high rRNA ratio and RNA integrity number. qPCR primers and TaqMan probes were optimized. Under optimized conditions, both qPCR gave high correlation coefficiency and similar amplification efficiency, but TaqMan qPCR generated higher y-intercepts than SYBR Green qPCR, which overestimated the expression levels regardless of the genes and tissues tested. This is validated using well-known Dgat2 and Fadx gene expression in tung tissues. The results demonstrate that both assays are reliable for determining gene expression in tung tissues and that the TaqMan assay is more sensitive but generates lower calculated expression levels than the SYBR Green assay. This study suggests that any discussion of gene expression levels needs to be linked to which qPCR method is used in the analysis.

  1. TaqMan real-time quantitative PCR assay for detection of fluoroquinolone-resistant Neisseria gonorrhoeae.

    PubMed

    Zhao, LiHong; Zhao, ShuPing

    2012-12-01

    It is noted that more than 99 % of fluoroquinolone resistance in Neisseria gonorrhoeae (QRNG) specimens have been shown to have the mutation of Ser91/Phe in the gyrA gene. In order to detect QRNG isolates as quickly as possible, the real-time TaqMan quantitative PCR assay was established for detection of the point mutation of Ser91/Phe in gyrA gene. The standard curve was generated automatically on ABI Prism PE7500. The correlation coefficient (r) of the standard curve was -0.9984 (R(2) = 0.9968), indicating a quietly precise log-linear relationship between the concentration of target DNA and the Ct value. Presently, correlated, cultured antimicrobial susceptibility testing of N. gonorrhoeae isolates continues to be the gold standard method for the detection of antimicrobial resistance. Comparison to the correlated, cultured antimicrobial susceptibility testing, the sensitivity and specificity of the established TaqMan assay for the detection of the QRNG specimens were 100 and 99 %, respectively. The TaqMan assay also allows for rapid detection of QRNG isolates without complex laboratory techniques. Therefore, real-time TaqMan quantitative PCR assay is a rapid, simple, highly sensitive, highly specific, and easy-to-perform method for the detection of the QRNG specimens. It can be applied as a quick screening method for QRNG isolates to help clinical determination of optimal treatment prescription.

  2. Development and Evaluation of a Broadly Reactive TaqMan Assay for Rapid Detection of Hepatitis A Virus

    PubMed Central

    Jothikumar, N.; Cromeans, T. L.; Sobsey, M. D.; Robertson, B. H.

    2005-01-01

    Primers and a TaqMan probe for the 5′-untranslated region (UTR) of the hepatitis A virus (HAV) genome were designed and evaluated. The assay detected 0.5 infectious units of HAV and 40 copies of a synthetic transcript and provides an important screening tool for rapid quantitative HAV detection in clinical or environmental samples. PMID:15933042

  3. Detection of Ralstonia solanacearum Strains with a Quantitative, Multiplex, Real-Time, Fluorogenic PCR (TaqMan) Assay

    PubMed Central

    Weller, S. A.; Elphinstone, J. G.; Smith, N. C.; Boonham, N.; Stead, D. E.

    2000-01-01

    A fluorogenic (TaqMan) PCR assay was developed to detect Ralstonia solanacearum strains. Two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (RS) detected all biovars of R. solanacearum, and a second more specific probe (B2) detected only biovar 2A. Amplification of the target was measured by the 5′ nuclease activity of Taq DNA polymerase on each probe, resulting in emission of fluorescence. TaqMan PCR was performed with DNA extracted from 42 R. solanacearum and genetically or serologically related strains to demonstrate the specificity of the assay. In pure cultures, detection of R. solanacearum to ≥102 cells ml−1 was achieved. Sensitivity decreased when TaqMan PCR was performed with inoculated potato tissue extracts, prepared by currently recommended extraction procedures. A third fluorogenic probe (COX), designed with the potato cytochrome oxidase gene sequence, was also developed for use as an internal PCR control and was shown to detect potato DNA in an RS-COX multiplex TaqMan PCR with infected potato tissue. The specificity and sensitivity of the assay, combined with high speed, robustness, reliability, and the possibility of automating the technique, offer potential advantages in routine indexing of potato tubers and other plant material for the presence of R. solanacearum. PMID:10877778

  4. Detection of Food Allergens by Taqman Real-Time PCR Methodology.

    PubMed

    García, Aina; Madrid, Raquel; García, Teresa; Martín, Rosario; González, Isabel

    2017-01-01

    Real-time PCR (polymerase chain reaction) has shown to be a very effective technology for the detection of food allergens. The protocol described herein consists on a real-time PCR assay targeting the plant ITS (Internal Transcribed Spacer) region, using species-specific primers and hydrolysis probes (Taqman) dual labeled with a reporter fluorophore at the 5' end (6-carboxyfluorescein, FAM) and a quencher fluorophore at the 3' end (Blackberry, BBQ). The species-specific real-time PCR systems (primers/probe) described in this work allowed the detection of different nuts (peanut, hazelnut, pistachio, almond, cashew, macadamia, walnut and pecan), common allergens present in commercial food products, with a detection limit of 0.1 mg/kg.

  5. Detection of Xanthomonas oryzae pv. oryzae in seeds using a specific TaqMan probe.

    PubMed

    Zhao, Wen-Jun; Zhu, Shui-Fang; Liao, Xiao-Lan; Chen, Hong-Yun; Tan, Tian-Wei

    2007-02-01

    Xanthomonas oryzae pv. oryzae is the pathogen that causes bacterial leaf blight in rice. Bacterial leaf blight is the main cause for severe rice underproduction in many countries. However, with conventional methods it is difficult to quickly and reliably distinguish this pathogen from other closely related pathogenic bacteria, especially X. oryzae pv. oryzicola, the causal organism of bacterial leaf streak in rice. We have developed a novel and highly sensitive real-time method for the identification of this specific bacteria based on a TaqMan probe. This probe is designed to recognize the sequence of a putative siderophore receptor gene cds specific to X. oryzae pv. oryzae, and can be identified from either a bacterial culture or naturally infected rice seeds and leaves in only 2 h. The sensitivity of the method is 100 times higher than that of the current polymerase chain reaction (PCR) gel electrophoresis method for diagnosis.

  6. Dietary Docosahexaenoic Acid and trans-10, cis-12-Conjugated Linoleic Acid Differentially Alter Oxylipin Profiles in Mouse Periuterine Adipose Tissue.

    PubMed

    Adkins, Yuriko; Belda, Benjamin J; Pedersen, Theresa L; Fedor, Dawn M; Mackey, Bruce E; Newman, John W; Kelley, Darshan S

    2017-05-01

    Diets containing high n-3 polyunsaturated fatty acids (PUFA) decrease inflammation and the incidence of chronic diseases including cardiovascular disease and nonalcoholic fatty liver disease while trans-fatty acids (TFA) intake increases the incidence of these conditions. Some health benefits of n-3 PUFA are mediated through the impact of their oxygenated metabolites, i.e. oxylipins. The TFA, trans-10, cis-12-conjugated linoleic acid (CLA; 18:2n-6) is associated with adipose tissue (AT) inflammation, oxidative stress, and wasting. We examined the impact of a 4-week feeding of 0, 0.5, and 1.5% docosahexaenoic acid (DHA; 22:6n-3) in the presence and absence of 0.5% CLA on AT oxylipin profiles in female C57BL/6N mice. Esterified oxylipins in AT derived from linoleic acid (LNA), alpha-linolenic acid (ALA), arachidonic acid (ARA), eicosapentaenoic acid (EPA), DHA, and putative from CLA were quantified. CLA containing diets reduced AT mass by ~62%. Compared with the control diet, the DHA diet elevated concentrations of EPA-and DHA-derived alcohols and epoxides and LNA-derived alcohols, reduced ARA-derived alcohols, ketones, epoxides, and 6-keto-prostaglandin (PG) F1α (P < 0.05), and had mixed effects on ALA-derived alcohols. Dietary CLA lowered EPA-, DHA-, and ALA-derived epoxides, ARA-derived ketones and epoxides, and ALA-derived alcohols. While dietary CLA induced variable effects in EPA-, DHA-, and LNA-derived alcohols and LNA-derived ketones, it elevated ARA-derived alcohols and PGF1α, PGF2α, and F2-isoprostanes. DHA counteracted CLA-induced effects in 67, 57, 43, and 29% of total DHA-, ARA-, EPA-, and ALA-derived oxylipins, respectively. Thus, CLA elevated proinflammatory oxylipins while DHA increased anti-inflammatory oxylipins and diminished concentration of CLA-induced pro-inflammatory oxylipins in AT.

  7. Effects of polyunsaturated fatty acids on the proliferation of mitogen stimulated bovine peripheral blood mononuclear cells.

    PubMed

    Thanasak, J; Müller, K E; Dieleman, S J; Hoek, A; Noordhuizen, J P T M; Rutten, V P M G

    2005-04-08

    The present study aimed at analysis of the effects of polyunsaturated fatty acid (PUFA), linoleic acid (LA, C18:2n - 6) and linolenic acid (LNA, C18:3n - 3) on bovine peripheral blood mononuclear cells (PBMC) in vitro. Both mitogen (ConA)-induced proliferative lymphocyte responsiveness during 4 days of culture and eicosanoid (prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4))) production during 36 h were determined in relation to the absence or presence of various concentrations of LA and LNA (0, 1, 5, 25, 125 and 250 microM). Mitogen-driven proliferative responses of lymphocytes tended to be uninfluenced in the presence of lower concentrations of LA, whereas significant inhibition was observed at the higher concentrations of LA (125 and 250 microM). However, increasing amounts of LNA did not affect the proliferation. ConA stimulation induced a clear PGE(2) response, which significantly decreased in the presence of 250 microM of LA. In addition, increasing amounts of LNA, but not LA, led to a significant decrease in LTB(4) levels. However, The production of LTB(4) did not alter due to mitogenic stimulation. In conclusion, the present study shows that bovine mononuclear cells may functionally be influenced by the presence of PUFA in their environment. Further studies need to be conducted to clarify in vivo consequences of these findings in a situation of PUFA enriched rations in ruminants.

  8. Development and evaluation of serotype- and group-specific fluorogenic reverse transcriptase PCR (TaqMan) assays for dengue virus.

    PubMed

    Callahan, J D; Wu, S J; Dion-Schultz, A; Mangold, B E; Peruski, L F; Watts, D M; Porter, K R; Murphy, G R; Suharyono, W; King, C C; Hayes, C G; Temenak, J J

    2001-11-01

    Five fluorogenic probe hydrolysis (TaqMan) reverse transcriptase PCR (RT-PCR) assays were developed for serotypes 1 to 4 and group-specific detection of dengue virus. Serotype- and group-specific oligonucleotide primers and fluorogenic probes were designed against conserved regions of the dengue virus genome. The RT-PCR assay is a rapid single-tube method consisting of a 30-min RT step linked to a 45-cycle PCR at 95 and 60 degrees C that generates a fluorogenic signal in positive samples. Assays were initially evaluated against cell culture-derived dengue stock viruses and then with 67 dengue viremic human sera received from Peru, Indonesia, and Taiwan. The TaqMan assays were compared to virus isolation using C6/36 cells followed by an immunofluorescence assay using serotype-specific monoclonal antibodies. Viral titers in sera were determined by plaque assay in Vero cells. The serotype-specific TaqMan RT-PCR assay detected 62 of 67 confirmed dengue virus-positive samples, for a sensitivity of 92.5%, while the group-specific assay detected 66 of 67 confirmed dengue virus-positive samples, for a sensitivity of 98.5%. The TaqMan RT-PCR assays have a specificity of 100% based on the serotype concordance of all assays compared to cell culture isolation and negative results obtained when 21 normal human sera and plasma samples were tested. Our results demonstrate that the dengue virus TaqMan RT-PCR assays may be utilized as rapid, sensitive, and specific screening and serotyping tools for epidemiological studies of dengue virus infections.

  9. Robust Inhibitory Effects of Conjugated Linolenic Acids on a Cyclooxygenase-related Linoleate 10S-Dioxygenase: Comparison with COX-1 and COX-2

    PubMed Central

    Mashhadi, Zahra; Boeglin, William E.; Brash, Alan R.

    2015-01-01

    There are many reports of the anti-inflammatory, anti-cancer, and anti-atherosclerotic activities of conjugated linolenic acids (cLNA). They constitute a small percentage of fatty acids in the typical human diet, although up to 80% of the fatty acids in certain fruits such as pomegranate. In the course of studying a bacterial fatty acid dioxygenase (Nostoc linoleate 10S-DOX, an ancient relative of mammalian cyclooxygenases), we detected strong inhibitory activity in a commercial sample of linoleic acid. We identified two cLNA isomers, β-eleostearic (9E,11E,13E-18:3) and β-calendic acid (8E,10E,12E-18:3), as responsible for that striking inhibition with a Ki of ~49 nM and ~125 nM, respectively, the most potent among eight cLNA tested. We also examined the effects of all eight cLNA on the activity of COX-1 and COX-2. Jacaric acid (8Z,10E,12Z-18:3) and its 12E isomer, 8Z,10E,12E-18:3, strongly inhibit the activity of COX-1 with a Ki of ~1.7 and ~1.1 μM, respectively. By contrast, COX-2 was ≤ 30% inhibited at 10μM concentrations of the cLNA. Identifying the activities of the naturally occurring fatty acids is of interest in terms of understanding their interaction with the enzymes, and for explaining the mechanistic basis of their biological effects. The study also highlights the potential presence of inhibitory fatty acids in commercial lipids prepared from natural sources. Analysis of seven commercial samples of linoleic acid by HPLC and UV spectroscopy is illustrated as supplementary data. PMID:26209563

  10. Robust inhibitory effects of conjugated linolenic acids on a cyclooxygenase-related linoleate 10S-dioxygenase: Comparison with COX-1 and COX-2.

    PubMed

    Mashhadi, Zahra; Boeglin, William E; Brash, Alan R

    2015-10-01

    There are many reports of the anti-inflammatory, anti-cancer, and anti-atherosclerotic activities of conjugated linolenic acids (cLNA). They constitute a small percentage of fatty acids in the typical human diet, although up to 80% of the fatty acids in certain fruits such as pomegranate. In the course of studying a bacterial fatty acid dioxygenase (Nostoc linoleate 10S-DOX, an ancient relative of mammalian cyclooxygenases), we detected strong inhibitory activity in a commercial sample of linoleic acid. We identified two cLNA isomers, β-eleostearic (9E,11E,13E-18:3) and β-calendic acid (8E,10E,12E-18:3), as responsible for that striking inhibition with a Ki of ~49nM and ~125nM, respectively, the most potent among eight cLNA tested. We also examined the effects of all eight cLNA on the activity of COX-1 and COX-2. Jacaric acid (8Z,10E,12Z-18:3) and its 12E isomer, 8Z,10E,12E-18:3, strongly inhibit the activity of COX-1 with a Ki of ~1.7 and ~1.1μM, respectively. By contrast, COX-2 was ≤30% inhibited at 10μM concentrations of the cLNA. Identifying the activities of the naturally occurring fatty acids is of interest in terms of understanding their interaction with the enzymes, and for explaining the mechanistic basis of their biological effects. The study also highlights the potential presence of inhibitory fatty acids in commercial lipids prepared from natural sources. Analysis of seven commercial samples of linoleic acid by HPLC and UV spectroscopy is illustrated as supplementary data.

  11. Accuracy of genotyping using the TaqMan PCR assay for single nucleotide polymorphisms responsible for thiopurine sensitivity in Japanese patients with inflammatory bowel disease.

    PubMed

    Osaki, Rie; Imaeda, Hirotsugu; Ban, Hiromitsu; Aomatsu, Tomoki; Bamba, Shigeki; Tsujikawa, Tomoyuki; Sasaki, Masaya; Fujiyama, Yoshihide; Andoh, Akira

    2011-09-01

    Thiopurine drugs are the most common drugs used to maintain clinical remission in inflammatory bowel disease (IBD). Three single-nucleotide polymorphisms (SNPs), TPMT A719G (rs1142345), inosine triphosphate pyrophosphatase (ITPase) C94A (rs1127354) and multidrug resistance protein 4 MRP4 G2269A (rs3765534), have been reported to account for heightened sensitivity to thiopurine drugs in the Japanese population. We investigated the usefulness of the TaqMan(®) PCR assay (Applied Biosystems) for the rapid detection of these SNPs to improve the safety of thiopurine therapy. We enrolled 44 healthy volunteers and 235 IBD patients. Genotyping of the SNPs was performed using Custom TaqMan SNP genotyping assays, direct sequencing and PCR-RFLP. Genotyping for MRP4 G2269A by the TaqMan PCR assay was successfully achieved in all samples. Comparison with our previous data using direct sequencing indicated one discordant result, and re-sequencing showed that the TaqMan PCR assay was correct. The overall accuracy of the TaqMan assay for MRP4 G2269A was 100%. The TaqMan PCR genotyping for TPMT A719G and ITPase C94A was successfully performed in all samples. The results of TPMT A719G by the TaqMan assay were identical with those of PCR-RFLP. In ITPase C94A, a comparison of the TaqMan assay and PCR-RFLP yielded 12 discordant results, and direct sequencing showed that the TaqMan PCR assay was correct. The allelic frequency determined by the TaqMan assay was 0.145 for MRP4 G2269A, 0.009 for TPMT A719G and 0.121 for ITPase C94A, respectively. In conclusion, the TaqMan(®) PCR assay is useful for genotyping of SNPs responsible for thiopurine sensitivity in Japanese IBD patients.

  12. Incorporation of n-3 fatty acids into plasma and liver lipids of rats: importance of background dietary fat.

    PubMed

    MacDonald-Wicks, Lesley K; Garg, Manohar L

    2004-06-01

    The health benefits of long-chain n-3 PUFA (20:5n-3 and 22:6n-3) depend on the extent of incorporation of these FA into plasma and tissue lipids. This study aimed to investigate the effect of the background dietary fat (saturated, monounsaturated, or n-6 polyunsaturated) on the quantitative incorporation of dietary 18:3n-3 and its elongated and desaturated products into the plasma and the liver lipids of rats. Female weanling Wistar rats (n = 54) were randomly assigned to six diet groups (n = 9). The fat added to the semipurified diets was tallow (SFA), tallow plus linseed oil (SFA-LNA), sunola oil (MUFA), sunola oil plus linseed oil (MUFA-LNA), sunflower oil (PUFA), or sunflower oil plus linseed oil (PUFA-LNA). At the completion of the 4-wk feeding period, quantitative FA analysis of the liver and plasma was undertaken by GC. The inclusion of linseed oil in the rat diets increased the level of 18:3n-3, 20:5n-3, and, to a smaller degree, 22:6n-3 in plasma and liver lipids regardless of the background dietary fat. The extent of incorporation of 18:3n-3, 20:5n-3, and 22:5n-3 followed the order SFA-LNA > MUFA-LNA > PUFA-LNA. Levels of 22:6n-3 were increased to a similar extent regardless of the type of major fat in the rat diets. This indicates that the background diet affects the incorporation in liver and plasma FA pools of the n-3 PUFA with the exception of 22:6n-3 and therefore the background diet has the potential to influence the already established health benefits of long-chain n-3 fatty acids.

  13. TaqMan probes as blocking agents for enriched PCR amplification and DNA melting analysis of mutant genes.

    PubMed

    Botezatu, Irina V; Panchuk, Irina O; Stroganova, Anna M; Senderovich, Anastasia I; Kondratova, Valentina N; Shelepov, Valery P; Lichtenstein, Anatoly V

    2017-02-01

    Asymmetric PCR and DNA melting analysis with TaqMan probes applied for mutation detection is effectively used in clinical diagnostics. The method is simple, cost-effective, and carried out in a closed-tube format, minimizing time, labor, and risk of sample cross-contamination. Although DNA melting analysis is more sensitive than Sanger sequencing (mutation detection thresholds are ~5% and 15%-20%, respectively), it is less sensitive than more labor-intensive and expensive techniques such as pyrosequencing and droplet digital PCR. Here, we demonstrate that, under specially selected conditions of asymmetric PCR, TaqMan probes can play the role of blocking agents. Preferential blocking of the wild-type allele brings about enriched amplification of mutant alleles. As a result, an ~10-fold increase in the detection sensitivity for mutant BRAF and NRAS genes was achieved.

  14. Development of TaqMan real-time polymerase chain reaction for the detection and identification of Penicillium marneffei.

    PubMed

    Pornprasert, Sakorn; Praparattanapan, Jutarat; Khamwan, Chantana; Pawichai, Sudjai; Pimsarn, Parichat; Samleerat, Tanawan; Leechanachai, Pranee; Supparatpinyo, Khunchai

    2009-11-01

    Penicillium marneffei is a dimorphic fungus, which is endemic in Southeast Asia and responsible for emerging opportunistic infections. Diagnosis of penicilliosis may be difficult when few yeast cells are present, while a gold standard diagnosis technique requires long-term culture. In order to provide a more rapid and accurate diagnosis, we developed a TaqMan real-time PCR to detect and identify P. marneffei DNA coding for 5.8S rRNA in purified yeast DNA and clinical samples. All P. marneffei DNA preparations could be detected using specific primers and TaqMan probe. The assay has a sensitivity to detect at least 10 yeast cells in seeded blood. Moreover, it can detect P. marneffei DNA in peripheral blood samples and blood-culture bottles. Therefore, the real-time PCR assay may represent a potential tool for early diagnosis of penicilliosis marneffei.

  15. Detection and quantification of apoptosis in primary cells using Taqman® protein assay.

    PubMed

    Pfister, Christina; Pfrommer, Heike; Tatagiba, Marcos S; Roser, Florian

    2015-01-01

    There are several methods to detect apoptosis using cleaved caspase-3 and each harbors its own advantages and disadvantages. When primary cell cultures are used, the disadvantages of the standard methods can make apoptosis detection difficult due to their slow growth rate and replicative senescence, thereby limiting the available cell number and experiment time span. In this chapter, we describe apoptosis detection and quantification using an innovative method named TaqMan(®) protein assay. TaqMan(®) protein assay uses antibodies and proximity ligation for quantitative real-time PCR. Biotinylated antibodies are labeled with oligonucleotides. When the labeled antibodies bind in close proximity, the oligonucleotides are connected using DNA ligase. The ligation product is amplified and detected using Taqman(®) based Real-Time PCR. Using this technique, we can not only detect apoptosis with a 1,000-fold higher sensitivity than western blot, but we can also exactly quantify cleaved caspase-3 expression. Thereby apoptosis can be determined and quantified in a fast reliable manner.

  16. An updated TaqMan real-time PCR for canine and feline parvoviruses.

    PubMed

    Streck, André Felipe; Rüster, Dana; Truyen, Uwe; Homeier, Timo

    2013-10-01

    Canine parvovirus type 2 (CPV-2) emerged in late 1970s from the feline panleukopenia virus (FPLV) and developed, since then, into novel genetic and antigenic variants (CPV-2a, -2b and -2c). Canine and feline parvoviruses cause an acute enteric disease in their hosts, with high level of viral shedding. In this study, a quantitative TaqMan PCR for detection and quantitation of canine and feline parvoviruses in serum and fecal samples was developed. The primers were designed based upon the entire GenBank content for CPV and FPLV. A standard curve was generated, and validation tests were performed using 10-fold serial dilutions of CPV-2 virus in CPV/FPLV-negative feces and CPV/FPLV-negative serum samples. As a result, the 100% detection limit of the PCR was 18 copies of the viral genome per μl of serum and fecal sample. All canine parvovirus types as well as FPLV were detected. In conclusion, the real-time PCR represents an upgraded and useful tool to identify and quantify canine and feline parvoviruses in different sample matrices.

  17. SNP genotyping using TaqMan technology: the CYP2D6*17 assay conundrum.

    PubMed

    Gaedigk, Andrea; Freeman, Natalie; Hartshorne, Toinette; Riffel, Amanda K; Irwin, David; Bishop, Jeffrey R; Stein, Mark A; Newcorn, Jeffrey H; Jaime, Lazara Karelia Montané; Cherner, Mariana; Leeder, J Steven

    2015-03-19

    CYP2D6 contributes to the metabolism of many clinically used drugs and is increasingly tested to individualize drug therapy. The CYP2D6 gene is challenging to genotype due to the highly complex nature of its gene locus. TaqMan technology is widely used in the clinical and research settings for genotype analysis due to assay reliability, low cost, and the availability of commercially available assays. The assay identifying 1023C>T (rs28371706) defining a reduced function (CYP2D6*17) and several nonfunctional alleles, produced a small number of unexpected diplotype calls in three independent sets of samples, i.e. calls suggested the presence of a CYP2D6*4 subvariant containing 1023C>T. Gene resequencing did not reveal any unknown SNPs in the primer or probe binding sites in any of the samples, but all affected samples featured a trio of SNPs on their CYP2D6*4 allele between one of the PCR primer and probe binding sites. While the phenomenon was ultimately overcome by an alternate assay utilizing a PCR primer excluding the SNP trio, the mechanism causing this phenomenon remains elusive. This rare and unexpected event underscores the importance of assay validation in samples representing a variety of genotypes, but also vigilance of assay performance in highly polymorphic genes such as CYP2D6.

  18. Performance of a Taqman Assay for Improved Detection and Quantification of Human Rhinovirus Viral Load

    PubMed Central

    Ng, Kim Tien; Chook, Jack Bee; Oong, Xiang Yong; Chan, Yoke Fun; Chan, Kok Gan; Hanafi, Nik Sherina; Pang, Yong Kek; Kamarulzaman, Adeeba; Tee, Kok Keng

    2016-01-01

    Human rhinovirus (HRV) is the major aetiology of respiratory tract infections. HRV viral load assays are available but limitations that affect accurate quantification exist. We developed a one-step Taqman assay using oligonucleotides designed based on a comprehensive list of global HRV sequences. The new oligonucleotides targeting the 5′-UTR region showed high PCR efficiency (E = 99.6%, R2 = 0.996), with quantifiable viral load as low as 2 viral copies/μl. Assay evaluation using an External Quality Assessment (EQA) panel yielded a detection rate of 90%. When tested on 315 human enterovirus-positive specimens comprising at least 84 genetically distinct HRV types/serotypes (determined by the VP4/VP2 gene phylogenetic analysis), the assay detected all HRV species and types, as well as other non-polio enteroviruses. A commercial quantification kit, which failed to detect any of the EQA specimens, produced a detection rate of 13.3% (42/315) among the clinical specimens. Using the improved assay, we showed that HRV sheds in the upper respiratory tract for more than a week following acute infection. We also showed that HRV-C had a significantly higher viral load at 2–7 days after the onset of symptoms (p = 0.001). The availability of such assay is important to facilitate disease management, antiviral development, and infection control. PMID:27721388

  19. [Detection of human enteroviruses with real-time PCR assay using TaqMan fluorescent probe].

    PubMed

    Leś, Katarzyna; Przybylski, Maciej; Dzieciatkowski, Tomasz; Młynarczyk, Grazyna

    2010-01-01

    Infections with human enteroviruses are common worldwide and cause a wide range of signs and symptoms. Nowadays in current diagnostics procedures older virological methods, such virus isolation in a cell cultures and seroneutralisation assay, are replaced with molecular biology tests. The aim of the study was development of real-time PCR assay for detection of human adenoviruses. DNA isolated from MK2 cell line infected with nineteen different enterovirus strains was used for development of a qualitative real-time PCR assay using primers targeting a conserved region of the 5'UTR region and a specific TaqMan probe. The analytical sensitivity of real-time PCR assay was tested using serial dilutions of Coxackie A9 cDNA in range between 10 degrees and 10(-8). For comparison typical end-point detected RT-PCR for enterovirus detection with the same cDNA dilutions was made. The sensitivity of novel method was about ten thousand-fold higher than older one. The conclusion is that real-time PCR is very advisable in diagnostics of diseases caused with enteroviruses. The high level of sensitivity, specificity, accuracy, and rapidity provided by this assay are favorable for the use in the detection of enteroviral RNA in clinical specimens, especially from neuroinfections.

  20. Identification of the main malaria vectors in the Anopheles gambiae species complex using a TaqMan real-time PCR assay.

    PubMed

    Bass, Chris; Williamson, Martin S; Wilding, Craig S; Donnelly, Martin J; Field, Linda M

    2007-11-22

    The Anopheles gambiae sensu lato species complex comprises seven sibling species of mosquitoes that are morphologically indistinguishable. Rapid identification of the two main species which vector malaria, Anopheles arabiensis and An. gambiae sensu stricto, from the non-vector species Anopheles quadriannulatus is often required as part of vector control programmes. Currently the most widely used method for species identification is a multiplex PCR protocol that targets species specific differences in ribosomal DNA sequences. While this assay has proved to be reasonably robust in many studies, additional steps are required post-PCR making it time consuming. Recently, a high-throughput assay based on TaqMan single nucleotide polymorphism genotyping that detects and discriminates An. gambiae s.s and An. arabiensis has been reported. A new TaqMan assay was developed that distinguishes between the main malaria vectors (An. arabiensis and An. gambiae s.s.) and the non-vector An. quadriannulatus after it was found that the existing TaqMan assay incorrectly identified An. quadriannulatus, An. merus and An. melas as An. gambiae s.s. The performance of this new TaqMan assay was compared against the existing TaqMan assay and the standard PCR method in a blind species identification trial of over 450 samples using field collected specimens from a total of 13 countries in Sub-Saharan Africa. The standard PCR method was found to be specific with a low number of incorrect scores (<1%), however when compared to the TaqMan assays it showed a significantly higher number of failed reactions (15%). Both the new vector-specific TaqMan assay and the exisiting TaqMan showed a very low number of incorrectly identified samples (0 and 0.54%) and failed reactions (1.25% and 2.96%). In tests of analytical sensitivity the new TaqMan assay showed a very low detection threshold and can consequently be used on a single leg from a fresh or silica-dried mosquito without the need to first extract

  1. A High-Fat, High-Oleic Diet, But Not a High-Fat, Saturated Diet, Reduces Hepatic α-Linolenic Acid and Eicosapentaenoic Acid Content in Mice.

    PubMed

    Picklo, Matthew J; Murphy, Eric J

    2016-05-01

    Considerable research has focused upon the role of linoleic acid (LNA; 18:2n-6) as a competitive inhibitor of α-linolenic (ALA; 18:3n-3) metabolism; however, little data exist as to the impact of saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) on ALA metabolism. We tested the hypothesis that a high SFA diet, compared to a high MUFA (oleic acid 18:1n-9) diet, reduces ALA conversion to long chain n-3 fatty acids. Mice were fed for 12 weeks on three diets: (1) a control, 16 % fat energy diet consisting of similar levels of SFA and MUFA (2) a 50 % fat energy high MUFA energy diet (35 % MUFA and 7 % SFA) or (3) a 50 % fat energy, high SFA energy diet (34 % SFA, 8 % MUFA). ALA and LNA content remained constant. Analysis of hepatic lipids demonstrated a selective reduction (40 %) in ALA but not LNA and a 35 % reduction in eicosapentaenoic acid (EPA; 20:5n-3) in the high MUFA mice compared to the other groups. Lower content of ALA was reflected in the neutral lipid fraction, while smaller levels of phospholipid esterified EPA and docosapentaenoic acid (DPA; 22:5n-3) were evident. Docosahexaenoic acid (DHA; 22:6n-3) content was elevated by the high SFA diet. Expression of Fads1 (Δ5 desaturase) and Fads2 (Δ6 desaturase) was elevated by the high MUFA and reduced by the high SFA diet. These data indicate that a high MUFA diet, but not a high SFA diet, reduces ALA metabolism and point to selective hepatic disposition of ALA versus LNA.

  2. Optimization of melting analysis with TaqMan probes for detection of KRAS, NRAS, and BRAF mutations.

    PubMed

    Botezatu, Irina V; Nechaeva, Irina O; Stroganova, Аnna М; Senderovich, Anastasia I; Kondratova, Valentina N; Shelepov, Valery P; Lichtenstein, Anatoly V

    2015-12-15

    The TaqMan probes that have been long and effectively used in real-time polymerase chain reaction (PCR) may also be used in DNA melting analysis. We studied some factors affecting efficiency of the approach such as (i) number of asymmetric PCR cycles preceding DNA melting analysis, (ii) choice of fluorophores for the multiplex DNA melting analysis, and (iii) choice of sense or antisense TaqMan probes for optimal resolution of wild-type and mutant alleles. We also determined ΔTm (i.e., the temperature shift of a heteroduplex relative to the corresponding homoduplex) as a means of preliminary identification of mutation type. In experiments with serial dilution of mutant KRAS DNA with wild-type DNA, the limit of detection of mutant alleles was 1.5-3.0%. Using DNA from both tumor and formalin-fixed paraffin-embedded tissues, we demonstrated a high efficiency of TaqMan probes in mono- and multiplex mutation scanning of KRAS, NRAS (codons 12, 13, and 61), and BRAF (codon 600) genes. This cost-effective method, which can be applied to practically any mutation hot spot in the human genome, combines simplicity, ease of execution, and high sensitivity-all of the qualities required for clinical genotyping.

  3. A high-fat, high-oleic diet, but not a high-fat, saturated diet, reduces hepatic n3 fatty acid content in mice

    USDA-ARS?s Scientific Manuscript database

    While considerable research has centered upon the role of linoleic acid (LNA; 18:2n6) as a competitive inhibitor of alpha-linolenic (ALA; 18:3n3) metabolism, a growing literature indicates that the amount of fat consumed can reduce the elongation and desaturation process. However, little data exist ...

  4. Stage of harvest and polyunsaturated essential fatty acid concentrations in purslane (Portulaca oleraceae) leaves.

    PubMed

    Palaniswamy, U R; McAvoy, R J; Bible, B B

    2001-07-01

    Purslane is a nutritious vegetable crop rich in the polyunsaturated essential fatty acids (PUEFA) alpha-linolenic acid (LNA) and linoleic acid (LA), which are essential for normal human growth, health promotion, and disease prevention. Total lipids and fatty acid concentrations at three stages of harvest (6-, 10-, and 14-true-leaf stages) were examined in a cultivated variety of purslane (Portulaca oleraceae L. var. sativa). The 14-true-leaf stage of growth was found to be ideal for harvest because at this stage the leaf area, shoot fresh weight, shoot dry weight, and PUEFA concentrations per gram of leaf fresh weight were higher (P < or = 0.05) than at the 6- and 10-true-leaf stages of growth. The LNA to LA ratio was also highest at the 14-true-leaf stage.

  5. Comparison of the ABI 7700 System (TaqMan) and Competitive PCR for Quantification of IS6110 DNA in Sputum during Treatment of Tuberculosis

    PubMed Central

    Desjardin, L. e.; Chen, Y.; Perkins, M. D.; Teixeira, L.; Cave, M. D.; Eisenach, K. D.

    1998-01-01

    Mycobacterium tuberculosis can persist in sputum for long periods of time after the initiation of antituberculosis chemotherapy. The purpose of this study was to determine whether quantitative estimates of M. tuberculosis DNA in sputum correlate with the numbers of viable bacilli and thus measure the therapeutic response of patients during treatment. Two methods of M. tuberculosis DNA quantification were examined by using DNA isolated from sputum specimens serially collected during the course of chemotherapy. A competitive PCR assay was compared to an automated system of real-time quantification with the ABI Prism 7700 Sequence Detection System (TaqMan). The ABI 7700 system uses standard PCR in conjunction with a fluorogenic probe in which the intensity of fluorescence is proportional to the amount of target DNA present. The results showed that both PCR systems are reproducible and accurate. The amounts of M. tuberculosis DNA quantified in sputum corresponded well with the numbers of acid-fast bacilli (AFB) counted by microscopy. Before initiation of antituberculosis therapy, measures of AFB, M. tuberculosis DNA, and cultivable bacilli were similar, suggesting that quantification of DNA is a good method for measuring the initial bacillary load. However, the rate of disappearance of both AFB and M. tuberculosis DNA did not correlate with the decline in cultivable bacilli in the specimen; therefore, these tests are not appropriate for monitoring treatment efficacy. PMID:9650945

  6. Assessment of environmental factors on Listeria monocytogenes Scott A inlA gene expression by relative quantitative Taqman real-time reverse transcriptase PCR.

    PubMed

    Hanna, Scott E; Wang, Hua H

    2006-11-01

    Several virulence factors are involved in Listeria monocytogenes pathogenicity. L. monocytogenes internalins, particularly internalin A, are required for bacterial adhesion to and invasion of human intestinal epithelial cells. The expression of internalins is thus related to virulence. Identification of conditions involved in regulating the expression of L. monocytogenes virulence factors is essential for developing targeted strategies to control listeriosis incidence and improving therapeutic approaches. The primary aim of this study was to develop a quantitative real-time reverse transcriptase PCR platform to study the impact of environmental factors on L. monocytogenes Scott A virulence factor expression, particularly in potentially complex ecosystems. A Taqman PCR-based, rapid quantitative gene expression evaluation method was established with the L. monocytogenes ribosomal protein L4 encoding gene used as an internal standard. Our data suggest that inlA expression is influenced by food composition and temperature, indicating that certain food processing or storage conditions, such as the use of lactic and acetic acids at common storage temperatures, could affect the expression of L. monocytogenes virulence factor.

  7. Application of a spotting sample preparation technique for the detection of pathogens in woody plants by RT-PCR and real-time PCR (TaqMan).

    PubMed

    Osman, Fatima; Rowhani, Adib

    2006-05-01

    An extraction technique for reverse transcription-PCR (RT-PCR) detection of plant pathogens including viruses, bacteria and phytoplasma is described. The total nucleic acid of these plant pathogens was obtained by direct spotting of crude sap derived from infected leaf, petiole or cambial tissue onto two different types of membranes, positively charged Hybond N(+) Nylon and FTA membranes, and processed for use in PCR. Thirteen different plant viruses, Xylella fastidiosa (causal agent of Pierce's disease) and phytoplasmas were included in the experiment. A thermal treatment (95 degrees C for 10 min) of the Hybond N(+) Nylon discs in a buffered solution improved the detection, but for FTA membrane discs the thermal treatment was not required and the discs were directly placed in the PCR reaction cocktail. Specific amplification of genomic or ribosomal RNA fragments of these pathogens was obtained by one-step RT-PCR except for X. fastidiosa in which a fragment of the genomic DNA was used for amplification. The same sample preparation methods also worked well for real-time RT-PCR (TaqMan). The sample preparation techniques reported here could be used to store samples for future PCR test or for long distance shipment to a detection laboratory.

  8. Molecular detection of harmful algal blooms (HABs) using locked nucleic acids and bead array technology.

    PubMed

    Diaz, Mara R; Jacobson, James W; Goodwin, Kelly D; Dunbar, Sherry A; Fell, Jack W

    2010-06-01

    Harmful algal blooms (HABs) are a serious public health risk in coastal waters. As the intensity and frequency of HABs continue to rise, new methods of detection are needed for reliable identification. Herein, we developed a high-throughput, multiplex, bead array technique for the detection of the dinoflagellates Karenia brevis and Karenia mikimotoi. The method combined the Luminex detection system with two novel technologies: locked nucleic acid-modified oligonucleotides (LNA) and Mirus Label IT(®) nucleic acid technology. To study the feasibility of the method, we evaluated the performance of modified and unmodified LNA probes with amplicon targets that were biotin labeled with two different strategies: direct chemical labeling (Mirus Label IT) versus enzymatic end-labeling (single biotinylated primer). The results illustrated that LNA probes hybridized to complementary single-stranded DNA with better affinity and displayed higher fluorescence intensities than unmodified oligonucleotide DNA probes. The latter effect was more pronounced when the assay was carried out at temperatures above 53°C degree. As opposed to the enzymatic 5' terminal labeling technique, the chemical-labeling method enhanced the level of fluorescence by as much as ~83%. The detection limits of the assay, which were established with LNA probes and Mirus Label IT system, ranged from 0.05 to 46 copies of rRNA. This high-throughput method, which represents the first molecular detection strategy to integrate Luminex technology with LNA probes and Mirus Label IT, can be adapted for the detection of other HABs and is well suited for the monitoring of red tides at pre-blooming and blooming conditions.

  9. Antisense 2'-Deoxy, 2'-Fluoroarabino Nucleic Acid (2'F-ANA) Oligonucleotides: In Vitro Gymnotic Silencers of Gene Expression Whose Potency Is Enhanced by Fatty Acids.

    PubMed

    Souleimanian, Naira; Deleavey, Glen F; Soifer, Harris; Wang, Sijian; Tiemann, Katrin; Damha, Masad J; Stein, Cy A

    2012-01-01

    Gymnosis is the process of the delivery of antisense oligodeoxynucleotides to cells, in the absence of any carriers or conjugation, that produces sequence-specific gene silencing. While gymnosis was originally demonstrated using locked nucleic acid (LNA) gapmers, 2'-deoxy-2'fluoroarabino nucleic acid (2'F-ANA) phosphorothioate gapmer oligonucleotides (oligos) when targeted to the Bcl-2 and androgen receptor (AR) mRNAs in multiple cell lines in tissue culture, are approximately as effective at silencing of Bcl-2 expression as the iso-sequential LNA congeners. In LNCaP prostate cancer cells, gymnotic silencing of the AR by a 2'F-ANA phosphorothioate gapmer oligo led to downstream silencing of cellular prostate-specific antigen (PSA) expression even in the presence of the androgenic steroid R1881 (metribolone), which stabilizes cytoplasmic levels of the AR. Furthermore, gymnotic silencing occurs in the absence of serum, and silencing by both LNA and 2'F-ANA oligos is augmented in serum-free (SF) media in some cell lines when they are treated with oleic acid and a variety of ω-6 polyunsaturated fatty acids (ω-6 PUFAs), but not by an aliphatic (palmitic) fatty acid. These results significantly expand our understanding of and ability to successfully manipulate the cellular delivery of single-stranded oligos in vitro.

  10. Mass Spectrometric Confirmation of γ-Linolenic Acid Ester-Linked Ceramide 1 in the Epidermis of Borage Oil Fed Guinea Pigs.

    PubMed

    Shin, Kyong-Oh; Kim, Kunpyo; Jeon, Sanghun; Seo, Cho-Hee; Lee, Yong-Moon; Cho, Yunhi

    2015-10-01

    Ceramide 1 (Cer1), a Cer species with eicosasphingenine (d20:1) amide-linked to two different ω-hydroxy fatty acids (C30wh:0:C32wh:1), which are, in turn, ester-linked to linoleic acid (LNA; 18:2n-6), plays a critical role in maintaining the structural integrity of the epidermal barrier. Prompted by the recovery of a disrupted epidermal barrier with dietary borage oil [BO: 36.5% LNA and 23.5% γ-linolenic acid (GLA; 18:3n-6)], in essential fatty acid (EFA)-deficient guinea pigs, we further investigated the effects of BO on the substitution of ester-linked GLA for LNA in these two epidermal Cer1 species by LC-MS in positive and negative modes. Dietary supplementation of BO for 2 weeks in EFA-deficient guinea pigs increased LNA ester-linked to C32wh:1/d20:1 and C30wh:0/d20:1 of Cer1. Moreover, GLA ester-linked to C32wh:1/d20:1, but not to C30wh:0/d20:1, of Cer1 was detected, which was further confirmed by the product ions of m/z 277.2 for ester-linked GLA and m/z 802.3 for the deprotonated C32wh:1/d20:1. C20-Metabolized fatty acids of LNA or GLA were not ester-linked to these Cer1 species. Dietary BO induced GLA ester-linked to C32wh:1/d20:1 of epidermal Cer1.

  11. Comparison of high-resolution melting analysis, TaqMan Allelic discrimination assay, and sanger sequencing for Clopidogrel efficacy genotyping in routine molecular diagnostics.

    PubMed

    Zhang, Lina; Cui, Guanglin; Li, Zongzhe; Wang, Haoran; Ding, Hu; Wang, Dao Wen

    2013-09-01

    Clopidogrel, as a routine antiplatelet drug, is widely used in patients to reduce cardiovascular events following percutaneous coronary intervention. Because of genetic variation, patients undergoing percutaneous coronary intervention show differing responses to clopidogrel therapy. Recently, five single nucleotide polymorphisms (SNPs) within CYP2C19 (rs4244285, rs4986893, rs12248560), ABCB1 (rs1045642), and ITGB3 (rs5918) were identified that contribute prominently to variability in response to clopidogrel. Given that Sanger sequencing is labor intensive and time consuming, rapid genotyping methods for SNP detection are urgently required before clopidogrel therapy. Accordingly, we developed a high-resolution melting analysis (HRMA) and TaqMan allelic discrimination assay (TaqMan) to genotype those five SNPs, and compared these two assays with Sanger sequencing on accuracy of genotyping as well as operational characteristics. These two assays showed high accuracy (0.995, 95% CI 0.991 to 0.998 for HRMA; 0.997, 95% CI 0.994 to 0.999 for TaqMan, respectively), sensitivity (0.996, 95% CI 0.989 to 1.002 for HRMA; 0.998, 95% CI 0.993 to 1.002 for TaqMan, respectively), and specificity (0.995, 95% CI 0.991 to 0.999 for HRMA; 0.996, 95% CI 0.993 to 1.000 for TaqMan, respectively). Our study indicates that HRMA and TaqMan are easier to operate and obviously faster than Sanger sequencing. In conclusion, HRMA and TaqMan are rapid, convenient, and reliable assays for clopidogrel efficacy genotyping.

  12. Inhibitors of 20-hydroxyeicosatetraenoic acid reduce renal vasoconstrictor responsiveness.

    PubMed

    Quilley, J; Qiu, Y; Hirt, J

    2003-10-01

    20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P450-derived constrictor eicosanoid produced by the preglomerular vasculature where it contributes to regulation of tone. Removal of the tonic inhibitory influence of nitric oxide (NO) has been reported to increase renal 20-HETE release. Because inhibition of NO synthesis enhances responses to vasoconstrictor agents, we examined a contribution for increased 20-HETE generation. In the rat kidney perfused with Krebs' buffer, responses to U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy PGF2alpha), a thromboxane A2 mimetic, were compared before and after 50 microM L-nitroarginine (L-NA) to inhibit NO synthase. L-NA raised perfusion pressure (PP) from 79 +/- 3 to 190 +/- 7 mm Hg and enhanced constrictor responsiveness to U46619. U46619 (10, 30, 100, and 300 ng) increased PP by 7 +/- 1, 17 +/- 2, 50 +/- 7, and 67 +/- 7 mm Hg, respectively, before L-NA and 15 +/- 1, 37 +/- 7, 68 +/- 10, and 85 +/- 11 mm Hg, respectively, after L-NA, which did not increase 20-HETE efflux from the kidney. Nonetheless, an inhibitor of omega-hydroxylase, dibromododecencyl methylsulfonimide (DDMS), which reduced 20-HETE release, normalized the enhanced responsiveness to U46619. When PP was elevated with phenylephrine, vasoconstrictor responses to U46619 were similarly enhanced, an effect that was also prevented by DDMS. DDMS and an antagonist of 20-HETE, 20-HEDE [20-hydroxyeicosa-6(Z), 15(Z)-dienoic acid], also reduced vasoconstrictor responses to U46619 in the absence of elevation of PP. Because 20-HETE inhibits K+ channels, we examined the effects of K+ channel inhibitors on vasoconstrictor responses and showed that both tetraethylammonium (TEA) and charybdotoxin enhanced renal vasoconstrictor responses to U46619. However, the inhibitory effects of 20-HEDE on vasoconstrictor responses remained after treatment with TEA. These results support a role for 20-HETE vasoconstrictor responses but suggest an action independent of K+ channels.

  13. Bacterial production of conjugated linoleic and linolenic Acid in foods: a technological challenge.

    PubMed

    Gorissen, Lara; Leroy, Frédéric; De Vuyst, Luc; De Smet, Stefaan; Raes, Katleen

    2015-01-01

    Conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA) isomers are present in foods derived from ruminants as a result of the respective linoleic acid (LA) and α-linolenic acid (LNA) metabolism by ruminal microorganisms and in animals' tissues. CLA and CLNA have isomer-specific, health-promoting properties, including anticarcinogenic, antiatherogenic, anti-inflammatory, and antidiabetic activity, as well as the ability to reduce body fat. Besides ruminal microorganisms, such as Butyrivibrio fibrisolvens, many food-grade bacteria, such as bifidobacteria, lactic acid bacteria (LAB), and propionibacteria, are able to convert LA and LNA to CLA and CLNA, respectively. Linoleate isomerase activity, responsible for this conversion, is strain-dependent and probably related to the ability of the producer strain to tolerate the toxic effects of LA and LNA. Since natural concentrations of CLA and CLNA in ruminal food products are relatively low to exert their health benefits, food-grade bacteria with linoleate isomerase activity could be used as starter or adjunct cultures to develop functional fermented dairy and meat products with increased levels of CLA and CLNA or included in fermented products as probiotic cultures. However, results obtained so far are below expectations due to technological bottlenecks. More research is needed to assess if bacterial production kinetics can be increased and can match food processing requirements.

  14. TaqMan qPCR for detection and quantification of mitochondrial DNA from toxic pufferfish species.

    PubMed

    Luekasemsuk, Tassanee; Panvisavas, Nathinee; Chaturongakul, Soraya

    2015-08-01

    Outbreaks of pufferfish food poisoning have been reported worldwide, most were from unsuitable food preparation and adulteration. In order to rapidly detect pufferfish adulterant in processed foods, we developed and proposed the use of a TaqMan probe-based quantitative PCR. The designed detection oligos targeted a unique region in 16S rDNA of toxic marine pufferfish in Tetraodontidae Family and gave a positive signal at ≥1.75 pg of genomic DNA. Non-target DNA samples from other fish, chicken, and beef were negative.

  15. Real-time TaqMan RT-PCR for detection of maize chlorotic mottle virus in maize seeds.

    PubMed

    Zhang, Yongjiang; Zhao, Wenjun; Li, Mingfu; Chen, Hongjun; Zhu, Shuifang; Fan, Zaifeng

    2011-01-01

    Maize chlorotic mottle virus (MCMV) causes corn lethal necrosis disease, and can be transmitted through infected maize seeds. It remains a challenge to detect this virus in the seeds to prevent its introduction and infection. For this purpose, a real-time TaqMan RT-PCR procedure for efficient detection of MCMV was developed. The sensitivity of the method was 4 fg of total RNA or 25 copies of RNA transcripts, which was approximately ten-fold higher than conventional RT-PCR gel electrophoresis method. The successful detection of MCMV in maize seeds suggested the feasibility of this procedure for routine testing.

  16. [Rapid detection of Listeria monocytogenes in pork samples by real-time PCR with Taqman probe].

    PubMed

    Yan, Lin; Wang, Xiaoying; Guo, Yunchang; Pei, Xiaoyan; Yu, Dongmin; Yang, Dajin

    2014-03-01

    To develop a real-time PCR method for detection Listeria monocytogenes in pork samples. Listeria monocytogenes specific primers and Taqman probe were chosen on the basis of hlyA gene. Real-time PCR method was developed and its specificity was proved. Serial 10-fold diluted pure suspension culture of CMCC 540004 were detected by real-time PCR, and standard curve was constructed. Artificially contaminated experiment was done, six artificially-inoculated samples containing final concentration of Listeria monocytogenes CMCC 540004 (1.3 x 10(0), 1.3 x 10(1), 1.3 x 10(2), 1.3 x 10(3), 1.3 x 10(4), 1.3 x 10(5) and 1.3 x 10(6) CFU per 25 g pork samples) were preparated respectively, meanwhile one sample without inoculation was as control of background value. All the samples were incubated in LB1 enrichment for 24 h and then take 0.1 ml culture solutions to 10 ml LB2 enrichment for 18 - 24 h. All the samples were incubated for 0, 4, 8, 12, 18, 24, 30, 36 and 46 h, and detected Listeria monocytogenes bacteria by PCR, respectively. Twenty-four samples of retail pork were collected from markets in Beijing and detected by the above three methods. Real-time PCR method established was specific for the detection of Listeria monocytogenes. The sensitivity was 1.3 x 10(3) CFU/ml for pure culture without enrichment. Real-time PCR detection limit for artificially contaminated samples after enriching for 24 h was 1.3 CFU/ 25 g, which is the same with the limit of PCR and traditional method after enrichment for 46 h. Standard curve of sample after enrichment for 24 h was established. The positive rate out of total 24 samples was 70.83% (17/24) by real-time PCR, which is the same with the result of PCR and traditional method. The positive ones were quantitative analyzed using standard curve of sample and determined the initial Listeria monocytogenes numbers of CFU/25 g. CONCLUSION; The established real-time PCR technology was simple, rapid, sensitive and specific, which was suitable to

  17. LNA units present in the (2'-OMe)-RNA strand stabilize parallel duplexes (2'-OMe)-RNA/[All-R(P)-PS]-DNA and parallel triplexes (2'-OMe)-RNA/[All-R(P)-PS]-DNA/RNA. An improved tool for the inhibition of reverse transcription.

    PubMed

    Maciaszek, Anna; Krakowiak, Agnieszka; Janicka, Magdalena; Tomaszewska-Antczak, Agnieszka; Sobczak, Milena; Mikołajczyk, Barbara; Guga, Piotr

    2015-02-28

    Homopurine phosphorothioate analogs of DNA, possessing all phosphorus atoms of RP configuration ([All-RP-PS]-DNA), when interact with appropriate complementary RNA or (2'-OMe)-RNA templates, form parallel triplexes or parallel duplexes of very high thermodynamic stability. The present results show that T-LNA or 5-Me-C-LNA units introduced into the parallel Hoogsteen-paired (2'-OMe)-RNA strands (up to four units in the oligomers of 9 or 12 nt in length) stabilize these parallel complexes. At neutral pH, dodecameric parallel duplexes have Tm values of 62-68 °C, which are by 4-10 °C higher than Tm for the reference duplex (with no LNA units present), while for the corresponding triplexes, Tm values exceeded 85 °C. For nonameric parallel duplexes, melting temperatures of 38-62 °C were found and (2'-OMe)-RNA oligomers containing 5-Me-C-LNA units stabilized the complexes more efficiently than the T-LNA containing congeners. In both series the stability of the parallel complexes increased with an increasing number of LNA units present. The same trend was observed in experiments of reverse transcription RNA→DNA (using AMV RT reverse transcriptase) where the formation of parallel triplexes (consisting of an RNA template, [All-RP-PS]-DNA nonamer and Hoogsteen-paired (2'-OMe)-RNA strands containing the LNA units) led to the efficient inhibition of the process. Under the best conditions checked (four 5-Me-C-LNA units, three-fold excess over the RNA template) the inhibition was 94% effective, compared to 71% inhibition observed in the reference system with the Hoogsteen-paired (2'-OMe)-RNA strand carrying no LNA units. This kind of complexation may "arrest" harmful RNA oligomers (e.g., viral RNA or mRNA of unwanted proteins) and, beneficially, exclude them from enzymatic processes, otherwise leading to viral or genetic diseases.

  18. The use of Taqman genotyping assays for rapid confirmation of β-thalassaemia mutations in the Malays: accurate diagnosis with low DNA concentrations.

    PubMed

    Teh, L-K; Lee, T-Y; Tan, J A M A; Lai, M-I; George, E

    2015-02-01

    In Malaysia, β-thalassaemia is a common inherited blood disorder in haemoglobin synthesis with a carrier rate of 4.5%. Currently, PCR-incorporating techniques such as amplification refractory mutation system (ARMS) or reverse dot blot hybridization (RDBH) are used in β-thalassaemia mutation detection. ARMS allows single-mutation identification using two reactions, one for wild type and another for mutant alleles. RDBH requires probe immobilization and optimization of hybridization and washing temperatures which is time consuming. The aim of our study was to investigate whether β-thalassaemia mutations can be identified in samples with low DNA concentrations. Genotype identification of common β-thalassaemia mutations in Malays was carried out using Taqman genotyping assays. Results show that the Taqman assays allow mutation detection with DNA template concentrations as low as 2-100 ng. In addition, consistent reproducibility was observed in the Taqman assays when repeated eight times and at different time intervals. The developed sensitive Taqman assays allow molecular characterization of β-thalassaemia mutations in samples with low DNA concentrations. The Taqman genotyping assays have potential as a diagnostic tool for foetal blood, chorionic villi or pre-implantation genetic diagnosis where DNA is limited and precious. © 2014 John Wiley & Sons Ltd.

  19. Comparison of Simplexa Flu A/B & RSV PCR with cytospin-immunofluorescence and laboratory-developed TaqMan PCR in predominantly adult hospitalized patients.

    PubMed

    Landry, Marie L; Ferguson, David

    2014-08-01

    To compare Simplexa Flu A/B & RSV PCR with cytospin-immunofluorescence and laboratory-developed TaqMan PCR methods, 445 nasopharyngeal samples were tested. Of these, 199 were positive (46 for respiratory syncytial virus [RSV], 120 for influenza A, and 33 for influenza B) and 246 were negative. The direct fluorescent-antibody assay (DFA) detected 132 (66.3%) positive samples, Simplexa direct detected 162 (81.4%), Simplexa using extracts detected 177 (88.9%), and lab-developed TaqMan PCR reference methods detected 199 (100%). The specificities were 99.6%, 100%, 100%, and 100%, respectively. The two Simplexa methods were more sensitive than the DFA (P = 0.0001) but less sensitive than the TaqMan reverse transcriptase PCR (P = 0.0001).

  20. Clinical Evaluation of COBAS TaqMan PCR for the Detection of Mycobacterium tuberculosis and M. avium Complex

    PubMed Central

    Ikegame, Satoshi; Sakoda, Yoritake; Fujino, Nao; Taguchi, Kazuhito; Kawasaki, Masayuki; Kajiki, Akira

    2012-01-01

    A retrospective observational study was performed to determine the sensitivity and limitation of PCR test for the detection of Mycobacterium tuberculosis and M. avium complex. We obtained clinical specimens collected from the respiratory tract, cultured M. tuberculosis or M. avium complex, and performed PCR analysis. A total of 299 samples (M. tuberculosis, 177; M. avium, 35; M. intracellulare, 87) were analyzed by COBAS TaqMan PCR from April 2007 to March 2011. The PCR positivity rates were 50–55%, 70–100%, 88–98%, and 100% in smear-negative, smear 1+, 2+, and 3+ groups, respectively. The PCR positivity of tuberculosis in smear 1+ was 80.6%, which was statistically significantly (P < 0.001) lower than that of smear 2+ (97.3%). From January 2005 to March 2007, we collected an additional 138 samples (M. tuberculosis, 74; M. avium, 21; M. intracellulare, 43), which were analyzed by COBAS Amplicor PCR. The PCR positivity rates obtained using COBAS TaqMan PCR and COBAS Amplicor PCR were not significantly different. The sensitivity of PCR test for mycobacteria is not sufficient in case of smear 1+. Careful consideration must be given to the interpretation of negative PCR test results in smear 1+, because smear-positive tuberculosis is the criterion for isolation. PMID:23029612

  1. TaqMan probe real-time polymerase chain reaction assay for the quantification of canine DNA in chicken nugget.

    PubMed

    Rahman, Md Mahfujur; Hamid, Sharifah Bee Abd; Basirun, Wan Jefrey; Bhassu, Subha; Rashid, Nur Raifana Abdul; Mustafa, Shuhaimi; Mohd Desa, Mohd Nasir; Ali, Md Eaqub

    2016-01-01

    This paper describes a short-amplicon-based TaqMan probe quantitative real-time PCR (qPCR) assay for the quantitative detection of canine meat in chicken nuggets, which are very popular across the world, including Malaysia. The assay targeted a 100-bp fragment of canine cytb gene using a canine-specific primer and TaqMan probe. Specificity against 10 different animals and plants species demonstrated threshold cycles (Ct) of 16.13 ± 0.12 to 16.25 ± 0.23 for canine DNA and negative results for the others in a 40-cycle reaction. The assay was tested for the quantification of up to 0.01% canine meat in deliberately spiked chicken nuggets with 99.7% PCR efficiency and 0.995 correlation coefficient. The analysis of the actual and qPCR predicted values showed a high recovery rate (from 87% ± 28% to 112% ± 19%) with a linear regression close to unity (R(2) = 0.999). Finally, samples of three halal-branded commercial chicken nuggets collected from different Malaysian outlets were screened for canine meat, but no contamination was demonstrated.

  2. Development of PCR and TaqMan PCR Assays to Detect Pseudomonas coronafaciens, a Causal Agent of Halo Blight of Oats.

    PubMed

    An, Ji-Hye; Noh, Young-Hee; Kim, Yong-Eon; Lee, Hyok-In; Cha, Jae-Soon

    2015-03-01

    Pseudomonas coronafaciens causes halo blight on oats and is a plant quarantine bacterium in many countries, including the Republic of Korea. Using of the certificated seed is important for control of the disease. Since effective detection method of P. coronafaciens is not available yet, PCR and TaqMan PCR assays for specific detection of P. coronafaciens were developed in this study. PCR primers were designed from the draft genome sequence of P. coronafaciens LMG 5060 which was obtained by the next-generation sequencing in this study. The PCR primer set Pc-12-F/Pc-12-R specifically amplified 498 bp from the 13 strains of P. coronafaciens isolated in the seven different countries (Canada, Japan, United Kingdom, Zimbabwe, Kenya, Germany, and New Zealand) and the nested primer set Pc-12-ne-F/Pc-12-ne-R specifically amplified 298 bp from those strains. The target-size PCR product was not amplified from the non-target bacteria with the PCR and nested primer sets. TaqMan PCR with Pc-12-ne-F/Pc-12-ne-R and a TaqMan probe, Pc-taqman, which were designed inside of the nested PCR amplicon, generated Ct values which in a dose-dependent manner to the amount of the target DNA and the Ct values of all the P. coronafaciens strains were above the threshold Ct value for positive detection. The TaqMan PCR generated positive Ct values from the seed extracts of the artificially inoculated oat seeds above 10 cfu/ml inoculation level. PCR and TaqMan PCR assays developed in this study will be useful tools to detect and identify the plant quarantine pathogen, P. coronafaciens.

  3. Application and development of a TaqMan real-time PCR for detecting infectious spleen and kidney necrosis virus in Siniperca chuatsi.

    PubMed

    Lin, Qiang; Fu, Xiaozhe; Liu, Lihui; Liang, Hongru; Guo, Huizhi; Yin, Shuwen; Kumaresan, Venkatesh; Huang, Zhibin; Li, Ningqiu

    2017-06-01

    Infectious spleen and kidney necrosis virus (ISKNV) is one of the major epidemiological agents that had caused great economic loss in Chinese perch (Siniperca chuatsi). In this study, a specific TaqMan real-time PCR was developed using a pair of primers and a TaqMan probe specific to the ORF007 gene of ISKNV to rapidly detect ISKNV copies in Chinese perch samples. This assay was optimized to produce linearity from 8.75 × 10(8) to 8.75 × 10(1) copies in standard curve with an efficiency of 98% and a R(2) value of 0.9999. Moreover, the minimum detection limit of this assay was 10,000 times more sensitive than that of conventional PCR method. The coefficients of variation of intra- and inter-assay repeatability were less than 2.4% and 3.3%, respectively. The viral distribution in different tissues of diseased Chinese perch was evaluated by TaqMan real-time PCR method and the highest level of viral copies was detected in spleen. Among the 76 diseased Chinese perch clinical samples, 35 and 29 were positive samples based on the TaqMan real-time PCR and conventional PCR methods, respectively, indicating that the TaqMan real-time PCR was more sensitive than conventional PCR. Therefore, the TaqMan real-time PCR should be a useful tool for the early surveillance and quantitation of ISKNV. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Development of PCR and TaqMan PCR Assays to Detect Pseudomonas coronafaciens, a Causal Agent of Halo Blight of Oats

    PubMed Central

    An, Ji-Hye; Noh, Young-Hee; Kim, Yong-Eon; Lee, Hyok-In; Cha, Jae-Soon

    2015-01-01

    Pseudomonas coronafaciens causes halo blight on oats and is a plant quarantine bacterium in many countries, including the Republic of Korea. Using of the certificated seed is important for control of the disease. Since effective detection method of P. coronafaciens is not available yet, PCR and TaqMan PCR assays for specific detection of P. coronafaciens were developed in this study. PCR primers were designed from the draft genome sequence of P. coronafaciens LMG 5060 which was obtained by the next-generation sequencing in this study. The PCR primer set Pc-12-F/Pc-12-R specifically amplified 498 bp from the 13 strains of P. coronafaciens isolated in the seven different countries (Canada, Japan, United Kingdom, Zimbabwe, Kenya, Germany, and New Zealand) and the nested primer set Pc-12-ne-F/Pc-12-ne-R specifically amplified 298 bp from those strains. The target-size PCR product was not amplified from the non-target bacteria with the PCR and nested primer sets. TaqMan PCR with Pc-12-ne-F/Pc-12-ne-R and a TaqMan probe, Pc-taqman, which were designed inside of the nested PCR amplicon, generated Ct values which in a dose-dependent manner to the amount of the target DNA and the Ct values of all the P. coronafaciens strains were above the threshold Ct value for positive detection. The TaqMan PCR generated positive Ct values from the seed extracts of the artificially inoculated oat seeds above 10 cfu/ml inoculation level. PCR and TaqMan PCR assays developed in this study will be useful tools to detect and identify the plant quarantine pathogen, P. coronafaciens. PMID:25774107

  5. Increased amplification success from forensic samples with locked nucleic acids.

    PubMed

    Ballantyne, Kaye N; van Oorschot, Roland A H; Mitchell, R John

    2011-08-01

    Inadequate sample quantities and qualities can commonly result in poor DNA amplification success rates for forensic case samples. In some instances, modifying the PCR protocol or components may assist profiling by overcoming inhibition, or reducing the threshold required for successful amplification and detection. Incorporation of locked nucleic acids (LNAs) into PCR primers has previously been shown to increase amplification success for a range of non-forensic sample types and applications. To investigate their use in a forensic context, the PCR primers for four commonly used STR loci have been redesigned to include LNA bases. The modified LNA primers provided significantly increased amplification success when compared to standard DNA primers, with both high-quality buccal samples and simulated forensic casework samples. Peak heights increased by as much as 5.75× for the singleplex amplifications. When incorporated into multiplexes, the LNA primers continued to outperform standard DNA primers, with increased ease of optimisation, and increased amplification success. The use of LNAs in PCR primers can greatly assist the profiling of a range of samples, and increase success rates from challenging forensic samples.

  6. Rapid genotyping using pyrene−perylene locked nucleic acid complexes

    PubMed Central

    Kumar, T. Santhosh; Myznikova, Anna; Samokhina, Evgeniya; Astakhova, Irina Kira

    2013-01-01

    We have developed an assay for single strand DNA and RNA detection which is based on novel pyrene−perylene FRET pairs attached to short LNA/DNA probes. The assay is based on ratiometric emission upon binding of target DNA/RNA by three combinations of fluorescent LNA/DNA reporter strands. Specific geometry of the pyrene fluorophore attached to the 2′-amino group of 2′-amino-LNA in position 4 allows for the first time to efficiently utilize dipole−dipole orientation parameter for sensing of single-nucleotide polymorphisms (SNPs) in nucleic acid targets by FRET. Using novel probes, SNP detection is achieved with advantages of large Stokes shift (115 nm), high fluorescence quantum yields and low limit of target detection values (< 5 nM). Rapid and accurate genotyping of highly polymorphic HIV Pol cDNA and RNA fragments performed herein proves the possibility for broad application of the novel pyrene−perylene FRET pairs, e.g., in imaging and clinical diagnostics. PMID:24044052

  7. Performance evaluation of Xpert MTB/RIF in a moderate tuberculosis incidence compared with TaqMan MTB and TRCRapid M.TB.

    PubMed

    Tsuyuguchi, Kazunari; Nagai, Hideaki; Ogawa, Kenji; Matsumoto, Tomoshige; Morimoto, Kozo; Takaki, Akiko; Mitarai, Satoshi

    2017-02-01

    Xpert MTB/RIF is an automated nucleic acid amplification test (NAT) that can detect the presence of Mycobacterium tuberculosis complex (MTC) in clinical specimens as well as rifampicin (RIF) resistance resulting from rpoB mutation. Despite its high sensitivity and specificity for diagnosing tuberculosis (TB) with or without RIF resistance, the clinical performance of the test is variable. In this study, we evaluated the performance of Xpert MTB/RIF in a setting of moderate TB burden and high medical resources. A total of 427 sputum specimens were obtained from 237 suspected TB cases. Of these, 159 were identified as active TB, while the other 78 were non-TB diseases. The overall sensitivity and specificity of MTC detection by Xpert MTB/RIF using culture results as a reference were 86.8% [95% confidence interval (CI): 81.8%-90.6%] and 96.8% (95% CI: 93.1%-98.5%), respectively. Among MTC-positive culture specimens, Xpert MTB/RIF positivity was 95.2% (95% CI: 91.2%-97.5%) in smear-positive and 44.7% (95% CI 30.1-60.3) in smear-negative specimens. Xpert MTB/RIF was similar to other NATs (TaqMan MTB and TRCRapid M.TB) in terms of performance. Xpert MTB/RIF detected 25 RIF-resistant isolates as compared to 22 with the mycobacterial growth indicator tube antimicrobial susceptibility testing system, yielding a sensitivity of 100% (95% CI: 85.1%-100%) and specificity of 98.3% (95% CI: 95.1%-99.4%). These results indicate that although sensitivity in smear-negative/culture-positive specimens was relatively low, Xpert MTB/RIF is a useful diagnostic tool for detecting TB and RIF resistance even in settings of moderate TB burden. Copyright © 2016. Published by Elsevier Ltd.

  8. Methods for recovery of hepatitis A virus (HAV) and other viruses from processed foods and detection of HAV by nested RT-PCR and TaqMan RT-PCR.

    PubMed

    Love, David C; Casteel, Michael J; Meschke, John S; Sobsey, Mark D

    2008-08-15

    Enteric viruses are important agents of foodborne disease. Unfortunately, robust, quantitative methods for sampling and analysis of enteric and other viruses in processed or complex foods are not well-established. As a result, epidemiologically determined etiologies or pathogen sources in foodborne outbreaks are rarely confirmed by virological analysis. In this study, an acid-adsorption elution concentration (AEC) method previously used to monitor virus occurrence and investigate enteric virus outbreaks in shellfish was adapted for examination of processed food items, namely tomato sauce and blended strawberries. Hepatitis A virus (HAV), poliovirus, and coliphage MS2 (MS2) were seeded in 10 or 30 g samples of tomato sauce or blended strawberries, recovered by AEC, and quantified by cell culture infectivity assay. In addition, nested reverse transcription-polymerase chain reaction (RT-PCR) and TaqMan RT-PCR assays were used to detect HAV RNA. Viruses were efficiently adsorbed to foods as an initial concentration step, with infectious HAV and MS2 adsorption of 67% and 93%, respectively, to tomato sauce, and 89% and 99%, respectively, to blended strawberries. Forty-three to 65% of HAV and poliovirus were subsequently eluted and recovered from tomato sauce using 0.5 M threonine, pH 7.2. The lower limits of HAV detection were at initial seeding levels of 14 PFU/g of tomato sauce and 33 PFU/g of blended strawberries. Unlike TaqMan RT-PCR, nested RT-PCR was not inhibited by undiluted final RNA extracts of tomato sauce or blended strawberries. The successful adaptation of the AEC method for enteric and other virus recovery, quantitation and detection in processed foods demonstrates its potential for use in the investigation of foodborne outbreaks of viral etiology and for validation of virus disinfection and sanitary processing procedures used by the food industry.

  9. Comparison of different real-time PCR chemistries and their suitability for detection and quantification of genetically modified organisms.

    PubMed

    Buh Gasparic, Meti; Cankar, Katarina; Zel, Jana; Gruden, Kristina

    2008-03-06

    The real-time polymerase chain reaction is currently the method of choice for quantifying nucleic acids in different DNA based quantification applications. It is widely used also for detecting and quantifying genetically modified components in food and feed, predominantly employing TaqMan and SYBR Green real-time PCR chemistries. In our study four alternative chemistries: Lux, Plexor, Cycling Probe Technology and LNA were extensively evaluated and compared using TaqMan chemistry as a reference system. Amplicons were designed on the maize invertase gene and the 5'-junction of inserted transgene and plant genomic DNA in MON 810 event. Real-time assays were subsequently compared for their efficiency in PCR amplification, limits of detection and quantification, repeatability and accuracy to test the performance of the assays. Additionally, the specificity of established assays was checked on various transgenic and non-transgenic plant species. The overall applicability of the designed assays was evaluated, adding practicability and costs issues to the performance characteristics. Although none of the chemistries significantly outperformed the others, there are certain characteristics that suggest that LNA technology is an alternative to TaqMan when designing assays for quantitative analysis. Because LNA probes are much shorter they might be especially appropriate when high specificity is required and where the design of a common TaqMan probe is difficult or even impossible due to sequence characteristics. Plexor on the other hand might be a method of choice for qualitative analysis when sensitivity, low cost and simplicity of use prevail.

  10. Synthesis, nucleic acid hybridization properties and molecular modelling studies of conformationally restricted 3'-O,4'-C-methylene-linked alpha-L-ribonucleotides.

    PubMed

    Madsen, Andreas S; Hrdlicka, Patrick J; Kumar, T Santhosh; Wengel, Jesper

    2006-07-24

    Nucleotides with conformationally restricted carbohydrate rings such as locked nucleic acid (LNA), alpha-L-LNA or 2',5'-linked 3'-O,4'-C-methyleribonucleotides exhibit significant potential as building blocks for antigene and antisense strategies. 2',5'-Linked alpha-L-ribo configured monomer X (termed alpha-L-ONA) was designed as a potential structural mimic of alpha-L-LNA. The corresponding phosphoramidite building block of monomer X was obtained in five steps (10% overall yield) from the easily obtainable thymine derivative 1. Incorporation of monomer X into oligodeoxyribonucleotides (ONs) results in dramatically decreased thermal stabilities with DNA/RNA complements (DeltaTm/mod=-11.5 to -17.0 degrees C) compared to unmodified reference ONs. Less pronounced decreases (DeltaTm/mod=-4.5 to -8.5 degrees C) are observed when monomer X is incorporated into triplex forming ONs and targeted against double-stranded DNA (parallel orientation, pyrimidine motif). This biophysical data, together with modelling studies, suggest that 2',5'-linked alpha-L-ONA is a poor structural mimic of alpha-L-LNA.

  11. CYP2D7 Sequence Variation Interferes with TaqMan CYP2D6 (*) 15 and (*) 35 Genotyping.

    PubMed

    Riffel, Amanda K; Dehghani, Mehdi; Hartshorne, Toinette; Floyd, Kristen C; Leeder, J Steven; Rosenblatt, Kevin P; Gaedigk, Andrea

    2015-01-01

    TaqMan™ genotyping assays are widely used to genotype CYP2D6, which encodes a major drug metabolizing enzyme. Assay design for CYP2D6 can be challenging owing to the presence of two pseudogenes, CYP2D7 and CYP2D8, structural and copy number variation and numerous single nucleotide polymorphisms (SNPs) some of which reflect the wild-type sequence of the CYP2D7 pseudogene. The aim of this study was to identify the mechanism causing false-positive CYP2D6 (*) 15 calls and remediate those by redesigning and validating alternative TaqMan genotype assays. Among 13,866 DNA samples genotyped by the CompanionDx® lab on the OpenArray platform, 70 samples were identified as heterozygotes for 137Tins, the key SNP of CYP2D6 (*) 15. However, only 15 samples were confirmed when tested with the Luminex xTAG CYP2D6 Kit and sequencing of CYP2D6-specific long range (XL)-PCR products. Genotype and gene resequencing of CYP2D6 and CYP2D7-specific XL-PCR products revealed a CC>GT dinucleotide SNP in exon 1 of CYP2D7 that reverts the sequence to CYP2D6 and allows a TaqMan assay PCR primer to bind. Because CYP2D7 also carries a Tins, a false-positive mutation signal is generated. This CYP2D7 SNP was also responsible for generating false-positive signals for rs769258 (CYP2D6 (*) 35) which is also located in exon 1. Although alternative CYP2D6 (*) 15 and (*) 35 assays resolved the issue, we discovered a novel CYP2D6 (*) 15 subvariant in one sample that carries additional SNPs preventing detection with the alternate assay. The frequency of CYP2D6 (*) 15 was 0.1% in this ethnically diverse U.S. population sample. In addition, we also discovered linkage between the CYP2D7 CC>GT dinucleotide SNP and the 77G>A (rs28371696) SNP of CYP2D6 (*) 43. The frequency of this tentatively functional allele was 0.2%. Taken together, these findings emphasize that regardless of how careful genotyping assays are designed and evaluated before being commercially marketed, rare or unknown SNPs underneath primer

  12. CYP2D7 Sequence Variation Interferes with TaqMan CYP2D6*15 and *35 Genotyping

    PubMed Central

    Riffel, Amanda K.; Dehghani, Mehdi; Hartshorne, Toinette; Floyd, Kristen C.; Leeder, J. Steven; Rosenblatt, Kevin P.; Gaedigk, Andrea

    2016-01-01

    TaqMan™ genotyping assays are widely used to genotype CYP2D6, which encodes a major drug metabolizing enzyme. Assay design for CYP2D6 can be challenging owing to the presence of two pseudogenes, CYP2D7 and CYP2D8, structural and copy number variation and numerous single nucleotide polymorphisms (SNPs) some of which reflect the wild-type sequence of the CYP2D7 pseudogene. The aim of this study was to identify the mechanism causing false-positive CYP2D6*15 calls and remediate those by redesigning and validating alternative TaqMan genotype assays. Among 13,866 DNA samples genotyped by the CompanionDx® lab on the OpenArray platform, 70 samples were identified as heterozygotes for 137Tins, the key SNP of CYP2D6*15. However, only 15 samples were confirmed when tested with the Luminex xTAG CYP2D6 Kit and sequencing of CYP2D6-specific long range (XL)-PCR products. Genotype and gene resequencing of CYP2D6 and CYP2D7-specific XL-PCR products revealed a CC>GT dinucleotide SNP in exon 1 of CYP2D7 that reverts the sequence to CYP2D6 and allows a TaqMan assay PCR primer to bind. Because CYP2D7 also carries a Tins, a false-positive mutation signal is generated. This CYP2D7 SNP was also responsible for generating false-positive signals for rs769258 (CYP2D6*35) which is also located in exon 1. Although alternative CYP2D6*15 and *35 assays resolved the issue, we discovered a novel CYP2D6*15 subvariant in one sample that carries additional SNPs preventing detection with the alternate assay. The frequency of CYP2D6*15 was 0.1% in this ethnically diverse U.S. population sample. In addition, we also discovered linkage between the CYP2D7 CC>GT dinucleotide SNP and the 77G>A (rs28371696) SNP of CYP2D6*43. The frequency of this tentatively functional allele was 0.2%. Taken together, these findings emphasize that regardless of how careful genotyping assays are designed and evaluated before being commercially marketed, rare or unknown SNPs underneath primer and/or probe regions can impact

  13. Biochemical responses to dietary α-linolenic acid restriction proceed differently among brain regions in mice.

    PubMed

    Miyazawa, Daisuke; Yasui, Yuko; Yamada, Kazuyo; Ohara, Naoki; Okuyama, Harumi

    2011-08-01

    Previously, we noted that the dietary restriction of α-linolenic acid (ALA, n-3) for 4 weeks after weaning brought about significant decreases in the BDNF content and p38 MAPK activity in the striatum of mice, but not in the other regions of the brain, compared with an ALA- and linoleic acid (LNA, n-6)-adequate diet. In this study, we examined whether a prolonged dietary manipulation induces biochemical changes in other regions of the brain as well. Mice were fed a safflower oil (SAF) diet (ALA-restricted, LNA-adequate) or a perilla oil (PER) diet (containing adequate amounts of ALA and LNA) for 8 weeks from weaning. The docosahexaenoic acid (DHA, 22:6n-3) contents and p38 MAPK activities in the cerebral cortex, striatum and hippocampus were significantly lower in the SAF group. The BDNF contents and protein kinase C (PKC) activities in the cerebral cortex as well as in the striatum, but not in the hippocampus, were significantly lower in the SAF group. These data indicate that the biochemical changes induced by the dietary restriction of ALA have a time lag in the striatum and cortex, suggesting that the signal is transmitted through decreased p38 MAPK activity and BDNF content and ultimately decreased PKC activity.

  14. Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis.

    PubMed

    Zhao, Yong; Li, Guilian; Sun, Chongyun; Li, Chao; Wang, Xiaochen; Liu, Haican; Zhang, Pingping; Zhao, Xiuqin; Wang, Xinrui; Jiang, Yi; Yang, Ruifu; Wan, Kanglin; Zhou, Lei

    2015-01-01

    Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA) probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP) resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a "probe dropout" manner (quantification cycle = 0); thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE)/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100%) were correctly detected through the assay. Of these isolates, 88/88 (100%) were determined as RFP susceptible and 52/54 (96.3%) were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories.

  15. LNA aptamer based multi-modal, Fe3O4-saturated lactoferrin (Fe3O4-bLf) nanocarriers for triple positive (EpCAM, CD133, CD44) colon tumor targeting and NIR, MRI and CT imaging.

    PubMed

    Roy, Kislay; Kanwar, Rupinder K; Kanwar, Jagat R

    2015-12-01

    This is the first ever attempt to combine anti-cancer therapeutic effects of emerging anticancer biodrug bovine lactoferrin (bLf), and multimodal imaging efficacy of Fe3O4 nanoparticles (NPs) together, as a saturated Fe3O4-bLf. For cancer stem cell specific uptake of nanocapsules/nanocarriers (NCs), Fe3O4-bLf was encapsulated in alginate enclosed chitosan coated calcium phosphate (AEC-CP) NCs targeted (Tar) with locked nucleic acid (LNA) modified aptamers against epithelial cell adhesion molecule (EpCAM) and nucleolin markers. The nanoformulation was fed orally to mice injected with triple positive (EpCAM, CD133, CD44) sorted colon cancer stem cells in the xenograft cancer stem cell mice model. The complete regression of tumor was observed in 70% of mice fed on non-targeted (NT) NCs, with 30% mice showing tumor recurrence after 30 days, while only 10% mice fed with Tar NCs showed tumor recurrence indicating a significantly higher survival rate. From tumor tissue analyses of 35 apoptotic markers, 55 angiogenesis markers, 40 cytokines, 15 stem cell markers and gene expression studies of important signaling molecules, it was revealed that the anti-cancer mechanism of Fe3O4-bLf was intervened through TRAIL, Fas, Fas-associated protein with death domain (FADD) mediated phosphorylation of p53, to induce activation of second mitochondria-derived activator of caspases (SMAC)/DIABLO (inhibiting survivin) and mitochondrial depolarization leading to release of cytochrome C. Induction of apoptosis was observed by inhibition of the Akt pathway and activation of cytokines released from monocytes/macrophages and dendritic cells (interleukin (IL) 27, keratinocyte chemoattractant (KC)). On the other hand, the recurrence of tumor in AEC-CP-Fe3O4-bLf NCs fed mice mainly occurred due to activation of alternative pathways such as mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinases (ERK) and Wnt signaling leading to an increase in expression of survivin

  16. A multiplex TaqMan qPCR assay for sensitive and rapid detection of phytoplasmas infecting Rubus species

    PubMed Central

    Krüger, Erika; Reineke, Annette

    2017-01-01

    Rubus stunt is an economically important disease in the production of raspberries, blackberries, and loganberries. A fast, sensitive, and reliable diagnosis of phytoplasmas, the causal agent of the disease, is of prime importance to stop its spread by vegetative propagation and by insect vectors. Therefore, multiplex qPCR assays using TaqMan probes with different kinds of fluorophores in one reaction were developed, allowing the detection of phytoplasmas in general as well as a more specific detection of phytoplasmas belonging to group 16SrV and host DNA (either plant or insect). This assay now provides a practical tool for the screening of motherplants and monitoring the presence and distribution of phytoplasmas in Rubus plants of different geographic origins, cultivars, and cultivation systems, as well as in putative insect vectors like leafhoppers. PMID:28545043

  17. A multiplex TaqMan qPCR assay for sensitive and rapid detection of phytoplasmas infecting Rubus species.

    PubMed

    Linck, Holger; Krüger, Erika; Reineke, Annette

    2017-01-01

    Rubus stunt is an economically important disease in the production of raspberries, blackberries, and loganberries. A fast, sensitive, and reliable diagnosis of phytoplasmas, the causal agent of the disease, is of prime importance to stop its spread by vegetative propagation and by insect vectors. Therefore, multiplex qPCR assays using TaqMan probes with different kinds of fluorophores in one reaction were developed, allowing the detection of phytoplasmas in general as well as a more specific detection of phytoplasmas belonging to group 16SrV and host DNA (either plant or insect). This assay now provides a practical tool for the screening of motherplants and monitoring the presence and distribution of phytoplasmas in Rubus plants of different geographic origins, cultivars, and cultivation systems, as well as in putative insect vectors like leafhoppers.

  18. Detection of parC mutations in Streptococcus pneumoniae by Real-time PCR and Taqman-MGB probes.

    PubMed

    Alonso, Rodrigo; Mateo, Estibaliz; Cisterna, Ramón

    2007-04-01

    A Real-time PCR assay was developed by using Taqman-MGB probes to screen mutations at codons Ser79 and Asp83 of Streptococcus pneumoniae parC. One hundred and thirty levofloxacin-susceptible and forty-two levofloxacin-resistant clinical strains were assayed. Mutations at codon 79 were found among all the levofloxacin-resistant strains. Mutations at codon 79 or 83 were found in ten levofloxacin-susceptible strains. This procedure is a reliable method for a rapid detection of mutations in the QRDRs of parC gene of S. pneumoniae and could be carried out in a diagnostic laboratory for some high-risk patients or in epidemiological surveys.

  19. Detection of elephant endotheliotropic herpesvirus type 1 in asymptomatic elephants using TaqMan real-time PCR.

    PubMed

    Hardman, K; Dastjerdi, A; Gurrala, R; Routh, A; Banks, M; Steinbach, F; Bouts, T

    2012-02-25

    This study assessed the feasibility of identifying asymptomatic viral shedders using a novel TaqMan real-time PCR on trunk washes and swabs from the conjunctiva, palate and vulva of elephants. Six elephants from a UK collection were sampled weekly over a period of 11 weeks for this study. The herd prevalence of elephant endotheliotropic herpesvirus-1 (EEHV-1) was 100 per cent by PCR. The virus DNA was detected in all the sampling sites; however, the prevalence of virus DNA in the conjunctiva swabs was higher. In addition, Asian elephants from two continental European collections were sampled once and one animal tested positive on a trunk wash. The virus from this animal was phylogenetically typed as EEHV-1A based on 231 nucleotides of the terminase gene.

  20. Comparison of on-treatment HCV RNA during direct antiviral therapy using two different COBAS TaqMan HCV assays.

    PubMed

    Vermehren, Johannes; Bourlière, Marc; Pol, Stanislas; Marcellin, Patrick; Hyland, Robert H; Jiang, Deyuan; Brainard, Diana M; Zeuzem, Stefan; Welzel, Tania M

    2017-04-01

    Repeated measurements of hepatitis C virus (HCV) RNA levels during antiviral therapy are recommended to monitor treatment efficacy and adherence. Throughout most direct antiviral agent (DAA) approval studies, HCV RNA cutoffs and endpoints were established with the COBAS TaqMan assay for use with the High Pure System (HPS/CTM). Different assays used in clinical practice may yield different quantitative results and possibly impact treatment decisions. The concordance of the fully-automated COBAS AmpliPrep/COBAS TaqMan assay (CAP/CTM) with HPS/CTM and its ability to predict response to DAA-treatment with ledipasvir/sofosbuvir was assessed in cirrhotic patients with HCV genotype-1-infection who had failed prior treatment with protease inhibitor-based regimens. Serum samples from patients (n=154) treated in the phase-2 SIRIUS-study were collected at baseline and during antiviral therapy (weeks 1-8), and were tested in parallel by both assays. The mean difference between HPS/CTM and CAP/CTM at baseline (n=153) was 0.32 log10 IU/mL HCV RNA. Discordant results were observed in 12% of samples collected at treatment weeks 1-8, with the greatest differences observed at weeks 2 and 4 (14% and 29%, respectively, for undetectable HCV RNA). SVR rates were 96%-97% in the study and were not significantly different between patients with detectable vs. undetectable HCV RNA according to both assays at weeks 1-4 of antiviral therapy. CAP/CTM and HPS/CTM showed significantly different response rates during the early stages of ledipasvir/sofosbuvir treatment. However, on-treatment response was not predictive of SVR with either assay, indicating that determination of on-treatment HCV RNA levels may not be useful to guide treatment decisions. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Development of a real-time PCR method (Taqman) for rapid identification and quantification of Prorocentrum donghaiense

    NASA Astrophysics Data System (ADS)

    Yuan, Jian; Mi, Tiezhu; Zhen, Yu; Yu, Zhigang

    2012-09-01

    Prorocentrum donghaiense is a dinoflagellate that is widely distributed in the East China Sea and has become increasingly involved in Harmful Algal Blooms (HABs). Therefore, it is necessary to study this dinoflagellate to monitor HABs. In this study, 13 pairs of primers specific to P. donghaiense (within its internal transcribed spacer (ITS) regions) were designed for SYBR Green I real-time PCR. As the SYBR Green I real-time PCR could not identify P. donghaiense in a specific manner, a Taqman real-time PCR method was developed by designing a set of specific primers and a Taqman probe. A 10-fold serial dilution of recombinant plasmid containing ITS regions of P. donghaiense was prepared as standard samples and the standard curve was established. Additionally, we quantified the genomic DNA in P. donghaiense cells and utilized this DNA to prepare another 10-fold serial dilution of standard sample and accordingly set up the standard curve. The mathematic correlation between the cell number and its corresponding plasmid copy number was also established. In order to test the efficiency of the real-time PCR method, laboratory samples and P. donghaiense HAB field samples were employed for identification and quantitative analysis. As to laboratory samples, as few as 102 cells of P. donghaiense could be quantified precisely utilizing both centrifugation and filtration techniques. The quantification results from field samples by real-time PCR were highly similar to those by light microscopy. In conclusion, the real-time PCR could be applied to identify and quantify P. donghaiense in HABs.

  2. Variability of HHV8 LNA-1 Immunohistochemical Staining Across the 3 Histologic Stages of HIV-Associated Mucocutaneous Kaposi Sarcoma: Is There a Relationship to Patients' CD4 Counts?

    PubMed

    Mohanlal, Reena D; Pather, Sugeshnee

    2015-07-01

    The histologic diagnosis of Kaposi sarcoma (KS) can be confirmed with human herpes virus 8 (HHV8) latency-associated nuclear antigen (LNA)-1 immunohistochemistry, which may show variability in distribution and intensity. This retrospective study was aimed at addressing the factors that may contribute to this variability. All cases of mucocutaneous KS diagnosed in a 5-year period at the histopathology department at a tertiary hospital in South Africa with available patients' CD4 counts and HHV8 LNA-1 immunohistochemically stained slides were reviewed, and the biopsy stages of KS (patch/plaque/nodular), CD4 counts, immunohistochemistry staining method (manual vs. automated), and distribution (diffuse/focal) and intensity (strong/weak) of HHV8 LNA-1 staining were recorded. A total of 127 cases were reviewed. No relationship was demonstrated between the median CD4 count and the histologic stages of KS (P = 0.701) or the intensity and distribution of HHV8 immunohistochemical staining using either staining method. Multivariate analysis showed that method of immunohistochemical staining was a significant predictor of distribution (P = 0.006) and intensity (P = 0.044) of staining, and that stage was a significant predictor of distribution of staining (P = 0.033).

  3. Quantification of hepatitis C virus in patients treated with peginterferon-alfa 2a plus ribavirin treatment by COBAS TaqMan HCV test.

    PubMed

    Kanda, T; Imazeki, F; Yonemitsu, Y; Mikami, S; Takada, N; Nishino, T; Takashi, M; Tsubota, A; Kato, K; Sugiura, N; Tawada, A; Wu, S; Tanaka, T; Nakamoto, S; Mikata, R; Tada, M; Chiba, T; Kurihara, T; Arai, M; Fujiwara, K; Kanai, F; Yokosuka, O

    2011-07-01

    Extremely low levels of serum hepatitis C virus (HCV) RNA can be detected by COBAS TaqMan HCV test. To investigate whether the COBAS TaqMan HCV test is useful for measuring rapid virological response (RVR) and early virological response (EVR) to predict sustained virological response (SVR), we compared the virological response to PEG-IFN-alfa 2a plus RBV in 76 patients infected with HCV genotype 1 when undetectable HCV RNA by the COBAS TaqMan HCV test was used, with those when below 1.7 log IU/mL HCV RNA by COBAS TaqMan HCV test was used, which corresponded to the use of traditional methods. Among the 76 patients, 28 (36.8%) had SVR, 13 (17.1%) relapsed, 19 (25.0%) did not respond, and 16 (21.0%) discontinued the treatment due to side effects. The positive predictive values for SVR based on undetectable HCV RNA by COBAS TaqMan HCV test at 24 weeks after the end of treatment [10/10 (100%) at week 4, 21/23 (91.3%) at week 8 and 26/33 (78.7%) at week 12] were superior to those based on <1.7 log IU/mL HCV RNA [17/19 (89.4%) at week 4, 27/38 (71.0%) at week 8, and 27/43 (62.7%) at week 12]. The negative predictive values for SVR based on <1.7 log IU/mL HCV RNA by COBAS TaqMan HCV test [46/57 (80.7%) at week 4, 37/38 (97.3%) at week 8, and 32/33 (96.9%) at week 12] were superior to those based on undetectable HCV RNA [48/66 (72.7%) at week 4, 46/53 (86.7%) at week 8, and 41/43 (95.3%) at week 12]. The utilization of both undetectable RNA and <1.7 log IU/mL HCV RNA by COBAS TaqMan HCV test is useful and could predict SVR and non-SVR patients with greater accuracy.

  4. Comparison of allelic discrimination by dHPLC, HRM, and TaqMan in the detection of BRAF mutation V600E.

    PubMed

    Carbonell, Pablo; Turpin, María C; Torres-Moreno, Daniel; Molina-Martínez, Irene; García-Solano, José; Perez-Guillermo, Miguel; Conesa-Zamora, Pablo

    2011-09-01

    The V600E mutation in the BRAF oncogene is associated with colorectal carcinomas, with mismatch-repair deficiency and, recently, with nonresponse to epidermal growth factor receptor inhibitor therapy. The use of reliable techniques for its detection is important. The aim of our study was to compare the performance characteristics in V600E detection of denaturing high-performance liquid chromatography (dHPLC) and high-resolution melting (HRM) with TaqMan allelic discrimination as well as direct-sequencing methods in a series of 195 colorectal paraffin-embedded specimens up to the age of 15 years. The effectiveness for obtaining results on mutation status was best using TaqMan (96.9%), followed by dHPLC (93.3%), HRM (88.7%), and sequencing (88.2%). In general, TaqMan was best for analyzing older tissues, whereas sequencing was the least efficient. Heterozygotic V600E was detected in 11.6%, 9.9%, 11.6%, and 9.9% of tissues using TaqMan, dHPLC, HRM, and sequencing, respectively. Result concordances between dHPLC and TaqMan or sequencing were excellent (κ = 0.9411 and κ = 0.8988, respectively); for HRM, the concordances were good (κ = 0.7973 and κ = 0.7488, respectively). By using DNA dilutions from tumor tissue, a minimum of 10% of V600E harboring cancer content was required for the analysis by dHPLC and HRM. dHPLC could detect four non-V600E mutations, whereas HRM detected one. Our results indicate that dHPLC and HRM are techniques that can be reliably used for the detection of the BRAFV600E mutation in archival paraffin-embedded tissues.

  5. The Optimization of TaqMan Real-Time RT-PCR Assay for Transcriptional Profiling of GABA-A Receptor Subunit Plasticity

    PubMed Central

    Gangisetty, Omkaram; Reddy, Doodipala Samba

    2009-01-01

    The GABA-A receptor plays a critical role in inhibitory neurotransmission in the brain. Quantitation of GABA-A receptor subunits in various brain regions is essential to understand their role in plasticity and brain disorders. However, conventional RNA assays are tedious and less sensitive for use in studies of subunit plasticity. Here we describe optimization of a sensitive assay of GABA-A receptor subunit gene expression by TaqMan real-time PCR. For each subunit gene, a set of primers and TaqMan fluorogenic probe were designed to specifically amplify the target template. The TaqMan methodology was optimized for quantification of mouse GABA-A receptor subunits (α1–6, β1–3, γ2, and δ) and GAPDH. The TaqMan reaction detected very low levels of gene expression (~100 template copies of cDNA). A standard curve for GAPDH and one of the target genes, constructed using the cDNA, revealed slopes around −3.4 (r2=0.990), reflecting similar optimum PCR efficiencies. The methodology was utilized for quantification of the GABA-A receptor α4 subunit, which is known to upregulate following withdrawal from chronic progesterone or neurosteroids. Our results show that the α4-subunit expression increased threefold in the hippocampus following neurosteroid withdrawal in mice. The TaqMan PCR assay allows sensitive, high-throughput transcriptional profiling of complete GABA-A receptor subunit family, and thus provides specific tool for studies of GABA-A receptor subunit plasticity in neurological and psychiatric animal models. PMID:19406150

  6. The free energy of locking a ring: Changing a deoxyribonucleoside to a locked nucleic acid.

    PubMed

    Xu, You; Villa, Alessandra; Nilsson, Lennart

    2017-01-19

    Locked nucleic acid (LNA), a modified nucleoside which contains a bridging group across the ribose ring, improves the stability of DNA/RNA duplexes significantly, and therefore is of interest in biotechnology and gene therapy applications. In this study, we investigate the free energy change between LNA and DNA nucleosides. The transformation requires the breaking of the bridging group across the ribose ring, a problematic transformation in free energy calculations. To address this, we have developed a 3-step (easy to implement) and a 1-step protocol (more efficient, but more complicated to setup), for single and dual topologies in classical molecular dynamics simulations, using the Bennett Acceptance Ratio method to calculate the free energy. We validate the approach on the solvation free energy difference for the nucleosides thymidine, cytosine, and 5-methyl-cytosine. © 2017 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.

  7. Role of Omega-3 Polyunsaturated Fatty Acids in the Production of Prostaglandin E2 and Nitric Oxide during Experimental Murine Paracoccidioidomycosis

    PubMed Central

    Sargi, S. C.; Dalalio, M. M. O.; Moraes, A. G.; Visentainer, J. E. L.; Morais, D. R.; Visentainer, J. V.

    2013-01-01

    There has recently been increased interest in the potential health effects of omega-3 polyunsaturated fatty acids on the immune system. Paracoccidioidomycosis is the most important endemic mycosis in Latin America. Macrophages have a fundamental role and act as first line of organism defense. The purpose of this study was to analyze the effect of n-3 fatty acids on the production of PGE2 and NO by mice infected with Pb18 and fed a diet enriched with LNA for 8 weeks. To study the effect of omega-3 fatty acids on macrophage activity during experimental paracoccidioidomycosis, mice were infected with Pb18 and fed a diet supplemented with LNA. PGE2 in the serum of animals was analyzed and NO in the supernatants of macrophages cultured and challenged in vitro with Pb18 was measured. Omega-3 fatty acids seemed to decrease the production of PGE2 in vivo in the infected group fed an LNA-supplemented diet during the 4th and 8th weeks of the experiment. At the same time, we observed an increase in synthesis of NO by peritoneal macrophages in this group. Omega-3 fatty acids thus appear to have an immunomodulatory effect in paracoccidioidomycosis. PMID:24455741

  8. Utilization of milk fatty acids by the suckling Iberian piglets.

    PubMed

    Aguinaga, M A; Haro, A; Lara, L; Gómez-Carballar, F; Nieto, R; Aguilera, J F

    2016-11-01

    A total of 16 pure-bred Iberian (IB) sows, all of them suckling six piglets, were used, eight of them in each of the two consecutive trials (1 and 2). Daily milk yield and composition were determined weekly over a 34-day lactation period. Within each litter, one piglet at birth and four piglets on day 35 of life were slaughtered. Milk intake per piglet tended to be greater in trial 2 (832 v. 893 g/day; P=0.066), but piglets grew at 168±3.3 g/day, irrespective of the trial. In the IB sow milk, the linoleic (LA) : linolenic (LNA) acid ratio averaged 14.6 and 15.2 in trial 1 and trial 2, respectively. A fivefold increase in piglet body fat content was observed over lactation (P<0.001). Most of this fat (81.4%) was present in the carcass. After 34 days of lactation, whole-body relative content of palmitic, palmitoleic, stearic and oleic acids were very close to those in the milk consumed, suggesting direct deposition. Daily deposition of LA derivatives and of LNA and its derivatives was found to be extremely low (<0.02 g, on average). Moreover, some of the arachidonic acid (ARA) in tissues of the IB piglet at birth disappeared throughout the lactating period. An overall fractional deposition for total fatty acids (FA) was 0.409. Fractional oxidation (disappearance) rates were 0.939 and 0.926 for n-6 and n-3 polyunsaturated FA. The overall rate of disappearance for the major non-essential FA (myristic, palmitic, palmitoleic, stearic and oleic acids), estimated as 1-the overall fractional deposition rate, was 0.546. It is concluded that the high degree of FA unsaturation, high oxidation rate of LA and LNA, and poor synthesis of ARA from LA and of docosahexaenoic acid from LNA found in the suckling piglet might increase the energy cost of whole-body fat accretion, a contributor to the observed low efficiency of use of milk energy for growth.

  9. Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India

    PubMed Central

    Dinoop, K.P.; Parija, Subhash Chandra; Mandal, Jharna; Swaminathan, R.P.; Narayanan, P.

    2016-01-01

    Background & objectives: Amoebiasis is a common parasitic infection caused by Entamoeba histolytica and amoebic liver abscess (ALA) is the most common extraintestinal manifestation of amoebiasis. The aim of this study was to standardise real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from liver abscess pus and stool samples and compare its results with nested-multiplex PCR. Methods: Liver abscess pus specimens were subjected to DNA extraction. The extracted DNA samples were subjected to amplification by nested-multiplex PCR, Taqman (18S rRNA) and SYBR Green real-time PCR (16S-like rRNA assays to detect E. histolytica/E. dispar/E. moshkovskii). The amplification products were further confirmed by DNA sequence analysis. Receiver operator characteristic (ROC) curve analysis was done for nested-multiplex and SYBR Green real-time PCR and the area under the curve was calculated for evaluating the accuracy of the tests to dignose ALA. Results: In all, 17, 19 and 25 liver abscess samples were positive for E. histolytica by nested-multiplex PCR, SYBR Green and Taqman real-time PCR assays, respectively. Significant differences in detection of E. histolytica were noted in the real-time PCR assays evaluated (P<0.0001). The nested-multiplex PCR, SYBR Green real-time PCR and Taqman real-time PCR evaluated showed a positivity rate of 34, 38 and 50 per cent, respectively. Based on ROC curve analysis (considering Taqman real-time PCR as the gold standard), it was observed that SYBR Green real-time PCR was better than conventional nested-multiplex PCR for the diagnosis of ALA. Interpretation & conclusions: Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the

  10. Neospora caninum DNA detection by TaqMan real-time PCR assay in experimentally infected pregnant heifers.

    PubMed

    Pereira, Gabriel Ribas; Vogel, Fernanda Silveira Flores; Bohrer, Rodrigo Camponogara; da Nóbrega, Janduí Escarião; Ilha, Gustavo Freitas; da Rosa, Paulo Roberto Antunes; Glanzner, Werner Giehl; Camillo, Giovana; Braunig, Patricia; de Oliveira, João Francisco Coelho; Gonçalves, Paulo Bayard Dias

    2014-01-31

    Neosporosis has been considered the main cause of abortion between the first and the second trimester of pregnancy in cattle. Therefore, the objective of this study was to identify the presence of Neospora caninum DNA obtained from experimental models based on the evaluation of different areas of the fetal nervous system and organs from heifers previously inoculated with NC-1 after or before insemination. This study was performed with Hereford × Nelore (n=29) heifers and all animals were considered free of diseases at the beginning of the experiment. All animals were bred by fixed-time artificial insemination (TAI) and allocated as follows: (a) seronegative heifers subjected to TAI (TAI, n=9), (b) heifers infected with N. caninun 60 days prior to TAI (NC-1+TAI, n=9), and (c) heifers submitted to TAI and infected with N. caninum 60 days later (TAI+NC-1, n=11). The pregnancy was confirmed by transrectal ultrasonography 35 days after TAI and evaluated every 30 days until the end of gestation. Fetuses were collected surgically at 170 days of gestation, and immediately necropsied to remove tissues aseptically. Samples of the central nervous system (CNS), heart, kidney, lung, liver, skeletal muscle and caruncle were collected for DNA extraction. Days of gestation at abortion and interval from abortion to first insemination were examined by Student's t-test. At 35 days of gestation the pregnancy rates in the group NC-1+TAI (4/9, 44.4%) was lower than in the control group (8/9, 88.8%, P<0.05). At 60 days, the pregnancy rates in the NC-1+TAI group (0/4, 0%) was lower compared to TAI+NC-1 (5/7, 71.4%) and control (6/8, 75.0%) groups (P<0.05). Animals from the group NC-1+TAI were re-inseminated 60 days after the first TAI. After pregnancy losses throughout the study, 5 animals (TAI), 3 animals (NC-1+TAI) and 5 animals (TAI+NC-1) maintained pregnancy until 170 days of gestation. TaqMan RT-PCR demonstrated the presence of N. caninum DNA in the medulla and right posterior

  11. Differentiation of the traditional Chinese medicinal plants Euphorbia humifusa and E. maculata from adulterants by TaqMan real-time polymerase chain reaction.

    PubMed

    Xue, Heng-Gang; Wang, Hong; Li, De-Zhu; Xue, Chun-Ying; Wang, Qing-Zhong

    2008-02-01

    DNA sequence analysis of the rDNA internal transcribed spacer 1 (ITS1) and TaqMan real-time polymerase chain reaction were exploited for their applications in the differentiation of the traditional chinese medicinal plants euphorbia humifusa and e. maculata from three related adulterants e. hypericifolia, E. atoto and E. prostrata. The data demonstrated that variations in the ITS1 regions were very low at the intra-species level but extremely high at the inter-species level, so that they could be easily distinguished at the DNA level. The sequence difference allowed an effective and reliable differentiation of E. humifusa and E. maculata from the adulterants by TaqMan real-time PCR.

  12. Identification and quantification of genetically modified Moonshade carnation lines using conventional and TaqMan real-time polymerase chain reaction methods.

    PubMed

    Li, Peng; Jia, Junwei; Bai, Lan; Pan, Aihu; Tang, Xueming

    2013-07-01

    Genetically modified carnation (Dianthus caryophyllus L.) Moonshade was approved for planting and commercialization in several countries from 2004. Developing methods for analyzing Moonshade is necessary for implementing genetically modified organism labeling regulations. In this study, the 5'-transgene integration sequence was isolated using thermal asymmetric interlaced (TAIL)-PCR. Based upon the 5'-transgene integration sequence, conventional and TaqMan real-time PCR assays were established. The relative limit of detection for the conventional PCR assay was 0.05 % for Moonshade using 100 ng total carnation genomic DNA, corresponding to approximately 79 copies of the carnation haploid genome, and the limits of detection and quantification of the TaqMan real-time PCR assay were estimated to be 51 and 254 copies of haploid carnation genomic DNA, respectively. These results are useful for identifying and quantifying Moonshade and its derivatives.

  13. Novel TaqMan real-time polymerase chain reaction assay for verifying the authenticity of meat and commercial meat products from game birds.

    PubMed

    Rojas, María; González, Isabel; Pavón, Miguel Angel; Pegels, Nicolette; Lago, Adriana; Hernández, Pablo E; García, Teresa; Martín, Rosario

    2010-06-01

    Species-specific real-time polymerase chain reaction (PCR) assays using TaqMan probes have been developed for verifying the labeling of meat and commercial meat products from game birds, including quail, pheasant, partridge, guinea fowl, pigeon, Eurasian woodcock and song thrush. The method combines the use of species-specific primers and TaqMan probes that amplify small fragments (amplicons <150 base pairs) of the mitochondrial 12S rRNA gene, and an endogenous control primer pair that amplifies a 141-bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. Analysis of experimental raw and heat-treated binary mixtures as well as of commercial meat products from the target species demonstrated the suitability of the assay for the detection of the target DNAs.

  14. Asymmetric real-time PCR and multiplex melting curve analysis with TaqMan probes for detecting PIK3CA mutations.

    PubMed

    Botezatu, Irina V; Nechaeva, Irina O; Stroganova, Аnna М; Senderovich, Anastasia I; Kondratova, Valentina N; Shelepov, Valery P; Lichtenstein, Anatoly V

    2015-12-01

    The data in this article are related to the research article entitled "Optimization of melting analysis with TaqMan probes for detection of KRAS, NRAS, and BRAF mutations" Botezatu et al. [1]. Somatic mutations in the PIK3CA gene ("hot spots" in exons 9 and 20) are found in many human cancers, and their presence can determine prognosis and a treatment strategy. An effective method of mutation scanning PIK3CA in clinical laboratories is DNA Melting Analysis (DMA) (Vorkas et al., 2010; Simi et al., 2008) [2], [3]. It was demonstrated recently that the TaqMan probes which have been long used in Real Time PCR may also be utilized in DMA (Huang et al., 2011) [4]. After optimization of this method Botezatu et al. [1], it was used for multiplex scanning PIK3CA hotspot mutations in formalin-fixed paraffin-embedded (FFPE) samples from patients with colorectal and lung cancer.

  15. Sensitive detection of Tomato ringspot virus by real-time TaqMan RT-PCR targeting the highly conserved 3'-UTR region.

    PubMed

    Tang, Joe; Khan, Subuhi; Delmiglio, Catia; Ward, Lisa I

    2014-06-01

    A real-time TaqMan RT-PCR assay was developed for the rapid and sensitive detection of Tomato ringspot virus (ToRSV), an important plant virus which infects a wide range of fruit and ornamental crops. Primers and a probe were designed based on the highly conserved 3'-untranslated region (UTR) sequences of ToRSV, to amplify a 182bp fragment of this region of RNA-1 and RNA-2. The assay was demonstrated to reliably amplify all ToRSV isolates tested. The detection limit was estimated to be about 12 copies of the ToRSV target region. No amplification was observed from the RNA of other nepoviruses or healthy host species. A comparison with a published conventional RT-PCR and a SYBR-based qRT-PCR indicated that both of the published assays lacked reliability and sensitivity, as neither were able to amplify all ToRSV isolates tested, and both were approximately 1000 times less sensitive than the novel TaqMan real-time assay. This TaqMan real-time assay was tested using four different reagent kits and was shown to be robust and stable, with no significant differences in sensitivity between kits. It is expected that the implementation of this TaqMan real-time RT-PCR assay will facilitate efficient phytosanitary certification of nursery stock requiring testing for ToRSV by regulatory agencies, and will also have wider uses for the general detection of ToRSV in a range of hosts.

  16. Detection of seasonal H3N2 influenza A virus by type-specific TaqMan minor groove binder probe assay.

    PubMed

    Wang, Ruixue; Schwartzman, Louis M; Memoli, Matthew J; Taubenberger, Jeffery K

    2011-06-01

    Despite the emergence of the pandemic H1N1 influenza A virus in 2009, seasonal H3N2 viruses continue to co-circulate in the population and may even predominate in the coming influenza season. We describe a specific minor groove binder TaqMan assay for H3N2 viruses with a detection limit of 16.5 standard DNA copies.

  17. Identification of Bacterial and Viral Codetections With Mycoplasma pneumoniae Using the TaqMan Array Card in Patients Hospitalized With Community-Acquired Pneumonia.

    PubMed

    Diaz, Maureen H; Cross, Kristen E; Benitez, Alvaro J; Hicks, Lauri A; Kutty, Preeta; Bramley, Anna M; Chappell, James D; Hymas, Weston; Patel, Anami; Qi, Chao; Williams, Derek J; Arnold, Sandra R; Ampofo, Krow; Self, Wesley H; Grijalva, Carlos G; Anderson, Evan J; McCullers, Jonathan A; Pavia, Andrew T; Wunderink, Richard G; Edwards, Kathryn M; Jain, Seema; Winchell, Jonas M

    2016-03-01

    Mycoplasma pneumoniae was detected in a number of patients with community-acquired pneumonia in a recent prospective study. To assess whether other pathogens were also detected in these patients, TaqMan Array Cards were used to test 216 M pneumoniae-positive respiratory specimens for 25 additional viral and bacterial respiratory pathogens. It is interesting to note that 1 or more codetections, predominantly bacterial, were identified in approximately 60% of specimens, with codetections being more common in children.

  18. Bat white-nose syndrome: A real-time TaqMan polymerase chain reaction test targeting the intergenic spacer region of Geomyces destructans

    Treesearch

    Laura K Muller; Jeffrey M. Lorch; Daniel L. Lindner; Michael O' Connor; Andrea Gargas; David S. Blehert

    2013-01-01

    The fungus Geomyces destructans is the causative agent of white-nose syndrome (WNS), a disease that has killed millions of North American hibernating bats. We describe a real-time TaqMan PCR test that detects DNA from G. destructans by targeting a portion of the multicopy intergenic spacer region of the rRNA gene complex. The...

  19. Detection of seasonal H3N2 influenza A virus by type-specific TaqMan minor groove binder probe assay

    PubMed Central

    Wang, Ruixue; Schwartzman, Louis M.; Memoli, Matthew J.; Taubenberger, Jeffery K.

    2011-01-01

    Despite the emergence of the pandemic H1N1 influenza A virus in 2009, seasonal H3N2 viruses continue to co-circulate in the population, and may even predominate in the coming influenza season. We describe a specific minor groove binder Taqman assay for H3N2 viruses with a detection limit of 16.5 standard DNA copies. PMID:21429691

  20. Identification of Bacterial and Viral Codetections With Mycoplasma pneumoniae Using the TaqMan Array Card in Patients Hospitalized With Community-Acquired Pneumonia

    PubMed Central

    Diaz, Maureen H.; Cross, Kristen E.; Benitez, Alvaro J.; Hicks, Lauri A.; Kutty, Preeta; Bramley, Anna M.; Chappell, James D.; Hymas, Weston; Patel, Anami; Qi, Chao; Williams, Derek J.; Arnold, Sandra R.; Ampofo, Krow; Self, Wesley H.; Grijalva, Carlos G.; Anderson, Evan J.; McCullers, Jonathan A.; Pavia, Andrew T.; Wunderink, Richard G.; Edwards, Kathryn M.; Jain, Seema; Winchell, Jonas M.

    2016-01-01

    Mycoplasma pneumoniae was detected in a number of patients with community-acquired pneumonia in a recent prospective study. To assess whether other pathogens were also detected in these patients, TaqMan Array Cards were used to test 216 M pneumoniae-positive respiratory specimens for 25 additional viral and bacterial respiratory pathogens. It is interesting to note that 1 or more codetections, predominantly bacterial, were identified in approximately 60% of specimens, with codetections being more common in children. PMID:27191004

  1. Quantitation of mule duck in goose foie gras using TaqMan real-time Polymerase Chain Reaction.

    PubMed

    Rodríguez, Miguel A; García, Teresa; González, Isabel; Asensio, Luis; Hernández, Pablo E; Martín, Rosario

    2004-03-24

    A real-time quantitative Polymerase Chain Reaction (PCR) method has been developed for the quantitation of mule duck (Anas platyrhynchos x Cairina moschata) in binary duck/goose foie gras mixtures. The method combines the use of real-time PCR with duck-specific and endogenous control "duck + goose" primers to measure duck content and total foie gras content, respectively. Both PCR systems (duck-specific and duck + goose) were designed on the mitochondrial 12S ribosomal RNA gene (rRNA). The duck-specific system amplifies a 96 bp fragment from duck DNA, whereas the duck + goose system amplifies a 120 bp fragment from duck and goose DNA. The method measures PCR product accumulation through a FAM-labeled fluorogenic probe (TaqMan). The C(t) (threshold cycle) values obtained from the duck + goose system are used to normalize the ones obtained from the duck-specific system. Analysis of experimental duck/goose foie gras binary mixtures demonstrated the suitability of the assay for the detection and quantitation of duck in the range of 1-25%. This genetic marker can be very useful to avoid mislabeling or fraudulent species substitution of goose by duck in foie gras.

  2. Identification of hantavirus infection by Western blot assay and TaqMan PCR in patients hospitalized with acute kidney injury.

    PubMed

    Oldal, Miklós; Németh, Viktória; Madai, Mónika; Kemenesi, Gábor; Dallos, Bianka; Péterfi, Zoltán; Sebők, Judit; Wittmann, István; Bányai, Krisztián; Jakab, Ferenc

    2014-06-01

    Hantaviruses, one of the causative agents of viral hemorrhagic fevers, represent a considerable healthcare threat. In Hungary, Dobrava-Belgrade virus (DOBV) and Puumala virus (PUUV) are the main circulating hantavirus species, responsible for the clinical picture known as hemorrhagic fever with renal syndrome, a disease that may be accompanied by acute kidney injury (AKI), requiring hospitalization with occasionally prolonged recovery phase. A total of 20 patient sera were collected over a 2-year period from persons hospitalized with AKI, displaying clinical signs and laboratory findings directly suggestive for hantavirus infection. Samples were tested using an immunoblot assay, based on complete viral nucleocapsid proteins to detect patients' IgM and IgG antibodies against DOBV and PUUV. In parallel, all specimens were also tested by 1-step real-time TaqMan reverse-transcriptase polymerase chain reaction to confirm infection and to determine the causative hantavirus genotype. We present here the first Hungarian clinical study spanning across 2 years and dedicated specifically to assess acute kidney injuries, in the context of hantavirus prevalence.

  3. Development a diagnostic pan-dermatophyte TaqMan probe real-time PCR assay based on beta tubulin gene.

    PubMed

    Mirhendi, Hossein; Motamedi, Marjan; Makimura, Koichi; Satoh, Kazuo

    2016-08-01

    Early differentiation of dermatophytosis from other cutaneous mycoses is essential to avoid inaccurate therapy. DNA-based techniques including real-time PCR have increasingly been considered for detection of fungal elements in clinical specimens. In this study, after partial sequence analysis of beta tubulin (BT2) gene in 13 common and rare pathogenic dermatophyte species, a pan-dermatophyte primer and probe set was designed in a TaqMan probe-based PCR format. The sensitivity and specificity of the system was tested with 22 reference strains of dermatophytes, 234 positive clinical specimens, 32 DNA samples extracted from normal nails, several fungi other than dermatophytes and human DNAs. Analytical detection limit of the designed PCR on serially diluted DNAs of prepared recombinant plasmid indicated that only five molecules per sample are the minimum number for reliable detection by the assay. A total of 226 out of 234 (96.5%) DNAs extracted from clinical samples, but none of the 32 nail samples, from healthy volunteers were positive in PCR. The real-time PCR targeted beta tubulin gene established in this study could be a sensitive diagnostic tool which is significantly faster than the conventional culture method and should be useful in the clinical settings, in large-scale epidemiological studies and in clinical trials of antifungal therapy.

  4. [Scanning for KRAS, NRAS, BRAF, and PIK3CA mutations by DNA melting analysis with TaqMan probes].

    PubMed

    Botezatu, I V; Panchuk, I O; Stroganova, A M; Senderovich, A I; Kondratova, V N; Shelepov, V P; Lichtenstein, A V

    2017-01-01

    Scanning for mutations by DNA melting analysis (DMA) is based on asymmetric PCR followed by the melting of duplexes formed by single-stranded amplicons with TaqMan probes. The method is optimally suited for clinical genetic testing; it is easy to perform, high-throughput, and sensitive. The detection limit of mutant alleles by the DMA method is about 3%, which is much higher than the sensitivity of Sanger sequencing. In addition, the DMA method is realized in a closed-tube format, while 2-h assay is carried out in a single tube without any intermediate or additional procedures thereby minimizing the risk of cross contamination of the samples. The validation of the DMA method was performed by scanning for mutations of clinically significant genes KRAS, NRAS, BRAF, and   PIK3CA in 324 DNA samples from tumors of patients with melanoma, colorectal and lung cancer. DNA was isolated either directly from tumor tissues, or from formalin-fixed paraffin-embedded tumor tissues. The detected mutations were verified by Sanger sequencing. The spectra of mutations identified in each tumor type correspond to the literature data and, thus, validate the use of DMA.

  5. Improved Detection of Respiratory Pathogens by Use of High-Quality Sputum with TaqMan Array Card Technology.

    PubMed

    Wolff, Bernard J; Bramley, Anna M; Thurman, Kathleen A; Whitney, Cynthia G; Whitaker, Brett; Self, Wesley H; Arnold, Sandra R; Trabue, Christopher; Wunderink, Richard G; McCullers, Jon; Edwards, Kathryn M; Jain, Seema; Winchell, Jonas M

    2017-01-01

    New diagnostic platforms often use nasopharyngeal or oropharyngeal (NP/OP) swabs for pathogen detection for patients hospitalized with community-acquired pneumonia (CAP). We applied multipathogen testing to high-quality sputum specimens to determine if more pathogens can be identified relative to NP/OP swabs. Children (<18 years old) and adults hospitalized with CAP were enrolled over 2.5 years through the Etiology of Pneumonia in the Community (EPIC) study. NP/OP specimens with matching high-quality sputum (defined as ≤10 epithelial cells/low-power field [lpf] and ≥25 white blood cells/lpf or a quality score [q-score] definition of 2+) were tested by TaqMan array card (TAC), a multipathogen real-time PCR detection platform. Among 236 patients with matched specimens, a higher proportion of sputum specimens had ≥1 pathogen detected compared with NP/OP specimens in children (93% versus 68%; P < 0.0001) and adults (88% versus 61%; P < 0.0001); for each pathogen targeted, crossing threshold (CT) values were earlier in sputum. Both bacterial (361 versus 294) and viral detections (245 versus 140) were more common in sputum versus NP/OP specimens, respectively, in both children and adults. When available, high-quality sputum may be useful for testing in hospitalized CAP patients.

  6. TaqMan real time RT-PCR assays for detecting ferret innate and adaptive immune responses.

    PubMed

    Carolan, Louise A; Butler, Jeff; Rockman, Steve; Guarnaccia, Teagan; Hurt, Aeron C; Reading, Patrick; Kelso, Anne; Barr, Ian; Laurie, Karen L

    2014-09-01

    The ferret is an excellent model for many human infectious diseases including influenza, SARS-CoV, henipavirus and pneumococcal infections. The ferret is also used to study cystic fibrosis and various cancers, as well as reproductive biology and physiology. However, the range of reagents available to measure the ferret immune response is very limited. To address this deficiency, high-throughput real time RT-PCR TaqMan assays were developed to measure the expression of fifteen immune mediators associated with the innate and adaptive immune responses (IFNα, IFNβ, IFNγ, IL1α, IL1β, IL2, IL4, IL6, IL8, IL10, IL12p40, IL17, Granzyme A, MCP1, TNFα), as well as four endogenous housekeeping genes (ATF4, HPRT, GAPDH, L32). These assays have been optimized to maximize reaction efficiency, reduce the amount of sample required (down to 1ng RNA per real time RT-PCR reaction) and to select the most appropriate housekeeping genes. Using these assays, the expression of each of the tested genes could be detected in ferret lymph node cells stimulated with mitogens or infected with influenza virus in vitro. These new tools will allow a more comprehensive analysis of the ferret immune responses following infection or in other disease states.

  7. Optimizing High Dimensional Gene Expression Studies for Immune Response Following Smallpox Vaccination Using Taqman® Low Density Immune Arrays

    PubMed Central

    Oberg, Ann L.; Dhiman, Neelam; Grill, Diane E.; Ryan, Jenna E.; Kennedy, Richard B.; Poland, Gregory A.

    2011-01-01

    Introduction We sought to determine the time and vaccinia virus dose combination that would maximize the number of acute immune response changes in response to vaccinia stimulation in preparation for a large gene expression microarray experiment. Methods PBMCs from ten subjects were exposed to five vaccinia virus doses for three lengths of time. Gene expression was measured for 90 immune response genes via Taqman® Low Density Immune Arrays. Expression data were normalized via model-based non-linear normalization. Linear mixed effects model results were used to standardize changes across genes and determine the time/multiplicity of infection (MOI) combination with the largest number of changes. Results The greatest number of changes occurred with a MOI of 5.0 and exposure time of 48 hours. Further inspection revealed that most changes had occurred earlier and faded at this combination. The second highest number of changes was found at a MOI of 0.5 PFU/cell and time of 18 hours. Conclusions We conclude a time of 18 hours with a MOI of 0.5 PFU/cell is the optimal time/MOI combination for the full scale gene expression study. The strategy described herein is a general and resource efficient way to make critical decisions regarding experimental parameters for studies utilizing expensive assays that interrogate a large number of variables. PMID:21277306

  8. Detection of banned ruminant-derived material in industrial feedstuffs by TaqMan real-time PCR Assay.

    PubMed

    Pegels, Nicolette; González, Isabel; García, Teresa; Martín, Rosario

    2011-08-01

    A ruminant-specific real-time PCR system was designed and applied for the detection of processed animal protein from ruminants in industrial feedstuffs. The assay includes a primer pair and a TaqMan probe selectively targeting mitochondrial 16S rRNA gene sequences from the ruminant group and another primer-probe set based on the eukaryotic nuclear 18S rRNA gene (positive amplification control). Both ruminant and eukaryotic PCR systems generated short PCR amplicons of 79 and 77 bp, respectively. To evaluate the suitability of the real-time PCR assay for the detection of banned by-products of ruminant origin, 126 feed samples subjected to rendering under current European legislation regulations were analyzed. The assay achieved 100% success in classifying the samples as positive or negative in terms of qualitative ruminant composition, with a detection limit of 0.1%. The quantitative ability of the assay is however restricted by variations in the composition and treatment of the feeds, which affect the amount and quality of amplifiable DNA.

  9. High-throughput gender identification of Accipitridae eagles with real-time PCR using TaqMan probes.

    PubMed

    Chang, H-W; Gu, D-L; Su, S-H; Chang, C-C; Cheng, C-A; Huang, H-W; Yao, C-T; Chou, T-C; Chuang, L-Y; Cheng, C-C

    2008-07-01

    The objective was to develop high-throughput gender identification of eagles. Based on BLAST and alignment analyses, the CHD-Z and CHD-W sequences of nine species of eagles were highly homologous with Spilornis cheela hoya (S. c. hoya); therefore, TaqMan probes were designed to target their CHD-ZW-common and CHD-W-specific regions. In S. c. hoya, genders were identified using TaqMan-based, real-time PCR (amplified by P2/P8 primers); this method was validated with anatomically confirmed controls (one of each gender). Both genders had high intensities of the HEX-labeled (CHD-ZW-common) probe, whereas only females had high intensity of the FAM-labeled (CHD-W-specific) probe. The sequence of the CHD-W-specific probe designed for S. c. hoya was completely homologous with the CHD-W-specific region in Circaetus gallicus, Gyps indicus, and Gyps bengalensis, and was only one nucleotide different from those of Accipiter nisus, Spizaetus nipalensis, Aquila chrysaetos, Circus spilonotus, and Milvus migrans. For the CHD-ZW-common probe, all species listed were completely conserved. Using real-time PCR software, we established auto-calling of the genders of 15 individuals of S. c. hoya. In conclusion, this method provided accurate, high-throughput gender identification for S. c. hoya, and has considerable potential for identifying the gender of several related species of eagles.

  10. Taqman Real-Time PCR Detects Avipoxvirus DNA in Blood of Hawaìi `Amakihi (Hemignathus virens)

    PubMed Central

    Farias, Margaret E. M.; LaPointe, Dennis A.; Atkinson, Carter T.; Czerwonka, Christopher; Shrestha, Rajesh; Jarvi, Susan I.

    2010-01-01

    Background Avipoxvirus sp. is a significant threat to endemic bird populations on several groups of islands worldwide, including Hawaìi, the Galapagos Islands, and the Canary Islands. Accurate identification and genotyping of Avipoxvirus is critical to the study of this disease and how it interacts with other pathogens, but currently available methods rely on invasive sampling of pox-like lesions and may be especially harmful in smaller birds. Methodology/Principal Findings Here, we present a nested TaqMan Real-Time PCR for the detection of the Avipoxvirus 4b core protein gene in archived blood samples from Hawaiian birds. The method was successful in amplifying Avipoxvirus DNA from packed blood cells of one of seven Hawaiian honeycreepers with confirmed Avipoxvirus infections and 13 of 28 Hawaìi `amakihi (Hemignathus virens) with suspected Avipoxvirus infections based on the presence of pox-like lesions. Mixed genotype infections have not previously been documented in Hawaìi but were observed in two individuals in this study. Conclusions/Significance We anticipate that this method will be applicable to other closely related strains of Avipoxvirus and will become an important and useful tool in global studies of the epidemiology of Avipoxvirus. PMID:20523726

  11. Taqman real-time PCR detects Avipoxvirus DNA in blood of Hawai'i 'amakihi (Hemignathus virens).

    PubMed

    Farias, Margaret E M; LaPointe, Dennis A; Atkinson, Carter T; Czerwonka, Christopher; Shrestha, Rajesh; Jarvi, Susan I

    2010-05-27

    Avipoxvirus sp. is a significant threat to endemic bird populations on several groups of islands worldwide, including Hawai'i, the Galapagos Islands, and the Canary Islands. Accurate identification and genotyping of Avipoxvirus is critical to the study of this disease and how it interacts with other pathogens, but currently available methods rely on invasive sampling of pox-like lesions and may be especially harmful in smaller birds. Here, we present a nested TaqMan Real-Time PCR for the detection of the Avipoxvirus 4b core protein gene in archived blood samples from Hawaiian birds. The method was successful in amplifying Avipoxvirus DNA from packed blood cells of one of seven Hawaiian honeycreepers with confirmed Avipoxvirus infections and 13 of 28 Hawai'i 'amakihi (Hemignathus virens) with suspected Avipoxvirus infections based on the presence of pox-like lesions. Mixed genotype infections have not previously been documented in Hawai'i but were observed in two individuals in this study. We anticipate that this method will be applicable to other closely related strains of Avipoxvirus and will become an important and useful tool in global studies of the epidemiology of Avipoxvirus.

  12. Development of TaqMan RT-qPCR for the detection of type A human respiratory syncytial virus.

    PubMed

    Abdel-Moneim, Ahmed S; Shehab, Gaber M; Alsulaimani, Adnan A; Al-Malky, Mater I R

    2017-02-20

    The human respiratory syncytial virus is a common respiratory pathogen in children. Improved diagnosis of the virus is dependent on the development of tools for the rapid detection and estimation of the viral loads. In the current study, RT-qPCR using TaqMan hydrolysis probe based on the F gene detection was developed to identify and quantify hRSV in clinical samples. The assay was validated by comparing the results with a commercially available RT-qPCR kit. The newly developed assay was sensitive in detecting hRSV positive samples (59/126) which were equivalent to those detected by the commercial kit (57/126) with a detection limit of 1 × 10(2) copies/mL. A high correlation was found between the results of the newly developed assay and the commercial one. It was concluded that the newly developed RT-qPCR assay can be used as a sensitive detection tool for hRSV-A.

  13. Detection of Bordetella avium by TaqMan real-time PCR in tracheal swabs from wildlife birds.

    PubMed

    Stenzel, T; Pestka, D; Tykałowski, B; Śmiałek, M; Koncicki, A; Bancerz-Kisiel, A

    2017-03-28

    Bordetella avium, the causing agent of bordetellosis, a highly contagious infection of the respiratory tract in young poultry, causes significant losses in poultry farming throughout the world. Wildlife birds can be a reservoir of various pathogens that infect farm animals. For this reason the studies were conducted to estimate the prevalence of Bordetella avium in wildlife birds in Poland. Tracheal swab samples were collected from 650 birds representing 27 species. The bacterial DNA was isolated directly from the swabs and screened for Bordetella avium by TaqMan real-time PCR. The assay specificity was evaluated by testing DNA isolated from 8 other bacteria that can be present in avian respiratory tract, and there was no amplification from non-Bordetella avium agents. Test sensitivity was determined by preparing standard tenfold serial dilutions of DNA isolated from positive control. The assay revealed to be sensitive, with detection limit of approximately 4.07x10^2 copies of Bordetella avium DNA. The genetic material of Bordetella avium was found in 54.54% of common pheasants, in 9.09% of Eurasian coots, in 3.22% of black-headed gulls and in 2.77% of mallard ducks. The results of this study point to low prevalence of Bordetella avium infections in wildlife birds. The results also show that described molecular assay proved to be suitable for the rapid diagnosis of bordetellosis in the routine diagnostic laboratory.

  14. Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides

    PubMed Central

    Kasuya, Takeshi; Hori, Shin-ichiro; Watanabe, Ayahisa; Nakajima, Mado; Gahara, Yoshinari; Rokushima, Masatomo; Yanagimoto, Toru; Kugimiya, Akira

    2016-01-01

    Gapmer antisense oligonucleotides cleave target RNA effectively in vivo, and is considered as promising therapeutics. Especially, gapmers modified with locked nucleic acid (LNA) shows potent knockdown activity; however, they also cause hepatotoxic side effects. For developing safe and effective gapmer drugs, a deeper understanding of the mechanisms of hepatotoxicity is required. Here, we investigated the cause of hepatotoxicity derived from LNA-modified gapmers. Chemical modification of gapmer’s gap region completely suppressed both knockdown activity and hepatotoxicity, indicating that the root cause of hepatotoxicity is related to intracellular gapmer activity. Gene silencing of hepatic ribonuclease H1 (RNaseH1), which catalyses gapmer-mediated RNA knockdown, strongly supressed hepatotoxic effects. Small interfering RNA (siRNA)-mediated knockdown of a target mRNA did not result in any hepatotoxic effects, while the gapmer targeting the same position on mRNA as does the siRNA showed acute toxicity. Microarray analysis revealed that several pre-mRNAs containing a sequence similar to the gapmer target were also knocked down. These results suggest that hepatotoxicity of LNA gapmer is caused by RNAseH1 activity, presumably because of off-target cleavage of RNAs inside nuclei. PMID:27461380

  15. Evaluation of point mutation detection in Mycobacterium tuberculosis with isoniazid resistance using real-time PCR and TaqMan probe assay.

    PubMed

    Riahi, F; Derakhshan, M; Mosavat, A; Soleimanpour, S; Rezaee, S A

    2015-03-01

    Rapid methods for diagnosis of Mycobacterium tuberculosis (Mtb) drug resistance and choosing appropriate antibiotic treatment are pivotal. Thirty isoniazid (INH)-resistant and 30 INH-susceptible Mtb isolates were evaluated using minimum inhibitory concentration (MIC) method followed by multiplex real-time PCR (RT-PCR). Amplification refractory mutation system (ARMS) for detection of mutation in 315 codon of katG gene and single-nucleotide polymorphism (SNP) for detection of mutation in -15 (C>T) in the regulatory zone of mabA-inhA were carried out using the TaqMan method. Primers and probe were used for IS6110 region of Mtb as an internal amplification control. The sensitivity and specificity of the RT-PCR TaqMan probe for detection of Mtb complex were 100 %. Detection of INH-resistant Mtb using the ARMS method for KatG had 69 % sensitivity and 100 % specificity. The sensitivity and specificity of SNP in mabA-inhA fragment for detection of INH-resistant Mtb were 53 and 100 %, respectively. Furthermore, considering both regions, the sensitivity of RT-PCR has increased to 75 %. This study revealed that the qPCR-TaqMan method can be used as a standard tool for diagnosis of Mtb. Moreover, ARMS and SNP RT-PCR TaqMan methods can be used as rapid screening methods for detection of INH-resistant Mtb.

  16. Comparison of SYBR Green and TaqMan real-time PCR methods for quantitative detection of residual CHO host-cell DNA in biopharmaceuticals.

    PubMed

    Soltany-Rezaee-Rad, Mohammad; Sepehrizadeh, Zargham; Mottaghi-Dastjerdi, Negar; Yazdi, Mojtaba Tabatabaei; Seyatesh, Neda

    2015-03-01

    The Chinese hamster ovary (CHO) cell line is one of the predominant hosts used in the bioproduction of pharmaceutical proteins. There have been many concerns about the use of animal cell lines in biopharm industries, and one of the most important concerns has been residual host-cell DNA. Improper integration of residual DNA into the recipient genomes could activate oncogenes or deactivate tumor suppressor genes. Real-time polymerase chain reaction (PCR) is a routine assay method used in the quantification of DNA. In this study, genomic CHO DNA was purified and subjected to real-time PCR. The efficiency of the reaction was calculated, and the limit of detection (LOD) was determined. The calculated efficiency for the primers using the SYBR Green method was 94.3% (r(2) = 0.998). A melting curve analysis showed neither unspecific products nor primer dimers. The calculated efficiency for the TaqMan assay was 96.6% (r(2) = 1). The results showed that the LOD of the SYBR Green and TaqMan assays were 100 fg and 10 fg, respectively. Since the LOD of the TaqMan assay showed a better sensitivity than the SYBR Green, this method could be used directly on the final products for the quantification of residual DNA, without prior DNA extraction.

  17. Calibration of quantitative real-time TaqMan PCR by correlation with hyphal biomass and ITS copies in mycelia of Piloderma croceum.

    PubMed

    Raidl, S; Bonfigli, R; Agerer, R

    2005-11-01

    DNA-based quantification methods such as real-time TaqMan PCR allow a rapid and highly sensitive species-specific quantification of isolated fungal DNA material, but most quantification systems are only able to measure relative amounts of biomass or biomass changes during different treatments. In this experiment, an already established DNA quantification system for the ectomycorrhizal fungus Piloderma croceum, based on the ITS region of ribosomal DNA, was calibrated to absolute biomass to obtain a direct correlation between mycelial biomass and isolated ITS copies. Thin layers of sterile mycelia were cultured on slides. The mycelial biomass was calculated from measurements of the total hyphal length using image analysis, followed by determination of the mycelial volume, and multiplied by the specific weight of hyphae obtained from literature data. Using the very same mycelium, the number of ITS copies was quantified by TaqMan PCR. The mean value of 1047 (+/- 185) copies per mm hypha results in possible data for a direct conversion: one billion (10 (9)) ITS copies corresponded to 0.79 mg hyphal dry weight. For the ribosomal ITS multi-copy genes, the number of ITS copies could be calculated to approx. 152 (+/- 26) copies per dikaryotic cell. These conversion data now allow determination of the mycelial biomass of Piloderma croceum using real-time TaqMan PCR, a prerequisite for competition experiments with Piloderma croceum.

  18. The predictive value of selected serum microRNAs for acute GVHD by TaqMan MicroRNA arrays.

    PubMed

    Zhang, Chunyan; Bai, Nan; Huang, Wenrong; Zhang, Pengjun; Luo, Yuan; Men, Shasha; Wen, Ting; Tong, Hongli; Wang, Shuhong; Tian, Ya-Ping

    2016-10-01

    Currently, the diagnosis of acute graft-versus-host disease (aGVHD) is mainly based on clinical symptoms and biopsy results. This study was designed to further explore new no noninvasive biomarkers for aGVHD prediction/diagnosis. We profiled miRNAs in serum pools from patients with aGVHD (grades II-IV) (n = 9) and non-aGVHD controls (n = 9) by real-time qPCR-based TaqMan MicroRNA arrays. Then, predictive models were established using related miRNAs (n = 38) and verified by a double-blind trial (n = 54). We found that miR-411 was significantly down regulated when aGVHD developed and recovered when aGVHD was controlled, which demonstrated that miR-411 has potential as an indicator for aGVHD monitoring. We developed and validated a predictive model and a diagnostic model for aGVHD. The predictive model included two miRNAs (miR-26b and miR-374a), which could predict an increased risk for aGVHD 1 or 2 weeks in advance, with an AUC, Positive Predictive Value (PPV), and Negative Predictive Value (NPV) of 0.722, 76.19 %, and 69.70 %, respectively. The diagnostic model included three miRNAs (miR-28-5p, miR-489, and miR-671-3p) with an AUC, PPV, and NPV of 0.841, 85.71 % and 83.33 %, respectively. Our results show that circulating miRNAs (miR-26b and miR-374a, miR-28-5p, miR-489 and miR-671-3p) may serve as biomarkers for the prediction and diagnosis of grades II-IV aGVHD.

  19. Multilocus sequence typing of Mycoplasma hyorhinis strains identified by a real-time TaqMan PCR assay.

    PubMed

    Tocqueville, Véronique; Ferré, Séverine; Nguyen, Ngoc Hong Phuc; Kempf, Isabelle; Marois-Créhan, Corinne

    2014-05-01

    A real-time TaqMan PCR assay based on the gene encoding the protein p37 was developed to detect Mycoplasma hyorhinis. Its specificity was validated with 29 epidemiologically unrelated M. hyorhinis strains (28 field strains and one reference strain) and other mycoplasma species or with other microorganisms commonly found in pigs. The estimated detection limit of this qPCR assay was 125 microorganism equivalents/μl. The same 29 epidemiologically unrelated M. hyorhinis strains and four previously fully sequenced strains were typed by two portable typing methods, the sequencing of the p37 gene and a multilocus sequence typing (MLST) scheme. The first method revealed 18 distinct nucleotide sequences and insufficient discriminatory power (0.934). The MLST scheme was developed with the sequenced genomes of the M. hyorhinis strains HUB-1, GDL-1, MCLD, and SK76 and based on the genes dnaA, rpoB, gyrB, gltX, adk, and gmk. In total, 2,304 bp of sequence was analyzed for each strain. MLST was capable of subdividing the 33 strains into 29 distinct sequence types. The discriminatory power of the method was >0.95, which is the threshold value for interpreting typing results with confidence (D=0.989). Population analysis showed that recombination in M. hyorhinis occurs and that strains are diverse but with a certain clonality (one unique clonal complex was identified). The new qPCR assay and the robust MLST scheme are available for the acquisition of new knowledge on M. hyorhinis epidemiology. A web-accessible database has been set up for the M. hyorhinis MLST scheme at http://pubmlst.org/mhyorhinis/.

  20. The significance of low-level plasma HIV viral load on COBAS TaqMan HIV-1 assays for patients with undetectable plasma viral load on COBAS Amplicor monitor version 1.5.

    PubMed

    Manavi, Kaveh

    2008-01-01

    COBAS TaqMan assay is a new HIV assay for measuring plasma viral load (VL). A significant number of patients with undetectable plasma VL on Amplicor assay were reported to have detectable VL with TaqMan in the study centre. The aim of the present study was to investigate the significance of detectable VL counts with TaqMan assay amongst patients who have had undetectable plasma VL with COBAS Amplicor assay. Observational study on patients who have had undetectable (TaqMan assay between June 1, 2006 and April 30, 2007. All patients were on highly active antiretroviral therapy (HAART) for longer than 6 months before use of COBAS TaqMan assay. Patients with detectable VL were followed up on a monthly basis until their VL was <40 copes/mL or there was confirmed new resistance to HAART using genotypic and Virco resistance assay. Plasma VL was detectable (>40 copies/mL) in 113 (14%) patients on 126 episodes using TaqMan assay. VL was less than 500 copies/mL in 90% of those episodes. All episodes ended with VL <40 copies/mL after a median of 117 (94-143) days without change in HAART regimes. The duration of those episodes was longer than 150 days in 75% of cases. No new mutation was detected amongst specimens with detectable VL. Short-term detectable VL may be common with using TaqMan assay. This phenomenon did not result in new mutations or failure of HAART in study patients in the short term.

  1. Fifteen weeks of dietary n-3 polyunsaturated fatty acid deprivation increases turnover of n-6 docosapentaenoic acid in rat-brain phospholipids

    PubMed Central

    Igarashi, Miki; Kim, Hyung-Wook; Gao, Fei; Chang, Lisa; Ma, Kaizong; Rapoport, Stanley I.

    2012-01-01

    Docosapentaenoic acid (DPAn-6, 22:5n-6) is an n-6 polyunsaturated fatty acid (PUFA) whose brain concentration can be increased in rodents by dietary n-3 PUFA deficiency, which may contribute to their behavioral dysfunction. We used our in vivo intravenous infusion method to see if brain DPAn-6 turnover and metabolism also were altered with deprivation. We studied male rats that had been fed for 15 weeks post-weaning an n-3 PUFA adequate diet containing 4.6% alpha-linolenic acid (α-LNA, 18:3n-3) or a deficient diet (0.2% α-LNA), each lacking docosahexaenoic acid (22:6n-3) and arachidonic acid (AA, 20:4n-6). [1-14C]DPAn-6 was infused intravenously for 5 min in unanesthetized rats, after which the brain underwent high-energy microwaving, and then was analyzed. The n-3 PUFA deficient compared with adequate diet increased DPAn-6 and decreased DHA concentrations in plasma and brain, while minimally changing brain AA concentration. Incorporation rates of unesterified DPAn-6 from plasma into individual brain phospholipids were increased 5.2–7.7 fold, while turnover rates were increased 2.1–4.7 fold. The observations suggest that increased metabolism and brain concentrations of DPAn-6 and its metabolites, together with a reduced brain DHA concentration, contribute to behavioral and functional abnormalities reported with dietary n-3 PUFA deprivation in rodents. PMID:22142872

  2. A 5′ Nuclease PCR (TaqMan) High-Throughput Assay for Detection of the mecA Gene in Staphylococci

    PubMed Central

    Killgore, George E.; Holloway, Brian; Tenover, Fred C.

    2000-01-01

    In an effort to find a rapid, efficient, and reliable method of screening large numbers of bacterial isolates for specific antimicrobial resistance genes, we compared conventional PCR results to the results generated using the TaqMan 5′ nuclease PCR kit in conjunction with an ABI Prism 7700 Sequence Detector for detecting the mecA gene in various species of staphylococci. DNA was extracted using two techniques. The first used a high-salt extraction method suitable for conventional PCR but resulted in a 7.2% rate of PCR inhibition with the TaqMan technique. PCR inhibition could be overcome by diluting samples 1:5 prior to testing. The second method used the Qiagen QIAamp Tissue Kit; no instances of PCR inhibition were encountered with this method. A total of 197 (96%) of the 206 samples with no inhibition showed agreement between the two methods. Eight of the nine disagreements were likely the result of low-level DNA cross contamination caused by frequent specimen handling. Target DNA in all eight of these samples was first detected in the initial tests only after >30 PCR cycles, and all were negative upon repeat testing even after 40 PCR cycles using freshly extracted DNA. Among those positive samples in agreement, target DNA was invariably detected before 30 PCR cycles. The TaqMan assay eliminated the need to load, run, stain, and read agarose gels and provided the advantage of instant detection of PCR product by laser-activated fluorescence. Thus, final results were obtained 2 h after PCR was initiated, as opposed to a requirement of 2 days to examine 96 samples by agarose gel electrophoresis. PMID:10878035

  3. Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria.

    PubMed

    Maeda, Hiroshi; Fujimoto, Chiyo; Haruki, Yasuhiro; Maeda, Takemasa; Kokeguchi, Susumu; Petelin, Millan; Arai, Hideo; Tanimoto, Ichiro; Nishimura, Fusanori; Takashiba, Shogo

    2003-10-24

    Accurate quantification of bacterial species in dental plaque is needed for microbiological diagnosis of periodontal diseases. The present study was designed to assess the sensitivity, specificity and quantitativity of the real-time PCR using the GeneAmp Sequence Detection System with two fluorescence chemistries. TaqMan probe with reporter and quencher dye, and SYBR Green dye were used for sources of the fluorescence. Primers and probes were designed for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and total bacteria based on the nucleotide sequences of the respective 16S ribosomal RNA genes. Since spread of antibiotic resistance genes is one of the crucial problems in periodontal therapy, quantitative detection of tetQ gene, which confers resistance to tetracycline, was included in the examination. The detection of P. gingivalis, P. intermedia and A. actinomycetemcomitans was linear over a range of 10-10(7) cells (10-10(7) copies for tetQ gene), while the quantitative range for total bacteria was 10(2)-10(7) cells. Species-specific amplifications were observed for the three periodontal bacteria, and there was no significant difference between the TaqMan and SYBR Green chemistry in their specificity, quantitativity and sensitivity. The SYBR Green assay, which was simpler than TaqMan assay in its manipulations, was applied to the clinical plaque samples. The plaque samples were obtained from eight patients (eight periodontal pockets) before and 1 week after the local drug delivery of minocycline. Although the number of P. gingivalis, P. intermedia and A. actinomycetemcomitans markedly decreased after the antibiotic therapy in most cases, higher copy numbers of the tetQ gene were detectable. The real-time PCR demonstrated sufficient sensitivity, specificity and quantitativity to be a powerful tool for microbiological examination in periodontal disease, and the quantitative monitoring of antibiotic resistance gene accompanied

  4. Development of SYBR Green and TaqMan quantitative real-time PCR assays for hepatopancreatic parvovirus (HPV) infecting Penaeus monodon in India.

    PubMed

    Yadav, Reena; Paria, Anutosh; Mankame, Smruti; Makesh, M; Chaudhari, Aparna; Rajendran, K V

    2015-12-01

    Hepatopancreatic parvovirus (HPV) infects Penaeus monodon and causes mortality in the larval stages. Further, it has been implicated in the growth retardation in cultured P. monodon. Though different geographical isolates of HPV show large sequence variations, a sensitive PCR assay specific to Indian isolate has not yet been reported. Here, we developed a sensitive SYBR Green-based and TaqMan real-time PCR for the detection and quantification of the virus. A 441-bp PCR amplicon was cloned in pTZ57 R/T vector and the plasmid copy number was estimated. A 10-fold serial dilution of the plasmid DNA from 1 × 10(9) copies to 1 copy was prepared and used as the standard. The primers were tested initially using the standard on a conventional PCR format to determine the linearity of detection. The standards were further tested on real-time PCR format using SYBR Green and TaqMan chemistry and standard curves were generated based on the Ct values from three well replicates for each dilution. The assays were found to be sensitive, specific and reproducible with a wide dynamic range (1 × 10(9) to 10 copies) with coefficient of regression (R(2)) > 0.99, calculated average slope -3.196 for SYBR Green assay whereas, for TaqMan assay it was >0.99 and -3.367, respectively. The intra- and inter-assay variance of the Ct values ranged from 0.26% to 0.94% and 0.12% to 0.81%, respectively, for SYBR Green assay, and the inter-assay variance of the Ct values for TaqMan assay ranged from 0.07% to 1.93%. The specificity of the assays was proved by testing other DNA viruses of shrimp such as WSSV, IHHNV and MBV. Standardized assays were further tested to detect and quantify HPV in the post-larvae of P. monodon. The result was further compared with conventional PCR to test the reproducibility of the test. The assay was also used to screen Litopeneaus vannamei, Macrobrachium rosenbergii and Scylla serrata for HPV.

  5. Phylogeny of the genus Synchytrium and the development of TaqMan PCR assay for sensitive detection of Synchytrium endobioticum in soil.

    PubMed

    Smith, Donna S; Rocheleau, Hélène; Chapados, Julie T; Abbott, Cathryn; Ribero, Sharon; Redhead, Scott A; Lévesque, C André; De Boer, Solke H

    2014-04-01

    Potato wart, caused by the fungal pathogen Synchytrium endobioticum, is a serious disease with the potential to cause significant economic damage. The small subunit (SSU) and internal transcribed spacer (ITS) ribosomal DNA (rDNA) were sequenced for several Synchytrium spp., showing a high rate of variability for both of these markers among the different species and monophyly of the genus within phylum Chytridiomycota. The intergenic nontranscribed spacer (IGS) of rDNA was sequenced for different pathotypes and showed no intraspecific variation within S. endobioticum, similar to the other rDNA markers from this study. To facilitate screening for the pathogen in soil, three TaqMan polymerase chain reaction (PCR) assays were developed from SSU, ITS, and IGS rDNA sequences to detect S. endobioticum sporangia in the chloroform-flotation fraction of sieved soil extracts. In the screening portion of the method, a first TaqMan assay targeting the SSU rDNA was developed with positive results that were further confirmed with amplicon melt analysis. A synthetic reaction control cloned into a plasmid was incorporated into the procedure, facilitating the validation of negative results. The presence of the reaction control did not adversely affect the efficiency of the SSU target amplification. A second TaqMan assay targeting the ITS-1 region was developed as a confirmatory test. There was 100% accordance between the SSU and ITS-1 TaqMan assays. Utilizing these two assays in tandem achieved good specificity for S. endobioticum, generating negative results with the cloned SSU and ITS-1 regions from all 14 other Synchytrium spp. considered. Spike recovery experiments indicated that these assays, targeting the SSU and ITS-1 rDNA regions, developed from a phylogeny dataset of the genus, could reliably detect a single sporangium in the chloroform flotation fraction of a soil extract. Good correlation between microscopic detection of sporangia and PCR results in both positive and

  6. A scientific note on the detection of honeybee viruses using real-time PCR (TaqMan) in Varroa mites collected from a Thai honeybee (Apis mellifera) apiary.

    PubMed

    Chantawannakul, P; Ward, L; Boonham, N; Brown, M

    2006-01-01

    Bee parasitic mite syndrome is a disease complex of colonies simultaneously infested with Varroa destructor mites and infected with viruses and accompanied by high mortality. By using real-time PCR (TaqMan), five out of seven bee viruses were detected in mite samples (V. destructor) collected from Thailand. Moreover, the results of this study provide an evidence for the co-existence of several bee viruses in a single mite. This is also the first report of bee viruses in mites from Thailand.

  7. [Comparative analysis of real-time quantitative PCR-Sanger sequencing method and TaqMan probe method for detection of KRAS/BRAF mutation in colorectal carcinomas].

    PubMed

    Zhang, Xun; Wang, Yuehua; Gao, Ning; Wang, Jinfen

    2014-02-01

    To compare the application values of real-time quantitative PCR-Sanger sequencing and TaqMan probe method in the detection of KRAS and BRAF mutations, and to correlate KRAS/BRAF mutations with the clinicopathological characteristics in colorectal carcinomas. Genomic DNA of the tumor cells was extracted from formalin fixed paraffin embedded (FFPE) tissue samples of 344 colorectal carcinomas by microdissection. Real-time quantitative PCR-Sanger sequencing and TaqMan probe method were performed to detect the KRAS/BRAF mutations. The frequency and types of KRAS/BRAF mutations, clinicopathological characteristics and survival time were analyzed. KRAS mutations were detected in 39.8% (137/344) and 38.7% (133/344) of 344 colorectal carcinomas by using real-time quantitative PCR-Sanger sequencing and TaqMan probe method, respectively. BRAF mutation was detected in 4.7% (16/344) and 4.1% (14/344), respectively. There was no significant correlation between the two methods. The frequency of the KRAS mutation in female was higher than that in male (P < 0.05). The frequency of the BRAF mutation in colon was higher than that in rectum. The frequency of the BRAF mutation in stage III-IV cases was higher than that in stageI-II cases. The frequency of the BRAF mutation in signet ring cell carcinoma was higher than that in mucinous carcinoma and nonspecific adenocarcinoma had the lowest mutation rate. The frequency of the BRAF mutation in grade III cases was higher than that in grade II cases (P < 0.05). The overall concordance for the two methods of KRAS/BRAF mutation detection was 98.8% (kappa = 0.976). There was statistic significance between BRAF and KRAS mutations for the survival time of colorectal carcinomas (P = 0.039). There were no statistic significance between BRAF mutation type and BRAF/KRAS wild type (P = 0.058). (1) Compared with real-time quantitative PCR-Sanger sequencing, TaqMan probe method is better with regard to handling time, efficiency, repeatability, cost

  8. Phylogenetic relationship of psychoactive fungi based on the rRNA gene for a large subunit and their identification using the TaqMan assay.

    PubMed

    Maruyama, Takuro; Yokoyama, Kazumasa; Makino, Yukiko; Goda, Yukihiro

    2003-06-01

    "Magic mushrooms" (MMs) are psychoactive fungi containing the and Psychotropics Control Law in Japan. Because there are many kinds of MMs and they are often sold even as dry powders in local markets, it is very difficult to identify the original species of the MMs by morphological observation. Therefore, we investigated the rRNA gene for a large subunit (LSU) of several MMs to classify them by a genetic approach. In this paper, we described the phylogeny of species of MMs based on the partial sequence (about 970 bp) of the LSU and the rapid identification of MMs using the TaqMan PCR assay.

  9. MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay

    PubMed Central

    Nagy, Alexander; Černíková, Lenka; Vitásková, Eliška; Křivda, Vlastimil; Dán, Ádám; Dirbáková, Zuzana; Jiřincová, Helena; Procházka, Bohumír; Sedlák, Kamil; Havlíčková, Martina

    2016-01-01

    In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence. PMID:27031831

  10. MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay.

    PubMed

    Nagy, Alexander; Černíková, Lenka; Vitásková, Eliška; Křivda, Vlastimil; Dán, Ádám; Dirbáková, Zuzana; Jiřincová, Helena; Procházka, Bohumír; Sedlák, Kamil; Havlíčková, Martina

    2016-01-01

    In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence.

  11. Bat white-nose syndrome: a real-time TaqMan polymerase chain reaction test targeting the intergenic spacer region of Geomyces destructans.

    PubMed

    Muller, Laura K; Lorch, Jeffrey M; Lindner, Daniel L; O'Connor, Michael; Gargas, Andrea; Blehert, David S

    2013-01-01

    The fungus Geomyces destructans is the causative agent of white-nose syndrome (WNS), a disease that has killed millions of North American hibernating bats. We describe a real-time TaqMan PCR test that detects DNA from G. destructans by targeting a portion of the multicopy intergenic spacer region of the rRNA gene complex. The test is highly sensitive, consistently detecting as little as 3.3 fg genomic DNA from G. destructans. The real-time PCR test specifically amplified genomic DNA from G. destructans but did not amplify target sequence from 54 closely related fungal isolates (including 43 Geomyces spp. isolates) associated with bats. The test was qualified further by analyzing DNA extracted from 91 bat wing skin samples, and PCR results matched histopathology findings. These data indicate the real-time TaqMan PCR method described herein is a sensitive, specific and rapid test to detect DNA from G. destructans and provides a valuable tool for WNS diagnostics and research.

  12. Development and Validation of a TaqMan Real-Time PCR Assay for the Specific Detection and Quantification of Fusarium fujikuroi in Rice Plants and Seeds.

    PubMed

    Carneiro, Greice Amaral; Matić, Slavica; Ortu, Giuseppe; Garibaldi, Angelo; Spadaro, Davide; Gullino, Maria Lodovica

    2017-07-01

    Bakanae disease, which is caused by the seedborne pathogen Fusarium fujikuroi, is found throughout the world on rice. A TaqMan real-time PCR has been developed on the TEF 1-α gene to detect F. fujikuroi in different rice tissues. Three primer/probe sets were tested. The selected set produced an amplicon of 84 bp and was specific for F. fujikuroi with respect to eight Fusarium species of rice and six other rice common pathogens. The assay was validated for specificity, selectivity, sensitivity, repeatability, and reproducibility. The detection limit was set at 27.5 fg of DNA, which is approximately equivalent to one haploid genome of F. fujikuroi. The developed TaqMan real-time assay was able to efficiently detect and quantify F. fujikuroi from rice culms, leaves, roots, and seeds. At 1 week post-germination (wpg), the pathogen was more diffused in the green tissues, while at 3 wpg it was uniformly spread also in the roots. The highest concentration of F. fujikuroi was measured in the M6 cultivar, which showed around 1,450 fungal cells/g. The assay was sufficiently sensitive to detect a few genomic equivalents in the rice seeds, corresponding to 9.89 F. fujikuroi cells/g. The assay permitted bakanae disease to be detected in asymptomatic tissues at the early rice development stages.

  13. Development of an on-site rapid real-time polymerase chain reaction system and the characterization of suitable DNA polymerases for TaqMan probe technology.

    PubMed

    Furutani, Shunsuke; Naruishi, Nahoko; Hagihara, Yoshihisa; Nagai, Hidenori

    2016-08-01

    On-site quantitative analyses of microorganisms (including viruses) by the polymerase chain reaction (PCR) system are significantly influencing medical and biological research. We have developed a remarkably rapid and portable real-time PCR system that is based on microfluidic approaches. Real-time PCR using TaqMan probes consists of a complex reaction. Therefore, in a rapid real-time PCR, the optimum DNA polymerase must be estimated by using actual real-time PCR conditions. In this study, we compared the performance of three DNA polymerases in actual PCR conditions using our rapid real-time PCR system. Although KAPA2G Fast HS DNA Polymerase has the highest enzymatic activity among them, SpeedSTAR HS DNA Polymerase exhibited better performance to rapidly increase the fluorescence signal in an actual real-time PCR using TaqMan probes. Furthermore, we achieved rapid detection of Escherichia coli in 7 min by using SpeedSTAR HS DNA Polymerase with the same sensitivity as that of a conventional thermal cycler.

  14. Detection and Quantification of Methyl tert-Butyl Ether-Degrading Strain PM1 by Real-Time TaqMan PCR

    PubMed Central

    Hristova, Krassimira R.; Lutenegger, Christian M.; Scow, Kate M.

    2001-01-01

    The fuel oxygenate methyl tert-butyl ether (MTBE), a widely distributed groundwater contaminant, shows potential for treatment by in situ bioremediation. The bacterial strain PM1 rapidly mineralizes and grows on MTBE in laboratory cultures and can degrade the contaminant when inoculated into groundwater or soil microcosms. We applied the TaqMan quantitative PCR method to detect and quantify strain PM1 in laboratory and field samples. Specific primers and probes were designed for the 16S ribosomal DNA region, and specificity of the primers was confirmed with DNA from 15 related bacterial strains. A linear relationship was measured between the threshold fluorescence (CT) value and the quantity of PM1 DNA or PM1 cell density. The detection limit for PM1 TaqMan assay was 2 PM1 cells/ml in pure culture or 180 PM1 cells/ml in a mixture of PM1 with Escherichia coli cells. We could measure PM1 densities in solution culture, groundwater, and sediment samples spiked with PM1 as well as in groundwater collected from an MTBE bioaugmentation field study. In a microcosm biodegradation study, increases in the population density of PM1 corresponded to the rate of removal of MTBE. PMID:11679339

  15. Bat white-nose syndrome: a real-time TaqMan polymerase chain reaction test targeting the intergenic spacer region of Geomyces destructanstructans.

    USGS Publications Warehouse

    Muller, Laura K.; Lorch, Jeffrey M.; Lindner, Daniel L.; O'Connor, Michael; Gargas, Andrea; Blehert, David S.

    2013-01-01

    The fungus Geomyces destructans is the causative agent of white-nose syndrome (WNS), a disease that has killed millions of North American hibernating bats. We describe a real-time TaqMan PCR test that detects DNA from G. destructans by targeting a portion of the multicopy intergenic spacer region of the rRNA gene complex. The test is highly sensitive, consistently detecting as little as 3.3 fg of genomic DNA from G. destructans. The real-time PCR test specifically amplified genomic DNA from G. destructans but did not amplify target sequence from 54 closely related fungal isolates (including 43 Geomyces spp. isolates) associated with bats. The test was further qualified by analyzing DNA extracted from 91 bat wing skin samples, and PCR results matched histopathology findings. These data indicate the real-time TaqMan PCR method described herein is a sensitive, specific, and rapid test to detect DNA from G. destructans and provides a valuable tool for WNS diagnostics and research.

  16. Development and application of a TaqMan single nucleotide polymorphism genotyping assay to study infectious laryngotracheitis virus recombination in the natural host.

    PubMed

    Loncoman, Carlos A; Hartley, Carol A; Coppo, Mauricio J C; Vaz, Paola K; Diaz-Méndez, Andrés; Browning, Glenn F; Lee, Sang-Won; Devlin, Joanne M

    2017-01-01

    To date, recombination between different strains of the avian alphaherpesvirus infectious laryngotracheitis virus (ILTV) has only been detected in field samples using full genome sequencing and sequence analysis. These previous studies have revealed that natural recombination is widespread in ILTV and have demonstrated that recombination between two attenuated ILTV vaccine strains generated highly virulent viruses that produced widespread disease within poultry flocks in Australia. In order to better understand ILTV recombination, this study developed a TaqMan single nucleotide polymorphism (SNP) genotyping assay to detect recombination between two field strains of ILTV (CSW-1 and V1-99 ILTV) under experimental conditions. Following in vivo co-inoculation of these two ILTV strains in specific pathogen free (SPF) chickens, recovered viruses were plaque purified and subjected to the SNP genotyping assay. This assay revealed ILTV recombinants in all co-inoculated chickens. In total 64/87 (74%) of the recovered viruses were recombinants and 23 different recombination patterns were detected, with some of them occurring more frequently than others. The results from this study demonstrate that the TaqMan SNP genotyping assay is a useful tool to study recombination in ILTV and also show that recombination occurs frequently during experimental co-infection with ILTV in SPF chickens. This tool, when used to assess ILTV recombination in the natural host, has the potential to greatly contribute to our understanding of alphaherpesvirus recombination.

  17. Development and application of a TaqMan single nucleotide polymorphism genotyping assay to study infectious laryngotracheitis virus recombination in the natural host

    PubMed Central

    Hartley, Carol A.; Coppo, Mauricio J. C.; Vaz, Paola K.; Diaz-Méndez, Andrés; Browning, Glenn F.; Lee, Sang-won; Devlin, Joanne M.

    2017-01-01

    To date, recombination between different strains of the avian alphaherpesvirus infectious laryngotracheitis virus (ILTV) has only been detected in field samples using full genome sequencing and sequence analysis. These previous studies have revealed that natural recombination is widespread in ILTV and have demonstrated that recombination between two attenuated ILTV vaccine strains generated highly virulent viruses that produced widespread disease within poultry flocks in Australia. In order to better understand ILTV recombination, this study developed a TaqMan single nucleotide polymorphism (SNP) genotyping assay to detect recombination between two field strains of ILTV (CSW-1 and V1-99 ILTV) under experimental conditions. Following in vivo co-inoculation of these two ILTV strains in specific pathogen free (SPF) chickens, recovered viruses were plaque purified and subjected to the SNP genotyping assay. This assay revealed ILTV recombinants in all co-inoculated chickens. In total 64/87 (74%) of the recovered viruses were recombinants and 23 different recombination patterns were detected, with some of them occurring more frequently than others. The results from this study demonstrate that the TaqMan SNP genotyping assay is a useful tool to study recombination in ILTV and also show that recombination occurs frequently during experimental co-infection with ILTV in SPF chickens. This tool, when used to assess ILTV recombination in the natural host, has the potential to greatly contribute to our understanding of alphaherpesvirus recombination. PMID:28350819

  18. Development of a TaqMan assay for sensitive detection of all pestiviruses infecting cattle, including the emerging HoBi-like strains.

    PubMed

    Losurdo, Michele; Mari, Viviana; Lucente, Maria Stella; Colaianni, Maria Loredana; Padalino, Iolanda; Cavaliere, Nicola; Buonavoglia, Canio; Decaro, Nicola

    2015-11-01

    A real-time RT-PCR assay based on the TaqMan technology was developed for rapid and sensitive detection of pestiviruses infecting cattle, i.e., bovine viral diarrhea virus (BVDV) 1, BVDV-2, and the emerging HoBi-like pestiviruses. The assay was linear and reproducible, being able to detect as few as 10 copies of viral RNA. By real-time RT-PCR analysis of 986 biological samples collected from cattle herd with clinical signs suggestive of pestivirus infection and from animals recruited in a pestivirus surveillance programme, 165 pestivirus positive samples were detected, including 6 specimens, 2 nasal swabs, and 4 EDTA-blood samples, that tested negative by a gel-based RT-PCR assay targeting the 5'UTR. The developed TaqMan assay represents a new reliable and effective tool for rapid and sensitive diagnosis of infections caused by all pestiviruses circulating in cattle, thus being useful for extensive surveillance programs in geographic areas where HoBi-like pestiviruses are co-circulating with BVDV-1 and BVDV-2.

  19. Development of a real-time RT-PCR assay with TaqMan probe for specific detection of acute bee paralysis virus.

    PubMed

    Jamnikar Ciglenečki, Urška; Toplak, Ivan

    2012-09-01

    Real-time polymerase chain reaction (real-time PCR) is an accurate, rapid and reliable method that can be used for the detection and also for the quantitation of specific DNA molecules. It can be non-specific, with intercalating dyes (SYBR Green I dye) able to bind to any dsDNA, or specific with a probe (TaqMan), whereby the probe is designed to bind within the amplified PCR fragment. A new real-time reverse transcription and polymerase chain reaction (real time RT-PCR) assay with TaqMan probe for specific detection of acute bee paralysis virus was designed. The assay was optimized to be highly sensitive and analytically specific and tested with a selection of genetically diverse ABPV strains originating from Slovenia, the United Kingdom (UK), Hungary and Germany. The detection limit of the assay and sensitivity comparisons with conventional RT-PCR were analyzed and this assay can detect a minimum of 44 copies of ABPV/reaction and is 230 times more sensitive than conventional RT-PCR. In addition, the assay is highly reproducible, with an average slope of standard curve made of ten-fold dilutions of standard copies/reaction -3.479±0.19 and an average slope of standard curve made of ten-fold dilutions of RNA -3.409±0.18.

  20. Comparison of the Xpert MTB/RIF test with an IS6110-TaqMan real-time PCR assay for direct detection of Mycobacterium tuberculosis in respiratory and nonrespiratory specimens.

    PubMed

    Armand, Sylvie; Vanhuls, Pascale; Delcroix, Guy; Courcol, René; Lemaître, Nadine

    2011-05-01

    The sensitivities of the Xpert MTB/RIF test and an in-house IS6110-based real-time PCR using TaqMan probes (IS6110-TaqMan assay) for the detection of Mycobacterium tuberculosis complex (MTBC) DNA were compared by use of 117 clinical specimens (97 culture positive and 20 culture negative for MTBC) that were frozen in sediment. The 97 clinical specimens included 60 respiratory and 37 nonrespiratory specimens distributed into 36 smear-positive and 61 smear-negative specimens. Among the 97 culture-positive specimens, 4 had rifampin-resistant isolates. Both methods were highly specific and exhibited excellent sensitivity (100%) with smear-positive specimens. The sensitivity of the Xpert MTB/RIF test with the whole smear-negative specimens was more reduced than that of the IS6110-TaqMan assay (48 versus 69%, P = 0.005). Both methods exhibited similar sensitivities with smear-negative respiratory specimens, but the Xpert MTB/RIF test had lower sensitivity with smear-negative nonrespiratory specimens than the IS6110-TaqMan assay (37 versus 71%, P = 0.013). Finally, the sensitivities of the Xpert MTB/RIF test and the IS6110-TaqMan assay were 79% and 84%, respectively, with respiratory specimens and 53% and 78%, respectively (P = 0.013), with nonrespiratory specimens. The Xpert MTB/RIF test correctly detected the rifampin resistance in smear-positive specimens but not in the one smear-negative specimen. The Xpert MTB/RIF test is a simple rapid method well adapted to a routine laboratory that appeared to be as sensitive as the IS6110-TaqMan assay with respiratory specimens but less sensitive with paucibacillary specimens, such as smear-negative nonrespiratory specimens.

  1. Effects of salt, polyethylene glycol, and locked nucleic acids on the thermodynamic stabilities of consecutive terminal adenosine mismatches in RNA duplexes.

    PubMed

    Gu, Xiaobo; Nguyen, Mai-Thao; Overacre, Abigail; Seaton, Samantha; Schroeder, Susan J

    2013-04-04

    Consecutive terminal mismatches add thermodynamic stability to RNA duplexes and occur frequently in microRNA-mRNA interactions. Accurate thermodynamic stabilities of consecutive terminal mismatches contribute to the development of specific, high-affinity siRNA therapeutics. Consecutive terminal adenosine mismatches (TAMS) are studied at different salt concentrations, with polyethylene glycol cosolutes, and with locked nucleic acid (LNA) substitutions. These measurements provide benchmarks for the application of thermodynamic predictions to different physiological or therapeutic conditions. The salt dependence for RNA duplex stability is similar for TAMS, internal loops, and Watson-Crick duplexes. A unified model for predicting the free energy of an RNA duplex with or without loops and mismatches at lower sodium concentrations is presented. The destabilizing effects of PEG 200 are larger for TAMS than internal loops or Watson-Crick duplexes, which may result from different base stacking conformations, dynamics, and water hydration. In contrast, LNA substitutions stabilize internal loops much more than TAMS. Surprisingly, the average per adenosine increase in stability for LNA substitutions in internal loops is -1.82 kcal/mol and only -0.20 kcal/mol for TAMS. The stabilities of TAMS and internal loops with LNA substitutions have similar favorable free energies. Thus, the unfavorable free energy of adenosine internal loops is largely an entropic effect. The favorable stabilities of TAMS result mainly from base stacking. The ability of RNA duplexes to form extended terminal mismatches in the absence of proteins such as argonaute and identifying the enthalpic contributions to terminal mismatch stabilities provide insight into the physical basis of microRNA-mRNA molecular recognition and specificity.

  2. Detection of North American eastern and western equine encephalitis viruses by nucleic acid amplification assays.

    PubMed

    Lambert, Amy J; Martin, Denise A; Lanciotti, Robert S

    2003-01-01

    We have developed nucleic acid sequence-based amplification (NASBA), standard reverse transcription PCR (RT-PCR), and TaqMan nucleic acid amplification assays for the rapid detection of North American eastern equine encephalitis (EEE) and western equine encephalitis (WEE) viral RNAs from samples collected in the field and clinical samples. The sensitivities of these assays have been compared to that of virus isolation. While all three types of nucleic acid amplification assays provide rapid detection of viral RNAs comparable to the isolation of viruses in Vero cells, the TaqMan assays for North American EEE and WEE viral RNAs are the most sensitive. We have shown these assays to be specific for North American EEE and WEE viral RNAs by testing geographically and temporally distinct strains of EEE and WEE viruses along with a battery of related and unrelated arthropodborne viruses. In addition, all three types of nucleic acid amplification assays have been used to detect North American EEE and WEE viral RNAs from mosquito and vertebrate tissue samples. The sensitivity, specificity, and rapidity of nucleic acid amplification demonstrate the usefulness of NASBA, standard RT-PCR, and TaqMan assays, in both research and diagnostic settings, to detect North American EEE and WEE viral RNAs.

  3. Conjugated linoleic acid (CLA) and fatty acid composition of milk, curd and Grana Padano cheese in conventional and organic farming systems.

    PubMed

    Prandini, Aldo; Sigolo, Samantha; Piva, Gianfranco

    2009-08-01

    CLA levels and fatty acid composition were measured to compare the fat composition in organic bulk milk, destined to the production of Grana Padano cheese, with those produced by conventional system. The curds and Grana Padano cheeses were also analysed to evaluate the effects of the production technology on the CLA content. All analysed organic samples were characterized by higher annual means of CLA, vaccenic acid (TVA) and linolenic acid (LNA) in comparison with conventional samples (with P<0.05). Nevertheless, no particular effect of the production technology was seen on the CLA content. The animal diet appears to be the factor which has the highest effect on the CLA concentration in milk and milk products and an organic diet based on fresh or dried forage, that is rich in CLA precursory fatty acids, may improve the yield of fatty acids with beneficial effects on health.

  4. Polyunsaturated fatty acids induce ovarian cancer cell death through ROS-dependent MAP kinase activation.

    PubMed

    Tanaka, Aiko; Yamamoto, Akane; Murota, Kaeko; Tsujiuchi, Toshifumi; Iwamori, Masao; Fukushima, Nobuyuki

    2017-09-04

    Free fatty acids not only play a role in cell membrane construction and energy production but also exert diverse cellular effects through receptor and non-receptor mechanisms. Moreover, epidemiological and clinical studies have so far suggested that polyunsaturated fatty acids (PUFAs) could have health benefits and the advantage as therapeutic use in cancer treatment. However, the underlying mechanisms of PUFA-induced cellular effects remained to be cleared. Here, we examined the effects of ω-3 and ω-6 PUFAs on cell death in ovarian cancer cell lines. ω-3 PUFA, docosahexaenoic acid (DHA) and ω-6 PUFA, γ-linolenic acid (γ-LNA) induced cell death in KF28 cells at the levels of physiological concentrations, but not HAC2 cells. Pharmacological and biochemical analyses demonstrated that cell death induced by DHA and γ-LNA was correlated with activation of JNK and p38 MAP kinases, and further an upstream MAP kinase kinase, apoptosis signal-regulating kinase 1, which is stimulated by reactive oxygen species (ROS). Furthermore, an antioxidant vitamin E attenuated PUFA-induced cell death and MAP kinase activation. These findings indicate that PUFA-induced cell death involves ROS-dependent MAP kinase activation and is a cell type-specific action. A further study of the underlying mechanisms for ROS-dependent cell death induced by PUFAs will lead to the discovery of a new target for cancer therapy or diagnosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Determination of essential fatty acid composition among mutant lines of Canola (Brassica napus), through high pressure liquid chromatography.

    PubMed

    Raza, Ghulam; Siddique, Aquil; Khan, Imtiaz Ahmad; Ashraf, Muhammed Yasin; Khatri, Abdullah

    2009-12-01

    The present study aimed to quantify the methyl esters of lenoleic acid (LA), gamma-lenolenic acid (LNA) and oleic acid (OL) in the oil of Brassica napus mutants. Five stable mutants (ROO-75/1, ROO-100/6, ROO-125/12, ROO-125/14, and ROO-125/17) of B. napus cv. 'Rainbow' (P) and three mutants (W97-95/16, W97-0.75/11 and W97-.075/13) of B. napus cv. 'Westar' (P) at M6 stage, exhibiting better yield and yield components, were analyzed for essential fatty acids. The highest seed yield was observed in the mutant (ROO-100/6) followed by ROO-125/14 of Rainbow, that is, 34% and 32% higher than their parent plants, respectively. Westar mutant W97-75/11 also showed 30% higher seed yield than its parent plant. High performance liquid chromatography analysis of the composition of fatty acids indicated that OL was the most dominant fatty acid, ranging from 39.1 to 66.3%; LA was second (15.3-41.6%) and LNA was third (18.1-28.9%). Mutant ROO-125/14 showed higher OL contents than parent (Rainbow). These results are expected to support the approval of ROO-125/14 in the National Uniform Varietal Yield Trials (NUVYT) as a new variety based on high oil quality.

  6. Spray-dried milk supplemented with alpha-linolenic acid or eicosapentaenoic acid and docosahexaenoic acid decreases HMG Co A reductase activity and increases biliary secretion of lipids in rats.

    PubMed

    Ramaprasad, Talahalli R; Srinivasan, Krishnapura; Baskaran, Vallikannan; Sambaiah, Kari; Lokesh, Belur R

    2006-05-01

    In our earlier study, we have shown that rats fed spray-dried milk containing alpha-linolenic acid (LNA 18:3 n-3) or eicosapentaenoic acid (EPA 20:5 n-3) and docosahexaenoic acid (DHA 22:6 n-3) had significantly lower amounts of serum and liver cholesterol. To evaluate the mechanism for hypocholesterolemic effect of n-3 fatty acids containing milk formulation, we fed male Wistar rats with spray-dried milk containing linseed oil (LSO) (source of LNA) or fish oil (FO) (source of EPA+DHA) for 8 weeks. Feeding n-3 fatty acid containing milk formulation lowered the hepatic 3-hydroxy-methylglutaryl coenzyme A (HMG Co A) activity by 17-22% compared to rats given control diet devoid of n-3 fatty acids. The cholesterol level in liver microsomes was found to be decreased by 16% and 20%, respectively, in LSO and FO containing formulation fed rats. The bile flow was enhanced to an extent of 19-23% in experimental groups compared to control animals. The biliary cholesterol and phospholipid secretion was increased to an extent of 49-55% and 140-146%, respectively, in rats fed n-3 fatty acid containing formulation. The increase in the total bile acids secretion in bile was mainly reflected on an increase in the levels of taurine conjugated bile acids. These results indicated that n-3 fatty acid containing spray-dried milk formulation would bring about the hypocholesterolemic effect by lowering HMG Co A reductase activity in liver and by increasing the secretion of bile constituents.

  7. Universal fluorescent labeling of amplification products using locked nucleic acids.

    PubMed

    Asari, Masaru; Oka, Kumiko; Omura, Tomohiro; Maseda, Chikatoshi; Tasaki, Yoshikazu; Shiono, Hiroshi; Matsubara, Kazuo; Matsuda, Mitsuyoshi; Shimizu, Keiko

    2013-02-01

    Amplification/hybridization-based genetic analyses using primers containing locked nucleic acids (LNAs) present many benefits. Here, we developed a novel design for universal fluorescent PCR using LNAs. Universal fluorescent PCR generates intermediate nonlabeled fragments and final fluorescent fragments in a two-step amplification process that uses locus-specific primers with universal tails and universal fluorescent primers. In this study, a few standard nucleotides were replaced with LNAs only in the fluorescent universal primers. The sequence of the fluorescent universal primer significantly affected the amplification efficiency. For primers with three LNAs, the fluorescent primers with stable M13(-47) sequences provided the most efficient signal (approximately tenfold higher than the primers with M13(-21) sequences at lower Tm values). Moreover, AT-rich LNA substitutions in the fluorescent primers produced much lower amplification efficiencies than GC-rich substitutions. GC-rich LNAs produced greater differences in Tm values among primers, and resulted in the preferential production of fluorescently labeled amplicons. The specificity and sensitivity of LNA-containing fluorescent primers were assessed by genotyping eight STRs in Japanese individuals, and full STR profiles could be generated using as little as 0.25 ng of genomic DNA. The method permitted clear discrimination of alleles and represents sensitive STR genotyping at a reduced cost. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Rapid quantification of hepatitis B virus DNA by real-time PCR using efficient TaqMan probe and extraction of virus DNA.

    PubMed

    Lu, Yan-Qin; Han, Jin-Xiang; Qi, Peng; Xu, Wei; Zu, Yan-Hui; Zhu, Bo

    2006-12-07

    To rapidly quantify hepatitis B virus (HBV) DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA. Three standards were prepared by cloning PCR products which targeted S, C and X region of HBV genome into pGEM-T vector respectively. A pair of primers and matched TaqMan probe were selected by comparing the copy number and the Ct values of HBV serum samples derived from the three different standard curves using certain serum DNA. Then the efficiency of six HBV DNA extraction methods including guanidinium isothiocyanate, proteinase K, NaI, NaOH lysis, alkaline lysis and simple boiling was analyzed in sample A, B and C by real-time PCR. Meanwhile, 8 clinical HBV serum samples were quantified. The copy number of the same HBV serum sample originated from the standard curve of S, C and X regions was 5.7 multiply 10(4)/mL, 6.3 multiply 10(2)/mL and 1.6 multiply 10(3)/mL respectively. The relative Ct value was 26.6, 31.8 and 29.5 respectively. Therefore, primers and matched probe from S region were chosen for further optimization of six extraction methods. The copy number of HBV serum samples A, B and C was 3.49 multiply 10(9)/mL, 2.08 multiply 10(6)/mL and 4.40 multiply 10(7)/mL respectively, the relative Ct value was 19.9, 30 and 26.2 in the method of NaOH lysis, which was the efficientest among six methods. Simple boiling showed a slightly lower efficiency than NaOH lysis. Guanidinium isothiocyanate, proteinase K and NaI displayed that the copy number of HBV serum sample A, B and C was around 10(5)/mL, meanwhile the Ct value was about 30. Alkaline failed to quantify the copy number of three HBV serum samples. Standard deviation (SD) and coefficient variation (CV) were very low in all 8 clinical HBV serum samples, showing that quantification of HBV DNA in triplicate was reliable and accurate. Real-time PCR based on optimized primers and TaqMan probe from S region in combination with NaOH lysis is a simple, rapid and accurate method for

  9. Identification and quantification of three genetically modified insect resistant cotton lines using conventional and TaqMan real-time polymerase chain reaction methods.

    PubMed

    Yang, Litao; Pan, Aihu; Zhang, Kewei; Guo, Jinchao; Yin, Changsong; Chen, Jianxiu; Huang, Cheng; Zhang, Dabing

    2005-08-10

    As the genetically modified organisms (GMOs) labeling policies are issued in many countries, qualitative and quantitative polymerase chain reaction (PCR) techniques are increasingly used for the detection of genetically modified (GM) crops in foods. Qualitative PCR and TaqMan real-time quantitative PCR methods to detect and identify three varieties of insect resistant cotton, i.e., Mon531 cotton (Monsanto Co.) and GK19 and SGK321 cottons (Chinese Academy of Agricultural Sciences), which were approved for commercialization in China, were developed in this paper. Primer pairs specific to inserted DNAs, such as Cowpea trypsin inhibitor (CpTI) gene of SGK321 cotton and the specific junction DNA sequences containing partial Cry1A(c) gene and NOS terminator of Mon531, GK19, and SGK321 cotton varieties were designed to conduct the identified PCR assays. In conventional specific identified PCR assays, the limit of detection (LOD) was 0.05% for Mon531, GK19, or SGK321 in 100 ng of cotton genomic DNA for one reaction. Also, the multiplex PCR method for screening the three GM cottons was also established, which could save time and cost in practical detection. Furthermore, a real-time quantitative PCR assay based on TaqMan chemistry for detection of insect resistant gene, Cry1A(c), was developed. This assay also featured the use of a standard plasmid as a reference molecule, which contained both a specific region of the transgene Cry1A(c) and an endogenous stearoyl-acyl carrier protein desaturase (Sad1) gene of the cotton. In quantitative PCR assay, the quantification range was from 0.01 to 100% in 100 ng of the genome DNA template, and in the detection of 1.0, 3.0, and 5.0% levels of three insect resistant cotton lines, respectively, all of the relative standard deviations (RSDs) were less than 8.2% except for the GM cotton samples with 1.0% Mon531 or GK19, which meant that our real-time PCR assays involving the use of reference molecule were reliable and practical for GM

  10. Digestion, milk production and milk fatty acid profile of dairy cows fed flax hulls and infused with flax oil in the abomasum.

    PubMed

    Côrtes, Cristiano; Kazama, Ricardo; da Silva-Kazama, Daniele; Benchaar, Chaouki; Zeoula, Lucia M; Santos, Geraldo T D; Petit, Hélène V

    2011-08-01

    Flax hull, a co-product obtained from flax processing, is a rich source of n-3 fatty acids (FA) but there is little information on digestion of flax hull based diets and nutritive value of flax hull for dairy production. Flax oil is rich in α-linolenic acid (LNA) and rumen bypass of flax oil contributes to increase n-3 FA proportions in milk. Therefore, the main objective of the experiment was to determine the effects of abomasal infusion of increasing amounts of flax oil on apparent digestibility, dry matter (DM) intake, milk production, milk composition, and milk FA profile with emphasis on the proportion of LNA when cows were supplemented or not with another source of LNA such as flax hull. Six multiparous Holstein cows averaging 650±36 kg body weight and 95±20 d in milk were assigned to a 6×6 Latin square design (21-d experimental periods) with a 2×3 factorial arrangement of treatments. Treatments were: 1) control, neither flax hull nor flax oil (CON), 2) diet containing (DM basis) 15·9% flaxseed hull (FHU); 3) CON with abomasal infusion of 250 g/d flax oil; 4) CON with abomasal infusion of 500 g/d flax oil; 5) FHU with abomasal infusion of 250 g/d flax oil; 6) FHU with abomasal infusion of 500 g/d flax oil. Infusion of flax oil in the abomasum resulted in a more pronounce decrease in DM intake for cows fed the CON diets than for those fed the FHU diets. Abomasal infusion of flax oil had little effect on digestibility and FHU supplementation increased digestibility of DM and crude protein. Milk yield was not changed by abomasal infusion of flax oil where it was decreased with FHU supplementation. Cows fed FHU had higher proportions of 18:0, cis9-18:1, trans dienes, trans monoenes and total trans in milk fat than those fed CON. Proportion of LNA was similar in milk fat of cows infused with 250 and 500 g/d flax oil in the abomasum. Independently of the basal diet, abomasal infusion of flax oil resulted in the lowest n-6:n-3 FA ratio in milk fat, suggesting

  11. Comparison of vero cell plaque assay, TaqMan reverse transcriptase polymerase chain reaction RNA assay, and VecTest antigen assay for detection of West Nile virus in field-collected mosquitoes.

    PubMed

    Nasci, Roger S; Gottfried, Kristy L; Burkhalter, Kristen L; Kulasekera, Varuni L; Lambert, Amy J; Lanciotti, Robert S; Hunt, Ann R; Ryan, Jeffrey R

    2002-12-01

    Mosquitoes collected during the epidemic of West Nile virus (WN) in Staten Island, NY, during 2000 were identified to species, grouped into pools of up to 50 individuals, and tested for the presence of WN by using TaqMan reverse transcriptase polymerase chain reaction (RT-PCR) to detect West Nile viral RNA, Vero cell plaque assay to detect infectious virus, and VecTest WNV/SLE Antigen Panel Assay. A total of 10,866 specimens was tested in 801 pools. Analysis of results indicated that TaqMan RT-PCR detected 34 WN-positive pools, more than either of the other techniques. The plaque assay detected 74% of the pools positive by TaqMan, and VecTest detected 60% of the pools positive by TaqMan. The VecTest assay detected evidence of West Nile viral antigen in 67% of the pools that contained live virus detected by plaque assay. A WN enzyme immunoassay performed similarly to the VecTest WN assay. Differences in performance were related to relative sensitivity of the tests. Infection rates of WN in Culex pipiens and Cx. salinarius calculated by the 3 techniques varied, but each estimate indicated a high infection rate in the population. Positive and negative attributes of each procedure, which may influence how and where they are used in surveillance programs, are discussed.

  12. Development a of multiplex TaqMan real-time RT-PCR assay for simultaneous detection of Asian prunus viruses, plum bark necrosis stem pitting associated virus, and peach latent mosaic virus

    USDA-ARS?s Scientific Manuscript database

    Asian prunus viruses (APV 1, APV 2 and APV 3) and Plum bark necrosis stem pitting associated virus (PBNSPaV) are two recently described viruses infecting Prunus spp., and Peach latent mosaic viroid (PLMVd) is a viroid that infects the same species. A single-tube multiplex, TaqMan real-time RT-PCR as...

  13. Comparison and evaluation of conventional RT-PCR, SYBR green I and TaqMan real-time RT-PCR assays for the detection of porcine epidemic diarrhea virus.

    PubMed

    Zhou, Xinrong; Zhang, Tiansheng; Song, Deping; Huang, Tao; Peng, Qi; Chen, Yanjun; Li, Anqi; Zhang, Fanfan; Wu, Qiong; Ye, Yu; Tang, Yuxin

    2017-06-01

    Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) is a highly contagious intestinal disease, resulting in substantial economic losses to the swine industry worldwide. In this study, three assays, namely a conventional reverse transcription-polymerase chain reaction (RT-PCR), a SYBR Green I real-time RT-PCR and a TaqMan real-time RT-PCR targeting the highly conserved M gene of PEDV, were developed and evaluated. Then, the analytical specificity, sensitivity and reproducibility of these assays were determined and compared. The TaqMan real-time RT-PCR was 100-fold and 10,000-fold more sensitive than that of the SYBR Green I real-time RT-PCR and the conventional RT-PCR, respectively. The analytical sensitivity of TaqMan real-time RT-PCR was 10 copies/μl of target gene and no cross amplification with other viruses tested was observed. With the features of high specificity, sensitivity, and reproducibility, the TaqMan real-time RT-PCR established in this study could be a useful tool for clinical diagnosis, epidemiological surveys and outbreak investigations of PED. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. TaqMan real-time PCR assays to assess arbuscular mycorrhizal responses to field manipulation of grassland biodiversity: effects of soil characteristics, plant species richness, and functional traits.

    PubMed

    König, Stephan; Wubet, Tesfaye; Dormann, Carsten F; Hempel, Stefan; Renker, Carsten; Buscot, François

    2010-06-01

    Large-scale (temporal and/or spatial) molecular investigations of the diversity and distribution of arbuscular mycorrhizal fungi (AMF) require considerable sampling efforts and high-throughput analysis. To facilitate such efforts, we have developed a TaqMan real-time PCR assay to detect and identify AMF in environmental samples. First, we screened the diversity in clone libraries, generated by nested PCR, of the nuclear ribosomal DNA internal transcribed spacer (ITS) of AMF in environmental samples. We then generated probes and forward primers based on the detected sequences, enabling AMF sequence type-specific detection in TaqMan multiplex real-time PCR assays. In comparisons to conventional clone library screening and Sanger sequencing, the TaqMan assay approach provided similar accuracy but higher sensitivity with cost and time savings. The TaqMan assays were applied to analyze the AMF community composition within plots of a large-scale plant biodiversity manipulation experiment, the Jena Experiment, primarily designed to investigate the interactive effects of plant biodiversity on element cycling and trophic interactions. The results show that environmental variables hierarchically shape AMF communities and that the sequence type spectrum is strongly affected by previous land use and disturbance, which appears to favor disturbance-tolerant members of the genus Glomus. The AMF species richness of disturbance-associated communities can be largely explained by richness of plant species and plant functional groups, while plant productivity and soil parameters appear to have only weak effects on the AMF community.

  15. Rapid detection of highly pathogenic avian influenza H5N1 virus by TaqMan reverse transcriptase-polymerase chain reaction.

    PubMed

    Heine, H G; Trinidad, L; Selleck, P; Lowther, S

    2007-03-01

    Highly pathogenic avian influenza (AI) H5N1 viruses have been spreading from Asia since late 2003. Early detection and classification are paramount for control of the disease because these viruses are lethal to birds and have caused fatalities in humans. Here, we described TaqMan reverse transcriptase-polymerase chain reaction assays for rapid detection of all AI viruses (influenza type A) and for identification of H5N1 of the Eurasian lineage. The assays were sensitive and quantitative over a 10(5)-10(6) linear range, detected all of the tested AI viruses, and enabled differentiation between H5 and H7 subtypes. These tests allow definitive confirmation of an AI virus as H5 within hours, which is crucial for rapid implementation of control measures in the event of an outbreak.

  16. [Method validation according to ISO 15189 and SH GTA 04: application for the detection of KRAS mutations using PCR TaqMan assay].

    PubMed

    Harlé, Alexandre; Dubois, Cindy; Rouyer, Marie; Merlin, Jean-Louis

    2013-01-01

    Since January 16(th) 2010, the French legislation requires that the medical laboratories must be accredited according to ISO 15189 standards. Thus, all medical laboratories in France must be accredited for at least part of their biological tests before the end of October 2013. Molecular biology tests are also concerned by the accreditation. Validation of molecular biology methods is made difficult, for reasons related to the methods, but also by the type of analytes that are basically rare. This article describes the validation of the qualitative detection of KRAS mutations in metastatic colorectal cancer using TaqMan PCR according to ISO 15189 and to the technical guide for accreditation in Human Health, SH-GTA-04, edited by the COFRAC.

  17. Second-generation Cobas AmpliPrep/Cobas TaqMan HCV quantitative test for viral load monitoring: a novel dual-probe assay design.

    PubMed

    Zitzer, Heike; Heilek, Gabrielle; Truchon, Karine; Susser, Simone; Vermehren, Johannes; Sizmann, Dorothea; Cobb, Bryan; Sarrazin, Christoph

    2013-02-01

    Hepatitis C virus (HCV) RNA viral load (VL) monitoring is a well-established diagnostic tool for the management of chronic hepatitis C patients. HCV RNA VL results are used to make treatment decisions with the goal of therapy to achieve an undetectable VL result. Therefore, a sensitive assay with high specificity in detecting and accurately quantifying HCV RNA across genotypes is critical. Additionally, a lower sample volume requirement is desirable for the laboratory and the patient. This study evaluated the performance characteristics of a second-generation real-time PCR assay, the Cobas AmpliPrep/Cobas TaqMan HCV quantitative test, version 2.0 (CAP/CTM HCV test, v2.0), designed with a novel dual-probe approach and an optimized automated extraction and amplification procedure. The new assay demonstrated a limit of detection and lower limit of quantification of 15 IU/ml across all HCV genotypes and was linear from 15 to 100,000,000 IU/ml with high accuracy (<0.2-log(10) difference) and precision (standard deviation of 0.04 to 0.22 log(10)). A specificity of 100% was demonstrated with 600 HCV-seronegative specimens without cross-reactivity or interference. Correlation to the Cobas AmpliPrep/Cobas TaqMan HCV test (version 1) was good (n = 412 genotype 1 to 6 samples, R(2) = 0.88; R(2) = 0.94 without 105 genotype 4 samples). Paired plasma and serum samples showed similar performance (n = 25, R(2) = 0.99). The sample input volume was reduced from 1 to 0.65 ml in the second version. The CAP/CTM HCV test, v2.0, demonstrated excellent performance and sensitivity across all HCV genotypes with a smaller sample volume. The new HCV RNA VL assay has performance characteristics that make it suitable for use with currently available direct-acting antiviral agents.

  18. Development and evaluation of a TaqMan duplex real-time PCR quantification method for reliable enumeration of Candidatus Microthrix.

    PubMed

    Vanysacker, Louise; Denis, Carla; Roels, Joris; Verhaeghe, Kirke; Vankelecom, Ivo F J

    2014-02-01

    Candidatus Microtrhix parvicella is one of the most common filamentous bacteria reported to be involved in bulking and foaming problems in activated sludge plants worldwide. In order to detect and quantify both M. parvicella and Microthrix calida by quantitative PCR (qPCR), primers targeting 16S rDNA genes were designed. The qPCR reaction was optimized by using the TaqMan technology and an internal positive control was included to ensure the absence of PCR inhibitors. A total of 29 samples originating from different wastewater treatment plants were analyzed and the results were compared by using conventional microscopy, fluorescent in situ hybridization and an existing SYBR Green-based assay. Our assay showed a 100% specificity for both M. parvicella and M. calida, a sensitivity of 2.93×10(9) to 29 copy numbers/reaction, an amplification efficiency of 93% and no PCR inhibition. By performing a spiking experiment including different Microthrix concentrations, recovery rates ranging from 65 to 98% were obtained. A positive correlation with the SYBR Green assay (R(2)=0.85) was found and most of the samples were in accordance with the microscopical observation. In comparison with SYBR Green assay, the probe-based TaqMan assay had a much lower detection limit. Compared with microscopy, some samples had a lower or higher enumeration when using qPCR. In conclusion, a qPCR method is forwarded here that could be useful as an early warning tool for fast and reliable detection of Microthrix in for instance sludge bulking events.

  19. TaqMan Real-Time PCR Assays for Single-Nucleotide Polymorphisms Which Identify Francisella tularensis and Its Subspecies and Subpopulations

    PubMed Central

    Birdsell, Dawn N.; Vogler, Amy J.; Buchhagen, Jordan; Clare, Ashley; Kaufman, Emily; Naumann, Amber; Driebe, Elizabeth; Wagner, David M.; Keim, Paul S.

    2014-01-01

    Francisella tularensis, the etiologic agent of tularemia and a Class A Select Agent, is divided into three subspecies and multiple subpopulations that differ in virulence and geographic distribution. Given these differences, there is a need to rapidly and accurately determine if a strain is F. tularensis and, if it is, assign it to subspecies and subpopulation. We designed TaqMan real-time PCR genotyping assays using eleven single nucleotide polymorphisms (SNPs) that were potentially specific to closely related groups within the genus Francisella, including numerous subpopulations within F. tularensis species. We performed extensive validation studies to test the specificity of these SNPs to particular populations by screening the assays across a set of 565 genetically and geographically diverse F. tularensis isolates and an additional 21 genetic near-neighbor (outgroup) isolates. All eleven assays correctly determined the genetic groups of all 565 F. tularensis isolates. One assay differentiates F. tularensis, F. novicida, and F. hispaniensis from the more genetically distant F. philomiragia and Francisella-like endosymbionts. Another assay differentiates F. tularensis isolates from near neighbors. The remaining nine assays classify F. tularensis-confirmed isolates into F. tularensis subspecies and subpopulations. The genotyping accuracy of these nine assays diminished when tested on outgroup isolates (i.e. non F. tularensis), therefore a hierarchical approach of assay usage is recommended wherein the F. tularensis-specific assay is used before the nine downstream assays. Among F. tularensis isolates, all eleven assays were highly sensitive, consistently amplifying very low concentrations of DNA. Altogether, these eleven TaqMan real-time PCR assays represent a highly accurate, rapid, and sensitive means of identifying the species, subspecies, and subpopulation of any F. tularensis isolate if used in a step-wise hierarchical scheme. These assays would be very

  20. Development of a Second Version of the Cobas AmpliPrep/Cobas TaqMan Hepatitis C Virus Quantitative Test with Improved Genotype Inclusivity▿

    PubMed Central

    Vermehren, Johannes; Colucci, Giuseppe; Gohl, Peter; Hamdi, Nabila; Abdelaziz, Ahmed Ihab; Karey, Ursula; Thamke, Diana; Zitzer, Heike; Zeuzem, Stefan; Sarrazin, Christoph

    2011-01-01

    Hepatitis C virus (HCV) RNA measurement has been facilitated by the introduction of real-time PCR-based assays with low limits of detection and broad dynamic ranges for quantification. In the present study, the performance of two second-version prototypes of the Cobas AmpliPrep/Cobas TaqMan HCV Quantitative Test (CAP/CTM v2) with decreased sample input volume and improved genotype inclusivity was investigated. A total of 232 serum and plasma samples derived from patients with chronic hepatitis C (genotype 1 [GT1], n = 108; GT2, n = 8; GT3, n = 24; GT4, n = 87; GT5, n = 3; and GT6, n = 2) were processed in parallel with the Cobas AmpliPrep/Cobas TaqMan HCV Test (CAP/CTM), Cobas Amplicor HCV Monitor Test v2.0 (CAM), and two second-version prototype formulations of CAP/CTM, Mastermix 1 (MMx1) and MMx2. In addition, three GT4 transcripts containing rare variant sequences were tested. The mean log10 HCV RNA differences for the best-performing CAP/CTM v2/MMx2 formulation in comparison to CAM were −0.05, 0.05, −0.12, −0.10, −0.44, and −0.29 for patients with GT1, GT2, GT3, GT4, GT5, and GT6 infections, respectively. GT1, GT2, and GT4 samples including isolates with known variants within the 5′ untranslated region (G145A, A165T) that were underquantified with CAP/CTM were correctly quantified with the second-version prototype. In addition, CAP/CTM v2 was able to accurately quantify the three transcripts with rare variant sequences. In conclusion, CAP/CTM v2 accurately quantifies HCV RNA across all HCV genotypes, including specimens with rare polymorphisms previously associated with underquantification. PMID:21752967

  1. Development of a second version of the Cobas AmpliPrep/Cobas TaqMan hepatitis C virus quantitative test with improved genotype inclusivity.

    PubMed

    Vermehren, Johannes; Colucci, Giuseppe; Gohl, Peter; Hamdi, Nabila; Abdelaziz, Ahmed Ihab; Karey, Ursula; Thamke, Diana; Zitzer, Heike; Zeuzem, Stefan; Sarrazin, Christoph

    2011-09-01

    Hepatitis C virus (HCV) RNA measurement has been facilitated by the introduction of real-time PCR-based assays with low limits of detection and broad dynamic ranges for quantification. In the present study, the performance of two second-version prototypes of the Cobas AmpliPrep/Cobas TaqMan HCV Quantitative Test (CAP/CTM v2) with decreased sample input volume and improved genotype inclusivity was investigated. A total of 232 serum and plasma samples derived from patients with chronic hepatitis C (genotype 1 [GT1], n = 108; GT2, n = 8; GT3, n = 24; GT4, n = 87; GT5, n = 3; and GT6, n = 2) were processed in parallel with the Cobas AmpliPrep/Cobas TaqMan HCV Test (CAP/CTM), Cobas Amplicor HCV Monitor Test v2.0 (CAM), and two second-version prototype formulations of CAP/CTM, Mastermix 1 (MMx1) and MMx2. In addition, three GT4 transcripts containing rare variant sequences were tested. The mean log(10) HCV RNA differences for the best-performing CAP/CTM v2/MMx2 formulation in comparison to CAM were -0.05, 0.05, -0.12, -0.10, -0.44, and -0.29 for patients with GT1, GT2, GT3, GT4, GT5, and GT6 infections, respectively. GT1, GT2, and GT4 samples including isolates with known variants within the 5' untranslated region (G145A, A165T) that were underquantified with CAP/CTM were correctly quantified with the second-version prototype. In addition, CAP/CTM v2 was able to accurately quantify the three transcripts with rare variant sequences. In conclusion, CAP/CTM v2 accurately quantifies HCV RNA across all HCV genotypes, including specimens with rare polymorphisms previously associated with underquantification.

  2. Second-Generation Cobas AmpliPrep/Cobas TaqMan HCV Quantitative Test for Viral Load Monitoring: a Novel Dual-Probe Assay Design

    PubMed Central

    Zitzer, Heike; Heilek, Gabrielle; Truchon, Karine; Susser, Simone; Vermehren, Johannes; Sizmann, Dorothea; Cobb, Bryan

    2013-01-01

    Hepatitis C virus (HCV) RNA viral load (VL) monitoring is a well-established diagnostic tool for the management of chronic hepatitis C patients. HCV RNA VL results are used to make treatment decisions with the goal of therapy to achieve an undetectable VL result. Therefore, a sensitive assay with high specificity in detecting and accurately quantifying HCV RNA across genotypes is critical. Additionally, a lower sample volume requirement is desirable for the laboratory and the patient. This study evaluated the performance characteristics of a second-generation real-time PCR assay, the Cobas AmpliPrep/Cobas TaqMan HCV quantitative test, version 2.0 (CAP/CTM HCV test, v2.0), designed with a novel dual-probe approach and an optimized automated extraction and amplification procedure. The new assay demonstrated a limit of detection and lower limit of quantification of 15 IU/ml across all HCV genotypes and was linear from 15 to 100,000,000 IU/ml with high accuracy (<0.2-log10 difference) and precision (standard deviation of 0.04 to 0.22 log10). A specificity of 100% was demonstrated with 600 HCV-seronegative specimens without cross-reactivity or interference. Correlation to the Cobas AmpliPrep/Cobas TaqMan HCV test (version 1) was good (n = 412 genotype 1 to 6 samples, R2 = 0.88; R2 = 0.94 without 105 genotype 4 samples). Paired plasma and serum samples showed similar performance (n = 25, R2 = 0.99). The sample input volume was reduced from 1 to 0.65 ml in the second version. The CAP/CTM HCV test, v2.0, demonstrated excellent performance and sensitivity across all HCV genotypes with a smaller sample volume. The new HCV RNA VL assay has performance characteristics that make it suitable for use with currently available direct-acting antiviral agents. PMID:23241371

  3. Development of a TaqMan Array Card for Acute-Febrile-Illness Outbreak Investigation and Surveillance of Emerging Pathogens, Including Ebola Virus

    PubMed Central

    Liu, Jie; Ochieng, Caroline; Wiersma, Steve; Ströher, Ute; Towner, Jonathan S.; Whitmer, Shannon; Nichol, Stuart T.; Moore, Christopher C.; Kersh, Gilbert J.; Kato, Cecilia; Sexton, Christopher; Petersen, Jeannine; Massung, Robert; Hercik, Christine; Crump, John A.; Kibiki, Gibson; Maro, Athanasia; Mujaga, Buliga; Gratz, Jean; Jacob, Shevin T.; Banura, Patrick; Scheld, W. Michael; Juma, Bonventure; Onyango, Clayton O.; Montgomery, Joel M.

    2015-01-01

    Acute febrile illness (AFI) is associated with substantial morbidity and mortality worldwide, yet an etiologic agent is often not identified. Convalescent-phase serology is impractical, blood culture is slow, and many pathogens are fastidious or impossible to cultivate. We developed a real-time PCR-based TaqMan array card (TAC) that can test six to eight samples within 2.5 h from sample to results and can simultaneously detect 26 AFI-associated organisms, including 15 viruses (chikungunya, Crimean-Congo hemorrhagic fever [CCHF] virus, dengue, Ebola virus, Bundibugyo virus, Sudan virus, hantaviruses [Hantaan and Seoul], hepatitis E, Marburg, Nipah virus, o'nyong-nyong virus, Rift Valley fever virus, West Nile virus, and yellow fever virus), 8 bacteria (Bartonella spp., Brucella spp., Coxiella burnetii, Leptospira spp., Rickettsia spp., Salmonella enterica and Salmonella enterica serovar Typhi, and Yersinia pestis), and 3 protozoa (Leishmania spp., Plasmodium spp., and Trypanosoma brucei). Two extrinsic controls (phocine herpesvirus 1 and bacteriophage MS2) were included to ensure extraction and amplification efficiency. Analytical validation was performed on spiked specimens for linearity, intra-assay precision, interassay precision, limit of detection, and specificity. The performance of the card on clinical specimens was evaluated with 1,050 blood samples by comparison to the individual real-time PCR assays, and the TAC exhibited an overall 88% (278/315; 95% confidence interval [CI], 84% to 92%) sensitivity and a 99% (5,261/5,326, 98% to 99%) specificity. This TaqMan array card can be used in field settings as a rapid screen for outbreak investigation or for the surveillance of pathogens, including Ebola virus. PMID:26491176

  4. Rapid Identification and Quantification of Aureococcus anophagefferens by qPCR Method (Taqman) in the Qinhuangdao Coastal Area: A Region for Recurrent Brown Tide Breakout in China.

    PubMed

    Wang, Li-Ping; Lei, Kun

    2016-12-01

    Since 2009, Aureococcus anophagefferens has caused brown tide to occur recurrently in Qinhuangdao coastal area, China. Because the algal cells of A. anophagefferens are so tiny (~3 µm) that it is very hard to identify exactly under a microscope for natural water samples, it is very urgent to develop a method for efficient and continuous monitoring. Here specific primers and Taqman probe are designed to develop a real-time quantitative PCR (qPCR) method for identification and quantification continually. The algal community and cell abundance of A. anophagefferens in the study area (E 119°20'-119°50' and N 39°30'-39°50') from April to October in 2013 are detected by pyrosequencing, and are used to validate the specification and precision of qPCR method for natural samples. Both pyrosequencing and qPCR shows that the targeted cells are present only in May, June and July, and the cell abundance are July > June > May. Although there are various algal species including dinoflagellata, diatom, Cryptomonadales, Chrysophyceae and Chlorophyta living in the natural seawater simultaneously, no disturbance happens to qPCR method. This qPCR method could detect as few as 10 targeted cells, indicating it is able to detect the algal cells at pre-bloom levels. Therefore, qPCR with Taqman probe provides a powerful and sensitive method to monitor the brown tide continually in Qinhuangdao coastal area, China. The results provide a necessary technology support for forecasting the brown tide initiation, in China.

  5. Nucleic acid programmed polymeric nanomaterials for biological communication

    NASA Astrophysics Data System (ADS)

    Rush, Anthony Michael

    A number of nucleic acid-polymer conjugates were synthesized, resulting in amphiphilic polymer-nucleic acid conjugates with the capability to self-assemble into a range of discrete nanoscale architectures. These nanomaterials, termed DNA-polymer amphiphile nanoparticles (DPA NPs), were studied with respect to their enzymatic processing by both endo- and exonucleases and further deployed as antisense genetic regulatory elements in live cultured human cells. DPA NPs were designed to act as substrates for both non sequence-specific exonucleases and a sequence-specific endonuclease. In all cases, nucleic acids arranged in the corona of spherical nanoparticles exhibited increased resistance to nucleolytic cleavage as compared to native single- or double-stranded analogues. For the exonucleases studied (Exonuclease III from E. Coli and phosphodiesterase I from Crotalus adamanteus), nanoparticle display retarded enzymatic processing by roughly a factor of five. For the endonuclease studied (Nt.CviPII), nanoparticle display prohibited virtually all enzyme activity on oligonucleotides within the nanoparticle shell. To test the ability of these materials to regulate mRNA levels in live cultured human cells, LPA (LNA-polymer amphiphile) NPs were designed to be perfectly complementary to a 20-base region of mRNA encoding the anti-apoptosis protein survivin. In this study two key observations were made. The first observation is that packaging LNA into spherical micellar nanoparticles serves to dramatically enhance cellular uptake of LNA based on flow cytometry and fluorescence microscopy data. The second observation is that LPA NPs are capable of regulating mRNA levels by what is hypothesized to be activation of target mRNA for catalytic RNase H-mediated degradation. These materials represent a unique class of DNA delivery system capable of rendering nucleic acids with natural backbone chemistry resistant to nuclease degradation and further serving to deliver DNA into cells to

  6. Development and evaluation of a TaqMan Real-Time PCR assay for Fusarium oxysporum f. sp. spinaciae

    USDA-ARS?s Scientific Manuscript database

    Fusarium oxysporum f. sp. spinaciae, causal agent of spinach Fusarium wilt, is an important soilborne pathogen in many areas of the world where spinach is grown. The pathogen is persistent in acid soils of maritime western Oregon and Washington, the only region of the USA suitable for commercial spi...

  7. Regulation of tissue LC-PUFA contents, Δ6 fatty acyl desaturase (FADS2) gene expression and the methylation of the putative FADS2 gene promoter by different dietary fatty acid profiles in Japanese seabass (Lateolabrax japonicus).

    PubMed

    Xu, Houguo; Dong, Xiaojing; Ai, Qinghui; Mai, Kangsen; Xu, Wei; Zhang, Yanjiao; Zuo, Rantao

    2014-01-01

    The present study was conducted to evaluate the influences of different dietary fatty acid profiles on the tissue content and biosynthesis of LC-PUFA in a euryhaline species Japanese seabass reared in seawater. Six diets were prepared, each with a characteristic fatty acid: Diet PA: Palmitic acid (C16:0); Diet SA: Stearic acid (C18:0); Diet OA: Oleic acid (C18:1n-9); Diet LNA: α-linolenic acid (C18:3n-3); Diet N-3 LC-PUFA: n-3 LC-PUFA (DHA+EPA); Diet FO: the fish oil control. A 10-week feeding trial was conducted using juvenile fish (29.53 ± 0.86 g). The results showed that Japanese seabass had limited capacity to synthesize LC-PUFA and fish fed PA, SA, OA and LNA showed significantly lower tissue n-3 LC-PUFA contents compared to fish fed N-3 LC-PUFA and FO. The putative gene promoter and full-length cDNA of FADS2 was cloned and characterized. The protein sequence was confirmed to be homologous to FADS2s of marine teleosts and possessed all the characteristic features of microsomal fatty acid desaturases. The FADS2 transcript levels in liver of fish fed N-3 LC-PUFA and FO were significantly lower than those in fish fed other diets except LNA while Diet PA significantly up-regulated the FADS2 gene expression compared to Diet LNA, N-3 LC-PUFA and FO. Inversely, fish fed N-3 LC-PUFA and FO showed significantly higher promoter methylation rates of FADS2 gene compared to fish fed the LC-PUFA deficient diets. These results suggested that Japanese seabass had low LC-PUFA synthesis capacity and LC-PUFA deficient diets caused significantly reduced tissue n-3 LC-PUFA contents. The liver gene expression of FADS2 was up-regulated in groups enriched in C16:0, C18:0 and C18:1n-9 respectively but not in the group enriched in C18:3n-3 compared to groups with high n-3 LC-PUFA contents. The FADS2 gene expression regulated by dietary fatty acids was significantly negatively correlated with the methylation rate of putative FADS2 gene promoter.

  8. Development of Dual TaqMan Based One-Step rRT-PCR Assay Panel for Rapid and Accurate Diagnostic Test of MERS-CoV: A Novel Human Coronavirus, Ahead of Hajj Pilgrimage

    PubMed Central

    Hashemzadeh, Mohammad Sadegh; Rasouli, Rahimeh; Zahraei, Bentolhoda; Izadi, Morteza; Tat, Mahdi; Saadat, Seyed Hassan; Najarasl, Mohammad; Khansari Nejad, Behzad; Dorostkar, Ruhollah

    2016-01-01

    Background Coronaviruses (CoVs) are large ribonucleic acid (RNA) viruses causing primarily respiratory disease in humans. A novel human coronavirus, subsequently named middle east respiratory syndrome coronavirus (MERS-CoV), was first reported in Saudi Arabia in September of 2012. With increasing numbers of infections and deaths from MERS-CoV, development of a rapid and reliable kit was crucial to prevent further spread of MERS-CoV. Objectives In this study, we present two real-time reverse-transcription polymerase chain reaction (rRT-PCR) assays for in-house rapid and sensitive diagnostic testing of MERS-CoV, detecting the regions upstream of the envelope gene (upE) and open reading frame (ORF) 1b, respectively, for initial screening and final confirmation of MERS-CoV infection, as recommended by the world health organization (WHO). Materials and Methods In this experimental study, acquiring patient samples was difficult; thus, according to WHO recommendations and standard protocols, we synthesized RNA sequences of upE and ORF1b genes as the template signatures and TaqMan based-diagnostic rRT-PCR assays were carried out using these synthetic genes for detection of MERS-CoV. In this research, we also inaugurated a cell-free system to transcribe these RNA sequences using the DNA templates synthesized. Results The upE and ORF1b based one-step rRT-PCR assays were optimized by testing several times via different synthetic RNAs, and validation results were highly successful. The sensitivity obtained for upE was fewer than ten copies of RNA template per reaction and for ORF1b was 50 or fewer copies per reaction. Conclusions This study showed that the developed rRT-PCR assays are rapid, reliable, reproducible, specific, sensitive, and simple tools for detection of MERS-CoV. Finally, a kit consisting of two assay signatures and controls was assembled, which can be distributed to public health laboratories in Iran to support international MERS-CoV surveillance and public

  9. Comparison of COBAS 4800 KRAS, TaqMan PCR and high resolution melting PCR assays for the detection of KRAS somatic mutations in formalin-fixed paraffin embedded colorectal carcinomas.

    PubMed

    Harlé, Alexandre; Busser, Benoit; Rouyer, Marie; Harter, Valentin; Genin, Pascal; Leroux, Agnès; Merlin, Jean-Louis

    2013-03-01

    Many studies documented the influence of KRAS mutation status on the response of patients with metastatic colorectal cancer (mCRC) to anti-EGFR monoclonal antibodies. The COBAS 4800 KRAS is an assay using real time PCR and TaqMelt technology, CE-IVD validated, for the detection of 19 KRAS somatic mutations in exons 2 and 3. We compared COBAS with previously validated PCR TaqMan and High Resolution Melting (HRM) assays on 156 formalin-fixed paraffin embedded (FFPE) specimens of colorectal carcinoma. DNA extraction procedures, using the Qiagen QiAMP kit and the Roche COBAS DNA kit, were also compared. Of the 156 samples, 132 were interpretable using COBAS and TaqMan and 92 using COBAS and HRM. No statistically significant difference was found between COBAS/TaqMan and COBAS/HRM (k = 0.937; p < 0.001 - four discordant cases were found, mostly concerning codon 61 mutations and k = 0.891; p < 0.001 - five discordant cases were found, three regarding codon 61 and two on codon 12/13, respectively). No difference was found between the two DNA extraction methods (t = 1.7185; dol = 39; α = 5 %). The three assays were found suitable to detect accurately KRAS mutations in colon FFPE specimens. COBAS and TaqMan were found to be more robust than HRM, as they yielded fewer non-interpretable results. DNA extraction kits were found to provide equivalent results. The present study shows that pre-screening using COBAS with further TaqMan mutation characterization constitutes an easy and reliable approach for routine diagnostic purposes.

  10. Development and Validation of TaqMan Real-Time Polymerase Chain Reaction Assays for the Quantitative and Differential Detection of Wild-Type Infectious Laryngotracheitis Viruses from a Glycoprotein G-Deficient Candidate Vaccine Strain.

    PubMed

    Shil, Niraj K; Legione, Alistair R; Markham, Philip F; Noormohammadi, Amir H; Devlin, Joanne M

    2015-03-01

    Infectious laryngotracheitis (ILT) is a significant upper respiratory tract disease of chickens with a worldwide distribution. Differentiating between wild-type and vaccine strains of ILT virus (ILTV) would be useful for enhancing disease control, and in the early stages of a disease outbreak molecular diagnostic tools for the detection and differentiation of the circulating virus could be applied. This study developed TaqMan real-time PCR (qPCR) assays to detect and differentiate the glycoprotein G (gG)-deficient (ΔgG) ILTV candidate vaccine strain of ILTV from ILTV strains that contain the gG gene. The gG+ve and gG-ve ILTV TaqMan assays were used in individual and multiplex format to detect, differentiate, and quantitate ILTV DNA in laboratory and clinical samples. The assays were highly sensitive and highly specific, with a detection limit of 10 viral template copies for each assay. Low interassay coefficients of variation were recorded (0.021-0.042 and 0.013-0.039) for gG+ve and gG-ve TaqMan assays, respectively. The multiplex assay was successfully used to examine the replication kinetics of wild-type and ΔgG strains of ILTV in cultured leghorn male hepatoma cells and embryonated hen eggs under coinfection conditions. The results showed that the TaqMan qPCR assay, along with the ΔgG ILTV vaccine, has the potential to be used in a "Differentiating Infected from Vaccinated Animals" strategy for the control and eradication of ILT.

  11. Development of an mlrA Gene-Directed TaqMan PCR Assay for Quantitative Assessment of Microcystin-Degrading Bacteria within Water Treatment Plant Sand Filter Biofilms▿

    PubMed Central

    Hoefel, Daniel; Adriansen, Caroline M. M.; Bouyssou, Magali A. C.; Saint, Christopher P.; Newcombe, Gayle; Ho, Lionel

    2009-01-01

    We report for the first time a quantitative mlrA gene-directed TaqMan PCR assay for the rapid detection of microcystin-degrading bacteria. This was applied, in combination with 16S ribosomal DNA-directed quantitative PCR and denaturing gradient gel electrophoresis, to study virgin sand filter column biofilm development and to correlate mlrA gene abundance with microcystin removal efficiency. PMID:19502429

  12. A new molecular approach to help conclude drowning as a cause of death: simultaneous detection of eight bacterioplankton species using real-time PCR assays with TaqMan probes.

    PubMed

    Uchiyama, Taketo; Kakizaki, Eiji; Kozawa, Shuji; Nishida, Sho; Imamura, Nahoko; Yukawa, Nobuhiro

    2012-10-10

    We developed a novel tool for concluding drowning as a cause of death. We designed nine primer pairs to detect representative freshwater or marine bacterioplankton (aquatic bacteria) and then used real-time PCR with TaqMan probes to rapidly and specifically detect them. We previously cultured the genus Aeromonas, which is a representative freshwater bacterial species, in blood samples from 94% of victims who drowned in freshwater and the genera Vibrio and/or Photobacterium that are representative marine bacteria in 88% of victims who drowned in seawater. Based on these results, we simultaneously detected eight species of bacterioplankton (Aeromonas hydrophila, A. salmonicida; Vibrio fischeri, V. harveyi, V. parahaemolyticus; Photobacterium damselae, P. leiognathi, P. phosphoreum) using three sets of triplex real-time PCR assays and TaqMan probes labelled with fluorophores (FAM, NED, Cy5). We assayed 266 specimens (109 blood, 157 tissues) from 43 victims, including 32 who had drowned in rivers, ditches, wells, sea or around estuaries. All lung samples of these 32 victims were TaqMan PCR-positive including the lung periphery into which water does not readily enter postmortem. On the other hand, findings in blood and/or closed organs (kidney or liver) were PCR-positive in 84% of the drowned victims (except for those who drowned in baths) although the conventional test detected diatoms in closed organs in only 44% of the victims. Thus, the results of the PCR assay reinforced those of diatom tests when only a few diatoms were detectable in organs due to the low density of diatoms in the water where they were found. Multiplex TaqMan PCR assays for bacterioplankton were rapid, less laborious and high-throughput as well as sensitive and specific. Therefore, these assays would be useful for routine forensic screening tests to estimate the amount and type of aspirated water.

  13. Development and validation of a real-time TaqMan assay for the detection and enumeration of Pseudomonas fluorescens ATCC 13525 used as a challenge organism in testing of food equipments.

    PubMed

    Saha, Ratul; Bestervelt, Lorelle L; Donofrio, Robert S

    2012-02-01

    Pseudomonas fluorescens ATCC 13525 is used as the challenge organism to evaluate the efficacy of the clean-in-place (CIP) process of food equipment (automatic ice-maker) as per NSF/ANSI Standard 12. Traditional culturing methodology is presently used to determine the concentration of the challenge organism, which takes 48 h to confirm the cell density. Storage of the challenge preparation in the refrigerator might alter the cell density as P. fluorescens is capable of growing at 4 °C. Also, background organism can grow on the Pseudomonas F agar (PFA) used for the recovery of P. fluorescens thus affecting the results of the test. Real-time TaqMan assay targeting the cpn60 gene was developed for the enumeration and the identification of P. fluorescens because of its specificity, accuracy, and shorter turnaround time. The TaqMan primer-probe pair developed using the Allele ID® 7.0 probe design software was highly specific and sensitive for the target organism. The sensitivity of the assay was 10 colony forming units (CFU)/mL. The assay was also successful in determining the concentration of the challenge preparation within 2 h. Based on these observations, TaqMan assay targeting the cpn60 gene can be efficiently used for strain level identification and enumeration of bacteria. Pseudomonas fluorescens ATCC 13525 is used as a challenge organism in the efficacy testing of clean-in-place process of food equipments. Currently, culturing technique is used for its identification and estimation, which is not only time-consuming but also prone to error. Real-time TaqMan assay is more specific, sensitive, and accurate along with a shorter turnaround time compared to culturing techniques, thereby increasing the overall quality of the testing methodology to evaluate the clean-in-place process critical for the food industry to protect public health and safety. © 2012 Institute of Food Technologists®

  14. Competitive allele-specific TaqMan PCR (Cast-PCR) is a sensitive, specific and fast method for BRAF V600 mutation detection in Melanoma patients

    PubMed Central

    Barbano, Raffaela; Pasculli, Barbara; Coco, Michelina; Fontana, Andrea; Copetti, Massimiliano; Rendina, Michelina; Valori, Vanna Maria; Graziano, Paolo; Maiello, Evaristo; Fazio, Vito Michele; Parrella, Paola

    2015-01-01

    BRAF codon 600 mutation testing of melanoma patients is mandatory for the choice of the most appropriate therapy in the clinical setting. Competitive allele specific TaqMan PCR (Cast-PCR) technology allows not only the selective amplification of minor alleles, but it also blocks the amplification of non-mutant allele. We genotyped codon 600 of the BRAF gene in 54 patients’ samples by Cast-PCR and bidirectional direct sequence analysis. All the mutations detected by sequencing were also identified by Cast-PCR. In addition, Cast-PCR assay detected four samples carrying mutations and was able to clearly identify two mutations of uncertain interpretation by Sanger sequencing. The limit of detection of Cast-PCR was evaluated by constructing dilution curves of BRAFV600E and BRAFV600K mutated clinical samples mixed with a not-mutated specimens. Both mutations could be detected until a 1:100 mutated/not mutated ratio. Cloning and sequencing of the clones was used to confirm mutations on representative discrepant cases. Cast PCR performances were not affected by intratumour heterogeneity, and less affected by melanin content. Our results indicate that Cast-PCR is a reliable diagnostic tool for the identification of melanoma patients as eligible to be treated with TKIs and might be implemented in the clinical setting as elective screening method. PMID:26690267

  15. Development of a real time PCR Taqman assay based on the TPI gene for simultaneous identification of Clostridium chauvoei and Clostridium septicum.

    PubMed

    Garofolo, G; Galante, D; Serrecchia, L; Buonavoglia, D; Fasanella, A

    2011-02-01

    In the present study, a Taqman allelic discrimination assay based on three SNPs of the TPI gene is described. It was used as a differential diagnostic tool to detect blackleg and malignant edema. Sudden deaths of grazing ruminants, such as cattle, sheep and goats, which show clinical signs related to hyperacute infective processes, encouraged the development of a rapid and precise diagnostic molecular method. Specific primers and probes for Clostridium septicum and Clostridium chauvoei were designed on the basis of the TPI gene sequence. The multiplex PCR was tested on the DNA of a total of 57 strains, including 24 Clostridium chauvoei, 20 Clostridium septicum, 1 Bacillus anthracis and 12 other Clostridium spp. The DNA samples from Clostridium chauvoei and Clostridium septicum strains were amplified. Amplification of other DNA samples was not observed, with the exception of Clostridium tertium, which showed a weak positive signal. To avoid misdiagnosis, a confirmatory assay based on a Sybr green real time PCR was proposed. The authors confirmed the efficacy and the specificity of the test used in this study, which proved to be a useful tool for the diagnosis of clostridiosis that are often diagnosed using only traditional tools.

  16. Accurate interrogation of FCGR3A rs396991 in European and Asian populations using a widely available TaqMan genotyping method

    PubMed Central

    Niederer, Heather A.; King, Karen S.; Harris, Elizabeth C.; Glass, Sarah M.; Cox, Charles J.

    2015-01-01

    A polymorphism in the receptor for the Fc region of IgG, Fc γ-receptor IIIa (FcγRIIIa, FCGR3A rs396991), has been inconsistently shown in the literature to have an effect on response to monoclonal antibody therapy in several indications. The rs396991 (T/G) polymorphism leads to an F176V substitution and increased affinity for IgG. This variant has proven difficult to genotype accurately, primarily because of extensive homology between the FCGR3A and FCGR3B genes. We have shown that rs396991 can be genotyped by PCR amplification, followed by direct Sanger sequencing of the product, without coamplification of FCGR3B, and that the rs396991 TaqMan assay (C__25815666_10) agrees with Sanger sequencing results in 100% of European and Asian samples tested, but it has a small error rate in African and American populations. C__25815666_10 is therefore suitable to interrogate rs396991 in studies involving Europeans and Asians; however for other populations, the default genotyping method should be PCR followed by Sanger sequencing. PMID:26367501

  17. Development of a TaqMan Probe-Based Insulated Isothermal Polymerase Chain Reaction (iiPCR) Assay for Detection of Fusarium oxysporum f. sp. cubense Race 4

    PubMed Central

    Lin, Yi-Jia; Chang, Tsai-De; Hong, Li-Ling; Chen, Tzu-Yu; Chang, Pi-Fang Linda

    2016-01-01

    This study developed a novel and inexpensive detection method based on a TaqMan probe-based insulated isothermal polymerase chain reaction (iiPCR) method for the rapid detection of Panama disease caused by Fusarium oxysporum f. sp. cubense (Foc) race 4, which is currently among the most serious fungal vascular diseases worldwide. By using the portable POCKIT™ device with the novel primer set iiFoc-1/iiFoc-2, the Foc race 4 iiPCR assay (including DNA amplification and signal monitoring) could be completed within one hour. The developed Foc race 4 iiPCR assay is thus a user-friendly and efficient platform designed specifically for the detection of Foc race 4. The detection limit of this optimized Foc iiPCR system was estimated to be 1 copy of the target standard DNA as well as 1 fg of the Foc genomic DNA. This approach can serve as a rapid detection method for in planta detection of Foc race 4 in field-infected banana. It was concluded that this molecular detection procedure based on iiPCR has good potential for use as an efficient detection method. PMID:27448242

  18. Development of a real-time quantitative PCR assay using a TaqMan minor groove binder probe for the detection of α-lactalbumin in food.

    PubMed

    Xiao, Guan; Qin, Cai; Wenju, Zhang; Qin, Chen

    2016-03-01

    Here, we report the development of a real-time PCR assay using a TaqMan minor groove binder (MGB, Genecore, NCBI: AF249896.1, 806-820) probe and primer sets designed to recognize the α-lactalbumin gene from the cow (Bos taurus). We evaluated the efficacy of this assay for detecting and quantifying cow α-lactalbumin in commercial foods. Our results demonstrated that the developed method was highly sensitive and showed high specificity for cow milk, with consistent detection of 0.05 ng of bovine DNA. We tested 42 commercial food samples with or without cow milk listed as an ingredient by using the developed assay. Among the 42 samples, 26 products that listed milk as an ingredient and 3 products might contain milk showed positive signals, whereas the other 9 products that did not contain milk and 4 products that might contain milk tested negative. Therefore, this method could be widely used for the detection of cow milk allergens in food.

  19. Development of a TaqMan MGB RT-PCR for the rapid detection of H3 subtype avian influenza virus circulating in China.

    PubMed

    Teng, Qiaoyang; Shen, Weixia; Yan, Dawei; Yan, Liping; Li, Xuesong; Li, Guoxin; Yang, Jianmei; Li, Zejun

    2015-06-01

    Previous studies demonstrated that the H3 avian influenza virus (AIV) in China is isolated most frequently from wild birds and live poultry markets. However, there is no subtype-specific real-time polymerase chain reaction (RT-PCR) available for the rapid and highly sensitive identification of H3 AIV. In this study, a TaqMan minor groove binder (MGB) probe and a pair of primers were designed based on a conserved region in the hemagglutinin gene of H3 AIV. These were used to generate an H3-MGB RT-PCR assay that recognizes only H3 AIV. The detection limit of the H3-MGB RT-PCR was 10 copies of DNA per reaction when 10-fold serial dilutions of T-H3HA plasmid were used as the template. This was 1000-times more sensitive than conventional RT-PCR. In experimental samples obtained from oropharyngeal swabs or cloacal swabs, the virus was detected in all ducks using H3-MGB RT-PCR, whereas only one duck tested positive for the virus in oropharyngeal swabs tested using conventional RT-PCR. The H3-MGB RT-PCR assay developed in this study is a sensitive and rapid tool for screening H3 AIV in China.

  20. The Malaria TaqMan Array Card: 87 assays for Plasmodium falciparum drug resistance, speciation, and genotyping in a single reaction.

    PubMed

    Pholwat, Suporn; Liu, Jie; Stroup, Suzanne; Jacob, Shevin; Banura, Patrick; Moore, Christopher; Huang, Fang; Laufer, Miriam; Houpt, Eric; Guler, Jennifer

    2017-03-06

    Antimalarial drug resistance exacerbates the global disease burden and complicates eradication efforts. To facilitate the surveillance of resistance markers in malaria-endemic countries, we developed a suite of TaqMan assays for known resistance markers and compartmentalized them into a single array card (TAC). We included 87 assays for species identification, the detection of Plasmodium falciparum mutations associated with chloroquine, atovaquone, pyrimethamine, sulfadoxine, and artemisinin resistance, and neutral single nucleotide polymorphism (SNP) genotyping. Assay performance was first optimized using DNA from common laboratory parasite lines and plasmid controls. The limit of detection was 0.1-10 pg of DNA and yielded 100% accuracy when compared to sequencing. The tool was then evaluated on 87 clinical blood samples from around the world and the malaria-TAC once again achieved 100% accuracy when compared to sequencing, plus detected the presence of mixed infections in clinical samples. With its streamlined protocol and high accuracy, this malaria-TAC should be a useful tool for large-scale antimalarial resistance surveillance.

  1. Development and validation of a range of endogenous controls to support the implementation of practical Taqman real-time PCR-based surveillance for fish diseases within aquaculture.

    PubMed

    Bland, F; McIntosh, R; Bain, N; Snow, M

    2012-06-01

    The use of Taqman real-time PCR-based technology has recently become more frequent in the detection of pathogens in the aquaculture industry. This interest has necessitated the development of robust and reliable pathogen-detection assays. The development of a range of endogenous control assays to be run alongside these diagnostic assays works to further increase confidence in the latter. This study describes the design of a range of endogenous control assays based on the elongation factor 1-α (EF1-α) gene specific to a range of fish species including Atlantic salmon, Salmo salar; rainbow trout, Oncorhynchus mykiss; brown trout, Salmo trutta; cod, Gadus morhua; haddock, Melanogrammus aeglefinus; saithe, Pollachius virens; whiting, Merlangius merlangus; Norway pout, Trisopterus esmarkii; carp (family Cyprinidae), roach, Rutilus rutilus; European eel, Anguilla anguilla; and herring, Clupea harengus, as well as a number of fish cell lines. Evidence is provided of the validation of these assays for specific species, a range of tissue types and cell lines as well as an example of the potential uses of these assays.

  2. Development and validation of TaqMan quantitative PCR for detection of frog virus 3-like virus in eastern box turtles (Terrapene carolina carolina).

    PubMed

    Allender, Matthew C; Bunick, David; Mitchell, Mark A

    2013-03-01

    Ranavirus has caused disease epidemics and mass mortality events globally in free-ranging fish, amphibian, and reptile populations. Viral isolation and conventional PCR are the most common methods for diagnosis. In this study, a quantitative real-time PCR (qPCR) assay was developed using a TaqMan probe-based assay targeting a highly conserved region of the major capsid protein of frog virus 3-like virus (FV3-like) (Family Iridoviridae, genera Ranavirus). Standard curves were generated from a viral DNA segment cloned within a plasmid. The assay detected viral DNA 1000 times lower than conventional PCR. Thirty-one clinical samples (whole blood and oral swabs) from box turtles were tested using these assays and the prevalence of the virus determined. Quantitative PCR allows for a superior, rapid, sensitive, and quantitative method for detecting FV3-like virus in box turtles, and this assay will be useful for early detection and disease monitoring. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. The contribution of respiratory pathogens to fatal and non-fatal respiratory hospitalizations: a pilot study of Taqman Array Cards (TAC) in Kenya.

    PubMed

    Njuguna, Henry N; Chaves, Sandra S; Emukule, Gideon O; Nyawanda, Bryan; Omballa, Victor; Juma, Bonventure; Onyango, Clayton O; Mott, Joshua A; Fields, Barry

    2017-08-25

    Respiratory diseases cause substantial morbidity and mortality worldwide, with sub-Saharan Africa bearing the greatest burden. Identifying etiologies of respiratory disease is important to inform cost effective treatment, prevention and control strategies. Testing for all of the different pathogens that are potentially associated with respiratory illnesses is challenging. We piloted the use of a multi-pathogen respiratory Taqman Array Cards (TAC) to identify pathogens in respiratory samples collected from non-fatal and fatal cases and their matched asymptomatic controls. This is a case control study comparing viral and bacterial pathogens detected among non-fatal and fatal cases to those detected among age and time matched asymptomatic controls. We used McNemar's test to compare proportions of pathogens detected among cases (non-fatal and fatal) to their matched asymptomatic controls. We used Mann-Whitney test to compare the distribution of median Cycle threshold (Ct) values among non-fatal and fatal cases to their corresponding asymptomatic controls. There were 72 fatal and 72 non-fatal cases matched to 72 controls. We identified at least one pathogen in 109/144 (76%) cases and 59/72 (82%) controls. For most pathogens, the median Ct values were lower among cases (fatal and non-fatal) compared to asymptomatic controls. Similar rates of pathogen detection among cases and controls make interpretation of results challenging. Ct-values might be helpful in interpreting clinical relevance of detected pathogens using multi-pathogen diagnostic tools.

  4. Development of a Taqman real-time PCR assay for rapid detection and quantification of Vibrio tapetis in extrapallial fluids of clams

    PubMed Central

    Richard, Gaëlle G.; Le Bris, Cédric; Paillard, Christine

    2015-01-01

    The Gram-negative bacterium Vibrio tapetis is known as the causative agent of Brown Ring Disease (BRD) in the Manila clam Venerupis (=Ruditapes) philippinarum. This bivalve is the second most important species produced in aquaculture and has a high commercial value. In spite of the development of several molecular methods, no survey has been yet achieved to rapidly quantify the bacterium in the clam. In this study, we developed a Taqman real-time PCR assay targeting virB4 gene for accurate and quantitative identification of V. tapetis strains pathogenic to clams. Sensitivity and reproducibility of the method were assessed using either filtered sea water or extrapallial fluids of clam injected with the CECT4600T V. tapetis strain. Quantification curves of V. tapetis strain seeded in filtered seawater (FSW) or extrapallial fluids (EF) samples were equivalent showing reliable qPCR efficacies. With this protocol, we were able to specifically detect V. tapetis strains down to 1.125 101 bacteria per mL of EF or FSW, taking into account the dilution factor used for appropriate template DNA preparation. This qPCR assay allowed us to monitor V. tapetis load both experimentally or naturally infected Manila clams. This technique will be particularly useful for monitoring the kinetics of massive infections by V. tapetis and for designing appropriate control measures for aquaculture purposes. PMID:26713238

  5. Integrated Microfluidic Card with TaqMan Probes and High-Resolution Melt Analysis To Detect Tuberculosis Drug Resistance Mutations across 10 Genes

    PubMed Central

    Pholwat, Suporn; Liu, Jie; Stroup, Suzanne; Gratz, Jean; Banu, Sayera; Rahman, S. M. Mazidur; Ferdous, Sara Sabrina; Foongladda, Suporn; Boonlert, Duangjai; Ogarkov, Oleg; Zhdanova, Svetlana; Kibiki, Gibson; Heysell, Scott

    2015-01-01

    ABSTRACT Genotypic methods for drug susceptibility testing of Mycobacterium tuberculosis are desirable to speed the diagnosis and proper therapy of tuberculosis (TB). However, the numbers of genes and polymorphisms implicated in resistance have proliferated, challenging diagnostic design. We developed a microfluidic TaqMan array card (TAC) that utilizes both sequence-specific probes and high-resolution melt analysis (HRM), providing two layers of detection of mutations. Twenty-seven primer pairs and 40 probes were designed to interrogate 3,200 base pairs of critical regions of the inhA, katG, rpoB, embB, rpsL, rrs, eis, gyrA, gyrB, and pncA genes. The method was evaluated on 230 clinical M. tuberculosis isolates from around the world, and it yielded 96.1% accuracy (2,431/2,530) in comparison to that of Sanger sequencing and 87% accuracy in comparison to that of the slow culture-based susceptibility testing. This TAC-HRM method integrates assays for 10 genes to yield fast, comprehensive, and accurate drug susceptibility results for the 9 major antibiotics used to treat TB and could be deployed to improve treatment outcomes. PMID:25714709

  6. Quantitative Tetraplex Real-Time Polymerase Chain Reaction Assay with TaqMan Probes Discriminates Cattle, Buffalo, and Porcine Materials in Food Chain.

    PubMed

    Hossain, M A Motalib; Ali, Md Eaqub; Sultana, Sharmin; Asing; Bonny, Sharmin Quazi; Kader, Md Abdul; Rahman, M Aminur

    2017-05-17

    Cattle, buffalo, and porcine materials are widely adulterated, and their quantification might safeguard health, religious, economic, and social sanctity. Recently, conventional polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) assays have been documented but they are just suitable for identification, cannot quantify adulterations. We described here a quantitative tetraplex real-time PCR assay with TaqMan Probes to quantify contributions from cattle, buffalo, and porcine materials simultaneously. Amplicon-sizes were very short (106-, 90-, and 146-bp for cattle, buffalo, and porcine) because longer targets could be broken down, bringing serious ambiguity in molecular diagnostics. False negative detection was eliminated through an endogenous control (141-bp site of eukaryotic 18S rRNA). Analysis of 27 frankfurters and 27 meatballs reflected 84-115% target recovery at 0.1-10% adulterations. Finally, a test of 36 commercial products revealed 71% beef frankfurters, 100% meatballs, and 85% burgers contained buffalo adulteration, but no porcine was found in beef products.

  7. Accurate interrogation of FCGR3A rs396991 in European and Asian populations using a widely available TaqMan genotyping method.

    PubMed

    Murphy, Kay E; Niederer, Heather A; King, Karen S; Harris, Elizabeth C; Glass, Sarah M; Cox, Charles J

    2015-11-01

    A polymorphism in the receptor for the Fc region of IgG, Fc γ-receptor IIIa (FcγRIIIa, FCGR3A rs396991), has been inconsistently shown in the literature to have an effect on response to monoclonal antibody therapy in several indications. The rs396991 (T/G) polymorphism leads to an F176V substitution and increased affinity for IgG. This variant has proven difficult to genotype accurately, primarily because of extensive homology between the FCGR3A and FCGR3B genes. We have shown that rs396991 can be genotyped by PCR amplification, followed by direct Sanger sequencing of the product, without coamplification of FCGR3B, and that the rs396991 TaqMan assay (C__25815666_10) agrees with Sanger sequencing results in 100% of European and Asian samples tested, but it has a small error rate in African and American populations. C__25815666_10 is therefore suitable to interrogate rs396991 in studies involving Europeans and Asians; however for other populations, the default genotyping method should be PCR followed by Sanger sequencing.

  8. Development and validation of a novel Taqman-based real-time RT-PCR assay suitable for demonstrating freedom from viral haemorrhagic septicaemia virus.

    PubMed

    Jonstrup, S P; Kahns, S; Skall, H F; Boutrup, T S; Olesen, N J

    2013-01-01

    Viral haemorrhagic septicaemia (VHS) is a serious disease in several fish species. VHS is caused by the rhabdovirus viral haemorrhagic septicaemia virus (VHSV). To prevent spreading of the pathogen, it is important to use a fast, robust, sensitive and specific diagnostic tool to identify the infected fish. Traditional diagnosis based on isolation in cell culture followed by identification using, for example, ELISA is sensitive and specific but slow. By switching to RT-PCR for surveillance and diagnosis of VHS the time needed before a correct diagnosis can be given will be considerably shortened and the need for maintaining expensive cell culture facilities reduced. Here we present the validation, according to OIE guidelines, of a sensitive and specific Taqman-based real-time RT-PCR. The assay detects all isolates in a panel of 79 VHSV isolates covering all known genotypes and subtypes, with amplification efficiencies of approximately 100%. The analytical and diagnostic specificity of the real-time RT-PCR is close to 1, and the analytical and diagnostic sensitivity is comparable with traditional cell-based methods. In conclusion, the presented real-time RT-PCR assay has the necessary qualities to be used as a VHSV surveillance tool on par with cell culture assays. © 2012 Blackwell Publishing Ltd.

  9. Application of TaqMan qPCR for the detection and monitoring of Naegleria species in reservoirs used as a source for drinking water.

    PubMed

    Kao, Po-Min; Hsu, Bing-Mu; Hsu, Tsui-Kang; Chiu, Yi-Chou; Chang, Chung-Liang; Ji, Wen-Tsai; Huang, Shih-Wei; Fan, Cheng-Wei

    2014-10-01

    Naegleria spp. can be found in the natural aquatic environments. Naegleria fowleri can cause fatal infections in the central nervous system in humans and animals, and the most important source of infection is through direct water contact. In this study, PCR of 5.8S ribosomal RNA (rRNA) gene and internal transcribed spacer (ITS) region was performed in order to identify Naegleria isolates and quantify the Naegleria spp. by TaqMan real-time quantitative PCR in reservoir water samples. The occurrence of Naegleria spp. was investigated in 57 water samples from reservoirs with culture and PCR positive in 2 of them (3.5%), respectively. The total detection rate was 7.0% (4/ 57) for Naegleria spp. The identified species included Naegleria spp., Naegleria canariensis, and Naegleria clarki. N. fowleri was not found in Taiwan's reservoirs used for drinking purposes. The concentrations of Naegleria spp. in detected positive reservoir water samples were in the range of 599 and 3.1 × 10(3) cells/L. The presence or absence of Naegleria spp. within the reservoir water samples showed significant difference with the levels of water temperature. The presence of Naegleria spp. in reservoirs considered a potential public health threat if pathogenic species exist in reservoirs.

  10. Rapid and Quantitative Detection of Leifsonia xyli subsp. xyli in Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan) PCR Assay

    PubMed Central

    Fu, Hua-Ying; Sun, Sheng-Ren; Wang, Jin-Da; Ahmad, Kashif; Wang, Heng-Bo; Chen, Ru-Kai

    2016-01-01

    Ratoon stunting disease (RSD) of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agent Leifsonia xyli subsp. xyli (Lxx). A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay was established in this study for the quantification of Lxx detection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR) and a fluorogenic probe (Pat1-QP) targeting the Part1 gene of Lxx were used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg of Lxx genomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7%) of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174) were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174) were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods for Lxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields. PMID:27725937

  11. Combining Real-Time COLD- and MAMA-PCR TaqMan Techniques to Detect and Quantify R201 GNAS Mutations in the McCune-Albright Syndrome
.

    PubMed

    de Sanctis, Luisa; Galliano, Ilaria; Montanari, Paola; Matarazzo, Patrizia; Tessaris, Daniele; Bergallo, Massimiliano

    2017-01-01

    The McCune-Albright syndrome (MAS) is a potentially severe disorder hallmarked by fibrous bone dysplasia, café-au-lait skin spots, and endocrine hyperfunction. It is caused by postzygotic activating mutations at the R201 codon of the GNAS gene, leading to a state of somatic mosaicism. Our aim was to improve the mutation detection rate and to quantify the presence of R201 GNAS mutations in different DNA samples from MAS patients. Real-time COLD- and MAMA-PCR TaqMan techniques were combined to search for R201 mutations in the DNA of blood or affected tissues from 16 previously molecularly characterized MAS patients, from a further 84 subjects with MAS signs who were R201 negative at RFLP analysis, and from 36 controls. The ability of this new method to provide quantitative data was tested in the serial dilution of wild-type, R201H, or R201C cloned plasmid DNA samples; the mutant abundance was measured by spectrophotometry. A linear correlation between the true and the relative mutant abundance was observed until 2.5%, indicating a reliable quantification of R201 mutations. The assay's sensitivity was 0.05%, similar to that of previously described molecular methods. The real-time COLD-MAMA-PCR approach is a rapid, efficient, and inexpensive molecular technique for the identification of mutant alleles poorly represented in DNA samples.
. © 2017 S. Karger AG, Basel.

  12. Production of Conjugated Linoleic and Conjugated α-Linolenic Acid in a Reconstituted Skim Milk-Based Medium by Bifidobacterial Strains Isolated from Human Breast Milk

    PubMed Central

    Villar-Tajadura, María Antonia; Rodríguez-Alcalá, Luis Miguel; Martín, Virginia; Gómez de Segura, Aránzazu; Rodríguez, Juan Miguel; Fontecha, Javier

    2014-01-01

    Eight bifidobacterial strains isolated from human breast milk have been tested for their abilities to convert linoleic acid (LA) and α-linolenic acid (LNA) to conjugated linoleic acid (CLA) and conjugated α-linolenic acid (CLNA), respectively. These bioactive lipids display important properties that may contribute to the maintenance and improvement human health. Three selected Bifidobacterium breve strains produced CLA from LA and CLNA from LNA in MRS (160–170 and 210–230 μg mL−1, resp.) and, also, in reconstituted skim milk (75–95 and 210–244 μg mL−1, resp.). These bifidobacterial strains were also able to simultaneously produce both CLA (90–105 μg mL−1) and CLNA (290–320 μg mL−1) in reconstituted skim milk. Globally, our findings suggest that these bifidobacterial strains are potential candidates for the design of new fermented dairy products naturally containing very high concentrations of these bioactive lipids. To our knowledge, this is the first study describing CLNA production and coproduction of CLA and CLNA by Bifidobacterium breve strains isolated from human milk in reconstituted skim milk. PMID:25110689

  13. Production of conjugated linoleic and conjugated α-linolenic acid in a reconstituted skim milk-based medium by bifidobacterial strains isolated from human breast milk.

    PubMed

    Villar-Tajadura, María Antonia; Rodríguez-Alcalá, Luis Miguel; Martín, Virginia; Gómez de Segura, Aránzazu; Rodríguez, Juan Miguel; Requena, Teresa; Fontecha, Javier

    2014-01-01

    Eight bifidobacterial strains isolated from human breast milk have been tested for their abilities to convert linoleic acid (LA) and α-linolenic acid (LNA) to conjugated linoleic acid (CLA) and conjugated α-linolenic acid (CLNA), respectively. These bioactive lipids display important properties that may contribute to the maintenance and improvement human health. Three selected Bifidobacterium breve strains produced CLA from LA and CLNA from LNA in MRS (160-170 and 210-230 μg mL(-1), resp.) and, also, in reconstituted skim milk (75-95 and 210-244 μg mL(-1), resp.). These bifidobacterial strains were also able to simultaneously produce both CLA (90-105 μg mL(-1)) and CLNA (290-320 μg mL(-1)) in reconstituted skim milk. Globally, our findings suggest that these bifidobacterial strains are potential candidates for the design of new fermented dairy products naturally containing very high concentrations of these bioactive lipids. To our knowledge, this is the first study describing CLNA production and coproduction of CLA and CLNA by Bifidobacterium breve strains isolated from human milk in reconstituted skim milk.

  14. Frequencies of HNA-1, HNA-3, HNA-4, and HNA-5 in the Danish and Zambian populations determined using a novel TaqMan real time polymerase chain reaction method.

    PubMed

    Nielsen, K R; Koelbaek, M D; Varming, K; Baech, J; Steffensen, R

    2012-09-01

    In this study, we report a novel real time polymerase chain reaction (Q-PCR) method using TaqMan probes for human neutrophil antigens (HNA)-1, -3, -4, and -5 genotyping. The method was validated in a Caucasian Danish population, a Zambian population, and in clinical samples using three different methods: an in-house polymerase chain reaction with sequence-specific primers (PCR-SSP) method, a commercial available PCR-SSP kit and a novel Q-PCR method. We observed no discrepancy in the genotype frequencies determined by the PCR-SSP methods and the TaqMan assay in the populations studied. In tests of a family of Nigerian origin and in samples carrying the rare SLC44A2*1:2 genotype, different results were produced by the commercial PCR-SSP kit and the real-time TaqMan assay. The TaqMan-based genotyping method was rapid and reproducible, allowing high-throughput HNA-1, -3, -4, and -5 genotyping.

  15. Differences in sheep and goats milk fatty acid profile between conventional and organic farming systems.

    PubMed

    Tsiplakou, Eleni; Kotrotsios, Vaios; Hadjigeorgiou, Ioannis; Zervas, George

    2010-08-01

    The objective of this study was to investigate whether there is a difference in chemical composition and particularly in fatty acid (FA) profile, with emphasis on cis-9, trans-11 CLA, of milk obtained from conventional and organic dairy sheep and goats farms under the farming conditions practiced in Greece. Four dairy sheep and four dairy goat farms, representing common conventional production systems and another four dairy sheep and four dairy goat farms, organically certified, representing organic production and feeding systems were selected from all over Greece. One hundred and sixty two individual milk samples were collected from those farms in January-February 2009, about three months after parturition. The milk samples were analyzed for their main chemical constituents and their FA profile. The results showed that the production system affected milk chemical composition: in particular fat content was lower in the organic sheep and goats milk compared with the corresponding conventional. Milk from organic sheep had higher content in MUFA, PUFA, alpha-LNA, cis-9, trans-11 CLA, and omega-3 FA, whereas in milk from organic goats alpha-LNA and omega-3 FA content was higher than that in conventional one. These differences are, mainly, attributed to different feeding practices used by the two production systems. The results of this study show that the organic milk produced under the farming conditions practiced in Greece has higher nutritional value, due to its FA profile, compared with the respective conventional milk.

  16. Variations of the abundance and nucleic acid content of heterotrophic bacteria in Beaufort Shelf waters during winter and spring

    NASA Astrophysics Data System (ADS)

    Belzile, Claude; Brugel, Sonia; Nozais, Christian; Gratton, Yves; Demers, Serge

    2008-12-01

    Depth profiles of heterotrophic bacteria abundance were measured weekly over a 6-month period from December to May in Franklin Bay, a 230 m-deep coastal Arctic Ocean site of the southeastern Beaufort Sea. Total bacteria, low nucleic acid (LNA) and high nucleic acid (HNA) bacteria abundances were measured using flow cytometry after SYBR Green I staining. The HNA bacteria abundance in surface waters started to increase 5-6 weeks after phytoplankton growth resumed in spring, increasing from 1 × 10 5 to 3 × 10 5 cells mL - 1 over an 8-week period, with a net growth rate of 0.018 d - 1 . LNA bacteria response was delayed by more than two months relative to the beginning of the phytoplankton biomass accumulation and had a lower net growth rate of 0.013 d - 1 . The marked increase in bacterial abundance occurred before any significant increase in organic matter input from river discharge (as indicated by the unchanged surface water salinity and DOC concentrations), and in the absence of water temperature increase. The abundance of bacteria below the halocline was relatively high until January (up to 5 × 10 5 cells mL - 1 ) but then decreased to values close to 2 × 10 5 cells mL - 1 . The three-fold bacterial abundance increase observed in surface waters in spring was mostly due to HNA bacteria, supporting the idea that these cells are the most active.

  17. Rapid and reliable genotyping technique for GM1 gangliosidosis in Shiba dogs by real-time polymerase chain reaction with TaqMan minor groove binder probes.

    PubMed

    Chang, Hye-Sook; Arai, Toshiro; Yabuki, Akira; Hossain, Mohammad A; Rahman, Mohammad M; Mizukami, Keijiro; Yamato, Osamu

    2010-03-01

    Real-time polymerase chain reaction (PCR) with TaqMan minor groove binder (MGB) probes was examined to establish a rapid and reliable genotyping technique for GM1 gangliosidosis in Shiba dogs. This technique was applied to DNA samples extracted from the blood, umbilical cord, or postmortem liver tissue specimens, and to DNA-containing solutions prepared from blood and saliva that had been applied to Flinders Technology Associates filter papers (FTA cards). The amplification of the targeted sequence in all the samples was sufficient to determine the genotypes of GM1 gangliosidosis. Forty-seven DNA samples that had previously been obtained from blood or tissue specimens of Shiba dogs were examined using this real-time PCR technique, and the findings were consistent with the data obtained by the earlier PCR-restriction fragment length polymorphism (RFLP) assay. In addition, the use of this new technique in combination with FTA cards for sampling could markedly shorten the time required for genotyping, as well as simplify the procedure. Furthermore, in the present study, the results of a previous epidemiological screening of 96 Shiba dogs in the Czech Republic were rechecked by this real-time PCR technique using stored crude buccal cell DNA-containing solutions directly as DNA templates. The results provided clear-cut genotyping in all the samples although the earlier PCR-RFLP assay could not determine the genotype in all cases. In conclusion, this new real-time PCR technique is a simple, rapid, and reliable choice for large-scale screening to detect an abnormal allele indicating GM1 gangliosidosis in Shiba dogs.

  18. Efficient Diagnosis and Treatment Follow-Up of Human Brucellosis by a Novel Quantitative TaqMan Real-Time PCR Assay: a Human Clinical Survey

    PubMed Central

    Sohrabi, Majid; Khoramabadi, Nima; Hosseini Doust, Reza; Behmanesh, Mehrdad

    2014-01-01

    Rapid and effective diagnosis of brucellosis is a challenge for clinicians. Even when diagnosis is on time and therapy is initiated, meticulous follow-up appointments are crucial for ensuring the efficacy of the treatment. Due to shortcomings of serological methods, molecular diagnosis, especially real-time PCR, is becoming a main approach in laboratory diagnostics. Thus, the development of efficient procedures and standardization of the PCR tests will have a great impact on the precise detection and quantification of bacterial DNA loads, which is valuable for the medical management of brucellosis patients. We developed a new TaqMan real-time PCR directed to bcsp31, a shared gene of the brucellae. The bcsp31 gene fragment was cloned into pJET1.2. Recombinant pJET1.2-bcsp31 was linearized by HindIII digestion, and the product was used for the preparation of a standard curve. A panel of Brucella spp. and non-Brucella pathogens was tested. No bacterial genomes other than those of the brucellae were detected. According to the results, specificity of the method was 100%. In a clinical assessment, the positive-control group comprised 37 patients with microbiologically confirmed brucellosis, and 25 healthy individuals served as the negative-control group. By the end of the treatment period, there was a significant decrease in the DNA load of the 37 brucellosis patients, which persisted for the 4 weeks of monitoring after treatment, suggesting that our proposed method is an efficient monitoring tool. Serum samples prior to any treatment were collected from the 25 serologically suspicious patients and assessed by our method; 72% of these patients tested positive for brucellosis. PMID:25275001

  19. Development of a rapid, sensitive TaqMan real-time RT-PCR assay for the detection of Rose rosette virus using multiple gene targets.

    PubMed

    Babu, Binoy; Jeyaprakash, Ayyamperumal; Jones, Debra; Schubert, Timothy S; Baker, Carlye; Washburn, Brian K; Miller, Steven H; Poduch, Kristina; Knox, Gary W; Ochoa-Corona, Francisco M; Paret, Mathews L

    2016-09-01

    Rose rosette virus (RRV), belonging to the genus Emaravirus, is a highly destructive pathogen that causes rose rosette disease. The disease is a major concern for the rose industry in the U.S. due to the lack of highly sensitive methods for early detection of RRV. This is critical, as early identification of the infected plants and eradication is necessary in minimizing the risks associated with the spread of the disease. A highly reliable, specific and sensitive detection assay is thus required to test and confirm the presence of RRV in suspected plant samples. In this study a TaqMan real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of RRV from infected roses, utilizing multiple gene targets. Four pairs of primers and probes; two of them (RRV_2-1 and RRV_2-2) based on the consensus sequences of the glycoprotein gene (RNA2) and the other two (RRV_3-2 and RRV_3-5) based on the nucleocapsid gene (RNA3) were designed. The specificity of the primers and probes was evaluated against other representative viruses infecting roses, belonging to the genera Alfamovirus, Cucumovirus, Ilarvirus, Nepovirus, Tobamovirus, and Tospovirus and one Emaravirus (Wheat mosaic virus). Dilution assays using the in vitro transcripts (spiked with total RNA from healthy plants, and non-spiked) showed that all the primers and probes are highly sensitive in consistently detecting RRV with a detection limit of 1 fg. Testing of the infected plants over a period of time (three times in monthly intervals) indicated high reproducibility, with the primer/probe RRV_3-5 showing 100% positive detection, while RRV_2-1, RRV_2-2 and RRV_3-2 showed 90% positive detection. The developed real-time RT-PCR assay is reliable, highly sensitive, and can be easily used in diagnostic laboratories for testing and confirmation of RRV.

  20. Development of an in-House TaqMan Real-Time PCR-Based Method to Detect Residual Host Cell DNA in HBV Vaccine.

    PubMed

    Paryan, Mahdi; Khodayar, Mana; Kia, Vahid; Mohammadi-Yeganeh, Samira; Kaghazian, Hooman

    2016-06-01

    Biological therapeutic products such as recombinant hepatitis B virus (HBV) vaccine, produced by microbial fermentation in complex media, should be evaluated for host cell DNA contamination in purification steps. Eliminating these contaminations increases the efficacy of the vaccine and decreases its side effects. The objective of the present study is to trace the residual host cell DNA (HCD) in recombinant HBV vaccine by developing a TaqMan Real-Time PCR method which is more sensitive, specific, and reproducible than traditional methods such as Picogreen analysis and Threshold DNA assay. Primers and a probe were designed for the most highly conserved regions of Pichia pastoris genome. To determine the specificity of the assay, in addition to performing a BLAST for the primers and the probe in NCBI nucleotide database, 20 different human genomes and 8 bacterial and viral genomes were used. Moreover, serial dilutions of plasmids, from 10(2) to 10(7) copies/μL (from 0.00064 to 6.4 pg/μL), were prepared to find the sensitivity and the limit of detection (LOD) of the assay. Using 28 different genome samples, the specificity of the assay was determined to be 100 %. In addition, the sensitivity and LOD of the method was 0.39 × 10(-5) pg/μL. Moreover, the reproducibility of the assay based on intra- and inter-assay was 1.03 and 1.06 %, respectively. Considering the suitable specificity and sensitivity, ease of use, relatively low cost, and rapidity of the assay, it can be a reproducible and sensitive method to examine recombinant vaccines for P. pastoris residual DNA.

  1. Development and evaluation of one-step TaqMan real-time reverse transcription-PCR assays targeting nucleoprotein, matrix, and hemagglutinin genes of equine influenza virus.

    PubMed

    Lu, Zhengchun; Chambers, Thomas M; Boliar, Saikat; Branscum, Adam J; Sturgill, Tracy L; Timoney, Peter J; Reedy, Stephanie E; Tudor, Lynn R; Dubovi, Edward J; Vickers, Mary Lynne; Sells, Stephen; Balasuriya, Udeni B R

    2009-12-01

    The objective of this study was to develop and evaluate new TaqMan real-time reverse transcription-PCR (rRT-PCR) assays by the use of the minor groove binding probe to detect a wide range of equine influenza virus (EIV) strains comprising both subtypes of the virus (H3N8 and H7N7). A total of eight rRT-PCR assays were developed, targeting the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of the two EIV subtypes. None of the eight assays cross-reacted with any of the other known equine respiratory viruses. Three rRT-PCR assays (EqFlu NP, M, and HA3) which can detect strains of the H3N8 subtype were evaluated using nasal swabs received for routine diagnosis and swabs collected from experimentally inoculated horses. All three rRT-PCR assays have greater specificity and sensitivity than virus isolation by egg inoculation (93%, 89%, and 87% sensitivity for EqFlu NP, EqFlu M, and EqFlu HA3 assays, respectively). These assays had analytical sensitivities of >or=10 EIV RNA molecules. Comparison of the sensitivities of rRT-PCR assays targeting the NP and M genes of both subtypes with egg inoculation and the Directigen Flu A test clearly shows that molecular assays provide the highest sensitivity. The EqFlu HA7 assay targeting the H7 HA gene is highly specific for the H7N7 subtype of EIV. It should enable highly reliable surveillance for the H7N7 subtype, which is thought to be extinct or possibly still circulating at a very low level in nature. The assays that we developed provide a fast and reliable means of EIV diagnosis and subtype identification of EIV subtypes.

  2. Molecular quantitation of H9N2 avian influenza virus in various organs of broiler chickens using TaqMan real time PCR.

    PubMed

    Mosleh, Najmeh; Dadras, Habibollah; Mohammadi, Ali

    2009-01-16

    During the past decade, H9N2 low pathogenic avian influenza virus (LPAI) has caused considerable economic loss due to decreased production, increased mortality and the cost of vaccination in Iranian poultry industry. Because of widespread occurrence of this disease and the virus potential to mutate to highly-pathogenic (HP) form and transmission to humans, it is, therefore, imperative to understand the pathogenesis and properties of these viruses. In this study, a two step TaqMan real time PCR assay was performed for the quantitation of A/chicken/Iran/772/1998(H(9)N(2)) virus in various organs of broiler chickens at different days post inoculation (DPI). Forty 5-week-old commercial broiler chickens were inoculated with the virus. Five chickens were randomly selected on days 1, 3, 6 and 9 PI. Their trachea, lungs, spleen, kidneys, pancreas, blood and faeces were collected for virus detection. A PCR test was performed and the positive samples were used for quantitative real time PCR assay. The result of RT-PCR assay showed the presence of the virus in trachea (40%, 33%), lungs (20%, 66.6%) and spleen (20%, 50%) of infected chickens on days 3 and 6 PI, respectively. The virus was also detected in the kidneys of inoculated chickens on 3 (40%), 6 (60%) and 9 (100%) DPI. In faecal samples the virus was only detected on day 6 PI (83.3%). The molecular quantitation of AIV showed that the AIV titre in the trachea, lungs and spleen of chickens at 3 DPI is lower than the AIV titre at 6 DPI in these organs. The highest titre was observed in the faeces. The AIV titre in all organs of the birds which died at 6 DPI was higher than those of the same organs in the other experimental birds.

  3. Development of a TaqMan real-time PCR assay for quantification of airborne conidia of Botrytis squamosa and management of botrytis leaf blight of onion.

    PubMed

    Carisse, O; Tremblay, D M; Lévesque, C A; Gindro, K; Ward, P; Houde, A

    2009-11-01

    The use of a DNA-based method for quantifying airborne inoculum of Botrytis squamosa, a damaging pathogen of onion, was investigated. A method for purifying DNA from conidia collected using rotating-arm samplers and quantifying it using a TaqMan real-time quantitative polymerase chain reaction (qPCR) assay is described. The sensitivity of the qPCR assay was high, with a detection limit of 2 conidia/rod. A linear relationship between numbers of conidia counted with a compound microscope and those determined with the qPCR assay was obtained. Receiver operating characteristic curve analysis was used to evaluate the reliability of the two methods of conidia quantification (microscope examination and qPCR assay) to predict the risk of disease being below or above a damage threshold (D(th)). In total, 142 field samples from commercial onion fields were analyzed. At damage thresholds of 5 or 10 lesions/leaf, conidia quantification with the qPCR assay was more reliable at predicting disease risk than conidia quantification based on microscope counts. The proportion of decisions where the disease was present and predicted was higher for the qPCR assay than for the microscope counts, with values of 0.95 and 0.89 compared with 0.79 and 0.81 for D(th) of 5 and 10 lesions/leaf, respectively. The proportion of decisions where the disease was present but not predicted was lower for the qPCR assay than for microscope counts, with values of 0.05 and 0.11 compared with 0.20 and 0.19 for D(th) of 5 and 10 lesions/leaf, respectively. The results demonstrated that this new qPCR assay was reliable for quantifying B. squamosa airborne inoculum in commercial onion fields and that molecular conidia quantification could be used as a component of a risk management system for Botrytis leaf blight.

  4. Analytical Performance of a Multiplex Real-Time PCR Assay Using TaqMan Probes for Quantification of Trypanosoma cruzi Satellite DNA in Blood Samples

    PubMed Central

    Abate, Teresa; Cayo, Nelly M.; Parrado, Rudy; Bello, Zoraida Diaz; Velazquez, Elsa; Muñoz-Calderon, Arturo; Juiz, Natalia A.; Basile, Joaquín; Garcia, Lineth; Riarte, Adelina; Nasser, Julio R.; Ocampo, Susana B.; Yadon, Zaida E.; Torrico, Faustino; de Noya, Belkisyole Alarcón; Ribeiro, Isabela; Schijman, Alejandro G.

    2013-01-01

    Background The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy. Methods/Principal Findings We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR) based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC) in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD) of 0.70 parasite equivalents/mL and a limit of quantification (LOQ) of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject. Conclusions/Significance The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment. PMID:23350002

  5. Frequent detection of Streptococcus tigurinus in the human oral microbial flora by a specific 16S rRNA gene real-time TaqMan PCR

    PubMed Central

    2014-01-01

    Background Many bacteria causing systemic invasive infections originate from the oral cavity by entering the bloodstream. Recently, a novel pathogenic bacterium, Streptococcus tigurinus, was identified as causative agent of infective endocarditis, spondylodiscitis and meningitis. In this study, we sought to determine the prevalence of S. tigurinus in the human oral microbial flora and analyzed its association with periodontal disease or health. Results We developed a diagnostic highly sensitive and specific real-time TaqMan PCR assay for detection of S. tigurinus in clinical samples, based on the 16S rRNA gene. We analyzed saliva samples and subgingival plaque samples of a periodontally healthy control group (n = 26) and a periodontitis group (n = 25). Overall, S. tigurinus was detected in 27 (53%) out of 51 patients. There is no significant difference of the frequency of S. tigurinus detection by RT-PCR in the saliva and dental plaque samples in the two groups: in the control group, 14 (54%) out of 26 individuals had S. tigurinus either in the saliva samples and/or in the plaque samples; and in the periodontitis group, 13 (52%) out of 25 patients had S. tigurinus in the mouth samples, respectively (P = 0.895). The consumption of nicotine was no determining factor. Conclusion Although S. tigurinus was a frequently detected species of the human oral microbial flora, it was not associated with periodontal disease. Further investigations are required to determine whether S. tigurinus is a commensal or an opportunistic oral pathogen with a potential for development of invasive infections. PMID:25170686

  6. Evaluation of the Upgraded Version 2.0 of the Roche COBAS® AmpliPrep/COBAS® TaqMan HIV-1 Qualitative Assay in Central African Children

    PubMed Central

    Mossoro-Kpinde, CD; Jenabian, MA; Gody, JC; Robin, L; Talla, P; Longo, JDD; Grésenguet, G; Belec, L

    2016-01-01

    Background: Several commercially available molecular techniques were developed based on subtype B of HIV-1, which represents only 10% of HIV strains worldwide. Indeed, in sub-Saharan Africa, non-B subtypes of HIV-1 are predominant. The aim of this study was to evaluate the performances of the COBAS® AmpliPrep/COBAS® (CAP/CTM) HIV-1 Qualitative assays to detect the broad range of HIV-1 variants circulating in Central Africa and compare to the outgoing CAP/CTM HIV-1 Quantitative test v2.0 (Roche Molecular Systems), chosen as reference gold standard molecular assay. Methods: The CAP/CTM HIV-1 Qualitative tests versions 1.0 and 2.0 (Roche Molecular Systems, Inc., Branchburg, NJ, USA) were evaluated compared to CAP/CTM TaqMan HIV-1 Quantitative test v2.0 (Roche Molecular Systems) on 239 dried plasma spot (DPS) from 133 HIV-1-infected (with detectable plasma HIV RNA load) and 106 uninfected children, followed-up at Complexe Pédiatrique, Bangui, Central African Republic. Results: The version 1.0 showed low sensitivity (93.2%), with 9 (6.8%) false negative results, demonstrating under-detection of non-B HIV-1 subtypes. In contrast, the upgraded version 2.0 showed 100%-sensitivity, 100%-specificity and perfect agreement (κ coefficient, 1.0). Conclusion: Our evaluation in the Central African Republic demonstrates the clinical implications of the accuracy and reliability of the CAP/CTM HIV-1 Qualitative assay for early diagnosis of HIV-1 in Central African children. PMID:27857825

  7. Multiplex one-step Real-time PCR by Taqman-MGB method for rapid detection of pan and H5 subtype avian influenza viruses.

    PubMed

    Zhang, Zhujun; Liu, Dong; Sun, Wenqiang; Liu, Jing; He, Lihong; Hu, Jiao; Gu, Min; Wang, Xiaoquan; Liu, Xiaowen; Hu, Shunlin; Chen, Sujuan; Peng, Daxin; Liu, Xiufan

    2017-01-01

    Avian influenza virus (AIV) can infect a variety of avian species and mammals, leading to severe economic losses in poultry industry and posing a substantial threat to public health. Currently, traditional virus isolation and identification is inadequate for the early diagnosis because of its labor-intensive and time-consuming features. Real-time RT-PCR (RRT-PCR) is an ideal method for the detection of AIV since it is highly specific, sensitive and rapid. In addition, as the new quencher MGB is used in RRT-PCR, it only needs shorter probe and helps the binding of target gene and probe. In this study, a pan-AIV RRT-PCR for the detection of all AIVs and H5-AIV RRT-PCR for detection of H5 AIV based on NP gene of AIV and HA gene of H5 AIV were successfully established using Taqman-MGB method. We tested 14 AIV strains in total and the results showed that the pan-AIV RRT-PCR can detect AIV of various HA subtypes and the H5-AIV RRT-PCR can detect H5 AIV circulating in poultry in China in recent three years, including H5 viruses of clade 7.2, clade 2.3.4.4 and clade 2.3.2.1. Furthermore, the multiplex detection limit for pan-AIV and H5-AIV RRT-PCR was 5 copies per reaction. When this multiplex method was applied in the detection of experimental and live poultry market samples, the detection rates of pan-AIV and H5 AIV in RRT-PCR were both higher than the routine virus isolation method with embryonated chicken eggs. The multiplex RRT-PCR method established in our study showed high sensitivity, reproducibility and specificity, suggesting the promising application of our method for surveillance of both pan AIV and prevalent H5 AIV in live poultry markets and clinical samples.

  8. Multiplex one-step Real-time PCR by Taqman-MGB method for rapid detection of pan and H5 subtype avian influenza viruses

    PubMed Central

    Liu, Dong; Sun, Wenqiang; Liu, Jing; He, Lihong; Hu, Jiao; Gu, Min; Wang, Xiaoquan; Liu, Xiaowen; Hu, Shunlin; Chen, Sujuan; Peng, Daxin; Liu, Xiufan

    2017-01-01

    Avian influenza virus (AIV) can infect a variety of avian species and mammals, leading to severe economic losses in poultry industry and posing a substantial threat to public health. Currently, traditional virus isolation and identification is inadequate for the early diagnosis because of its labor-intensive and time-consuming features. Real-time RT-PCR (RRT-PCR) is an ideal method for the detection of AIV since it is highly specific, sensitive and rapid. In addition, as the new quencher MGB is used in RRT-PCR, it only needs shorter probe and helps the binding of target gene and probe. In this study, a pan-AIV RRT-PCR for the detection of all AIVs and H5-AIV RRT-PCR for detection of H5 AIV based on NP gene of AIV and HA gene of H5 AIV were successfully established using Taqman-MGB method. We tested 14 AIV strains in total and the results showed that the pan-AIV RRT-PCR can detect AIV of various HA subtypes and the H5-AIV RRT-PCR can detect H5 AIV circulating in poultry in China in recent three years, including H5 viruses of clade 7.2, clade 2.3.4.4 and clade 2.3.2.1. Furthermore, the multiplex detection limit for pan-AIV and H5-AIV RRT-PCR was 5 copies per reaction. When this multiplex method was applied in the detection of experimental and live poultry market samples, the detection rates of pan-AIV and H5 AIV in RRT-PCR were both higher than the routine virus isolation method with embryonated chicken eggs. The multiplex RRT-PCR method established in our study showed high sensitivity, reproducibility and specificity, suggesting the promising application of our method for surveillance of both pan AIV and prevalent H5 AIV in live poultry markets and clinical samples. PMID:28575115

  9. Detection of viable Salmonella in ice cream by TaqMan real-time polymerase chain reaction assay combining propidium monoazide.

    PubMed

    Wang, Yuexia; Yang, Ming; Liu, Shuchun; Chen, Wanyi; Suo, Biao

    2015-09-01

    Real-time polymerase chain reaction (PCR) allows rapid detection of Salmonella in frozen dairy products, but it might cause a false positive detection result because it might amplify DNA from dead target cells as well. In this study, Salmonella-free frozen ice cream was initially inoculated with heat-killed Salmonella Typhimurium cells and stored at -18°C. Bacterial DNA extracted from the sample was amplified using TaqMan probe-based real-time PCR targeting the invA gene. Our results indicated that DNA from the dead cells remained stable in frozen ice cream for at least 20 days, and could produce fluorescence signal for real-time PCR as well. To overcome this limitation, propidium monoazide (PMA) was combined with real-time PCR. PMA treatment can effectively prevent PCR amplification from heat-killed Salmonella cells in frozen ice cream. The PMA real-time PCR assay can selectively detect viable Salmonella at as low as 10(3) CFU/mL. Combining 18 hours of pre-enrichment with the assay allows for the detection of viable Salmonella at 10(0) CFU/mL and avoiding the false-positive result of dead cells. The PMA real-time PCR assay provides an alternative specifically for detection of viable Salmonella in ice cream. However, when the PMA real-time PCR assay was evaluated in ice cream subjected to frozen storage, it obviously underestimated the contamination situation of viable Salmonella, which might lead to a false negative result. According to this result, the use of enrichment prior to PMA real-time PCR analysis remains as the more appropriate approach. Copyright © 2015. Published by Elsevier B.V.

  10. Frequent detection of Streptococcus tigurinus in the human oral microbial flora by a specific 16S rRNA gene real-time TaqMan PCR.

    PubMed

    Zbinden, Andrea; Aras, Fatma; Zbinden, Reinhard; Mouttet, Forouhar; Schmidlin, Patrick R; Bloemberg, Guido V; Bostanci, Nagihan

    2014-08-24

    Many bacteria causing systemic invasive infections originate from the oral cavity by entering the bloodstream. Recently, a novel pathogenic bacterium, Streptococcus tigurinus, was identified as causative agent of infective endocarditis, spondylodiscitis and meningitis. In this study, we sought to determine the prevalence of S. tigurinus in the human oral microbial flora and analyzed its association with periodontal disease or health. We developed a diagnostic highly sensitive and specific real-time TaqMan PCR assay for detection of S. tigurinus in clinical samples, based on the 16S rRNA gene. We analyzed saliva samples and subgingival plaque samples of a periodontally healthy control group (n = 26) and a periodontitis group (n = 25). Overall, S. tigurinus was detected in 27 (53%) out of 51 patients. There is no significant difference of the frequency of S. tigurinus detection by RT-PCR in the saliva and dental plaque samples in the two groups: in the control group, 14 (54%) out of 26 individuals had S. tigurinus either in the saliva samples and/or in the plaque samples; and in the periodontitis group, 13 (52%) out of 25 patients had S. tigurinus in the mouth samples, respectively (P = 0.895). The consumption of nicotine was no determining factor. Although S. tigurinus was a frequently detected species of the human oral microbial flora, it was not associated with periodontal disease. Further investigations are required to determine whether S. tigurinus is a commensal or an opportunistic oral pathogen with a potential for development of invasive infections.

  11. Quantitative identification of fecal water pollution sources by TaqMan real-time PCR assays using Bacteroidales 16S rRNA genetic markers.

    PubMed

    Lee, Dae-Young; Weir, Susan C; Lee, Hung; Trevors, Jack T

    2010-12-01

    PCR-based analysis of Bacteroidales 16S rRNA genes has emerged as a promising tool to identify sources of fecal water pollution. In this study, three TaqMan real-time PCR assays (BacGeneral, BacHuman, and BacBovine) were developed and evaluated for their ability to quantitatively detect general (total), human-specific, and bovine-specific Bacteroidales 16S rRNA genetic markers. The detection sensitivity was determined to be 6.5 copies of 16S rRNA gene for the BacGeneral and BacHuman assays and 10 copies for the BacBovine assay. The assays were capable of detecting approximately one to two cells per PCR. When tested with 70 fecal samples from various sources (human, cattle, pig, deer, dog, cat, goose, gull, horse, and raccoon), the three assays positively identified the target markers in all samples without any false-negative results. The BacHuman and BacBovine assays exhibited false-positive reactions with non-target samples in a few cases. However, the level of the false-positive reactions was about 50 times smaller than that of the true-positive ones, and therefore, these cross-reactions were unlikely to cause misidentifications of the fecal pollution sources. Microbial source-tracking capability was tested at two freshwater streams of which water quality was influenced by human and cattle feces, respectively. The assays accurately detected the presence of the corresponding host-specific markers upon fecal pollution and the persistence of the markers in downstream areas. The assays are expected to reliably determine human and bovine fecal pollution sources in environmental water samples.

  12. Detection of rabies viral RNA by TaqMan real-time RT-PCR using non-neural specimens from dogs infected with rabies virus.

    PubMed

    Wacharapluesadee, Supaporn; Tepsumethanon, Veera; Supavonwong, Pornpun; Kaewpom, Thongchai; Intarut, Nirun; Hemachudha, Thiravat

    2012-09-01

    To determine the burden of rabies in developing countries, a reliable and accurate diagnostic test for the examination of the brains of animals is needed. Recently, the number of samples and carcasses submitted to rabies diagnostic units has been declining. Methods for obtaining tissues from different regions of the brain are even more difficult, and direct florescent antibody examination may fail if the samples decomposed. The spread of rabies virus to peripheral non-nervous tissues starts early during the pre-clinical phase. It has been shown that saliva and skin biopsies taken at the neck and containing hair follicles can be used in the ante-mortem diagnosis of rabies in humans. Obtaining oral swab samples, whisker or hair follicles from the heads of canines is easy and practical and can be performed without special equipment. The objective of this study was to determine whether these non-neural specimens can be used for the detection of rabies viral RNA. The RNAs extracted from these specimens were tested using a real-time reverse transcriptase polymerase chain reaction (RT-PCR). The sensitivity of the TaqMan real-time RT-PCR analysis using samples from dogs confirmed to be infected with rabies virus was 84.6% (55/65), 81.8% (54/66) and 66.7% (44/66) when using oral swab samples, extracted whisker follicles and extracted hair follicles; the specificity of all specimen types was 100%. The negative predictive values were 77.8%, 74.4% and 61.4%, respectively. Although the rate of positivity when combining the three non-neural specimen types was increased to 86.4%, this level of sensitivity was not sufficient to help physicians whether to administer post exposure prophylaxis. However, these oral swab and whisker specimens may serve to enhance epidemiological surveillance; such data will contribute in the planning of rabies control programs. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Genome Analysis and Development of a Multiplex TaqMan Real-Time PCR for Specific Identification and Detection of Clavibacter michiganensis subsp. nebraskensis.

    PubMed

    Tambong, James T; Xu, Renlin; Daayf, Fouad; Brière, Stephan; Bilodeau, Guillaume J; Tropiano, Raymond; Hartke, Allison; Reid, Lana M; Cott, Morgan; Cote, Tammy; Agarkova, Irina

    2016-12-01

    The reemergence of the Goss's bacterial wilt and blight disease in corn in the United States and Canada has prompted investigative research to better understand the genome organization. In this study, we generated a draft genome sequence of Clavibacter michiganensis subsp. nebraskensis strain DOAB 395 and performed genome and proteome analysis of C. michiganensis subsp. nebraskensis strains isolated in 2014 (DOAB 397 and DOAB 395) compared with the type strain, NCPPB 2581 (isolated over 40 years ago). The proteomes of strains DOAB 395 and DOAB 397 exhibited a 99.2% homology but had 92.1 and 91.8% homology, respectively, with strain NCPPB 2581. The majority (99.9%) of the protein sequences had a 99.6 to 100% homology between C. michiganensis subsp. nebraskensis strains DOAB 395 and DOAB 397, with only four protein sequences (0.1%) exhibiting a similarity <70%. In contrast, 3.0% of the protein sequences of strain DOAB 395 or DOAB 397 showed low homologies (<70%) with the type strain NCPPB 2581. The genome data were exploited for the development of a multiplex TaqMan real-time polymerase chain reaction (PCR) tool for rapid detection of C. michiganensis subsp. nebraskensis. The specificity of the assay was validated using 122 strains of Clavibacter and non-Clavibacter spp. A blind test and naturally infected leaf samples were used to confirm specificity. The sensitivity (0.1 to 1.0 pg) compared favorably with previously reported real-time PCR assays. This tool should fill the current gap for a reliable diagnostic technique.

  14. Efficient diagnosis and treatment follow-up of human brucellosis by a novel quantitative TaqMan real-time PCR assay: a human clinical survey.

    PubMed

    Sohrabi, Majid; Mohabati Mobarez, Ashraf; Khoramabadi, Nima; Hosseini Doust, Reza; Behmanesh, Mehrdad

    2014-12-01

    Rapid and effective diagnosis of brucellosis is a challenge for clinicians. Even when diagnosis is on time and therapy is initiated, meticulous follow-up appointments are crucial for ensuring the efficacy of the treatment. Due to shortcomings of serological methods, molecular diagnosis, especially real-time PCR, is becoming a main approach in laboratory diagnostics. Thus, the development of efficient procedures and standardization of the PCR tests will have a great impact on the precise detection and quantification of bacterial DNA loads, which is valuable for the medical management of brucellosis patients. We developed a new TaqMan real-time PCR directed to bcsp31, a shared gene of the brucellae. The bcsp31 gene fragment was cloned into pJET1.2. Recombinant pJET1.2-bcsp31 was linearized by HindIII digestion, and the product was used for the preparation of a standard curve. A panel of Brucella spp. and non-Brucella pathogens was tested. No bacterial genomes other than those of the brucellae were detected. According to the results, specificity of the method was 100%. In a clinical assessment, the positive-control group comprised 37 patients with microbiologically confirmed brucellosis, and 25 healthy individuals served as the negative-control group. By the end of the treatment period, there was a significant decrease in the DNA load of the 37 brucellosis patients, which persisted for the 4 weeks of monitoring after treatment, suggesting that our proposed method is an efficient monitoring tool. Serum samples prior to any treatment were collected from the 25 serologically suspicious patients and assessed by our method; 72% of these patients tested positive for brucellosis.

  15. High resolution TaqMan real-time PCR approach to detect hazelnut DNA encoding for ITS rDNA in foods.

    PubMed

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

    2013-12-01

    A broad range of foods have been described as causing allergies, but the majority of allergic reactions can be ascribed to a limited number of food components. Recent extensive surveys showed how tree nuts, particularly hazelnut (Corylus avellana L.) seeds, rank amongst the most important sources of food allergy. In order to protect the allergic consumer, efficient and reliable methods are required for the detection of allergenic ingredients. For this purpose, we have developed a real-time polymerase chain reaction (PCR) for detection of hazelnut in commercial food products. In this way a specific hazelnut primer pair based on the ITS marker (70 bp) and a nuclease (TaqMan) probe labelled with FAM and BHQ were designed. Sensibility of real-time PCR was determined by analysis of raw and heat treated hazelnut-wheat flour mixtures with a range of detection of 0.1-100,000 ppm. Practical applicability of the real-time PCR assay developed for determining hazelnut in different food matrices was investigated by analyzing 179 commercial foodstuffs comprising snacks, biscuits, chocolates, bonbons, creams, nut bars, ice creams, precooked meals, breads, beverages, yogurts, cereals, meat products, rice cake and nougat. From the total of samples analyzed, 40 commercial food products that didn't declare hazelnut nor traces on the label were found to contain hazelnut. The real-time PCR method proposed herein due to its high sensitivity facilitates the detection of hazelnut traces in commercial food products and can also be useful for monitoring the effectiveness of cleaning processes and as consequence, can help to prevent the food allergic consumer from unintentional ingestion of hidden allergens.

  16. TaqMan 5'-nuclease human immunodeficiency virus type 1 PCR assay with phage-packaged competitive internal control for high-throughput blood donor screening.

    PubMed

    Drosten, C; Seifried, E; Roth, W K

    2001-12-01

    Screening of blood donors for human immunodeficiency virus type 1 (HIV-1) infection by PCR permits the earlier diagnosis of HIV-1 infection compared with that by serologic assays. We have established a high-throughput reverse transcription (RT)-PCR assay based on 5'-nuclease PCR. By in-tube detection of HIV-1 RNA with a fluorogenic probe, the 5'-nuclease PCR technology (TaqMan PCR) eliminates the risk of carryover contamination, a major problem in PCR testing. We outline the development and evaluation of the PCR assay from a technical point of view. A one-step RT-PCR that targets the gag genes of all known HIV-1 group M isolates was developed. An internal control RNA detectable with a heterologous 5'-nuclease probe was derived from the viral target cDNA and was packaged into MS2 coliphages (Armored RNA). Because the RNA was protected against digestion with RNase, it could be spiked into patient plasma to control the complete sample preparation and amplification process. The assay detected 831 HIV-1 type B genome equivalents per ml of native plasma (95% confidence interval [CI], 759 to 936 HIV-1 B genome equivalents per ml) with a >or=95% probability of a positive result, as determined by probit regression analysis. A detection limit of 1,195 genome equivalents per ml of (individual) donor plasma (95% CI, 1,014 to 1,470 genome equivalents per ml of plasma pooled from individuals) was achieved when 96 samples were pooled and enriched by centrifugation. Up to 4,000 plasma samples per PCR run were tested in a 3-month trial period. Although data from the present pilot feasibility study will have to be complemented by a large clinical validation study, the assay is a promising approach to the high-throughput screening of blood donors and is the first noncommercial test for high-throughput screening for HIV-1.

  17. Performance of Version 2.0 of the Cobas AmpliPrep/Cobas TaqMan Real-Time PCR Assay for Hepatitis B Virus DNA Quantification ▿

    PubMed Central

    Chevaliez, Stéphane; Bouvier-Alias, Magali; Laperche, Syria; Hézode, Christophe; Pawlotsky, Jean-Michel

    2010-01-01

    The detection and quantification of hepatitis B virus (HBV) DNA are essential for the diagnosis and treatment of chronic HBV infection. The use of real-time PCR assays for HBV DNA quantification is strongly recommended. The goal of this study was to evaluate the intrinsic characteristics and clinical performance of version 2.0 (v2.0) of the Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) assay, a fully automated platform for HBV DNA quantification in serum or in plasma with a claimed lower limit of detection of 20 IU/ml and a claimed upper limit of quantification of 1.7 × 108 IU/ml. The specificity of the assay was 99% (95% confidence interval, 94.7 to 100%). Intra-assay and interassay coefficients of variation ranged from 0.21% to 2.67% and from 0.65% to 2.25%, respectively. The calibration of the assay was found to be satisfactory. Study of blood specimens from patients infected with HBV genotypes A to F showed good correspondence between HBV DNA levels measured by the CAP/CTM v2.0 assay, version 1.0 of the same assay, and the third-generation “branched DNA” assay. The CAP/CTM v2.0 assay quantified HBV DNA levels in serum or plasma from the same patients equally. In conclusion, the new version of the CAP/CTM assay is sensitive, specific, and reproducible. It accurately quantifies HBV DNA levels in patients chronically infected with HBV genotypes A to F. Improvements made to ensure equal quantification of HBV DNA in serum and plasma have been successful. Overall, the CAP/CTM assay, version 2.0, is well suited to monitoring clinical HBV DNA levels according to current clinical practice guidelines. PMID:20720031

  18. Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples.

    PubMed

    Cura, Carolina I; Duffy, Tomas; Lucero, Raúl H; Bisio, Margarita; Péneau, Julie; Jimenez-Coello, Matilde; Calabuig, Eva; Gimenez, María J; Valencia Ayala, Edward; Kjos, Sonia A; Santalla, José; Mahaney, Susan M; Cayo, Nelly M; Nagel, Claudia; Barcán, Laura; Málaga Machaca, Edith S; Acosta Viana, Karla Y; Brutus, Laurent; Ocampo, Susana B; Aznar, Christine; Cuba Cuba, Cesar A; Gürtler, Ricardo E; Ramsey, Janine M; Ribeiro, Isabela; VandeBerg, John L; Yadon, Zaida E; Osuna, Antonio; Schijman, Alejandro G

    2015-05-01

    Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI-TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.

  19. Quantification of Human Polyomaviruses JC Virus and BK Virus by TaqMan Quantitative PCR and Comparison to Other Water Quality Indicators in Water and Fecal Samples▿

    PubMed Central

    McQuaig, Shannon M.; Scott, Troy M.; Lukasik, Jerzy O.; Paul, John H.; Harwood, Valerie J.

    2009-01-01

    In the United States, total maximum daily load standards for bodies of water that do not meet bacterial water quality standards are set by each state. The presence of human polyomaviruses (HPyVs) can be used as an indicator of human-associated sewage pollution in these waters. We have developed and optimized a TaqMan quantitative PCR (QPCR) assay based on the conserved T antigen to both quantify and simultaneously detect two HPyVs; JC virus and BK virus. The QPCR assay was able to consistently quantify ≥10 gene copies per reaction and is linear over 5 orders of magnitude. HPyVs were consistently detected in human waste samples (57 of 64) and environmental waters with known human fecal contamination (5 of 5) and were not amplified in DNA extracted from 127 animal waste samples from 14 species. HPyV concentrations in sewage decreased 81.2 and 84.2% over 28 days incubation at 25 and 35°C, respectively. HPyVs results were compared to Escherichia coli, fecal coliform, and enterococci concentrations and the presence of three other human-associated microbes: Bacteroidetes, Methanobrevibacter smithii, and adenovirus. HPyVs were the most frequently detected of these in human and contaminated environmental samples and were more human specific than the Bacteroidetes (HF183) or M. smithii. HPyVs and M. smithii more closely mimicked the persistence of adenovirus in sewage than the other microbes. The use of this rapid and quantitative assay in water quality research could help regulatory agencies to identify sources of water pollution for improved remediation of contaminated waters and ultimately protect humans from exposure to pathogens. PMID:19346361

  20. A duplex qPCR for the simultaneous detection of Escherichia coli O157:H7 and Listeria monocytogenes using LNA probes.

    PubMed

    Nitecki, S S; Teape, N; Carney, B F; Slater, J W; Brück, W M

    2015-07-01

    In this study, a duplex qPCR assay was developed for the needs of the Irish fish industry to screen for the two major food-borne pathogens of fish, Listeria monocytogenes and Escherichia coli O157:H7. The assay can claim positive or negative results for two pathogens in one go in only 20 h including 16 h universal pre-enrichment and compared to traditional ISO approved plate culture methods the labour and the cost involved in testing of one sample is reduced to minimum. The highly specific genomic areas targeted for PCR amplification in the assay are the hly gene for listeriolysin O (LLO) of L. monocytogenes and the stx gene for Shiga-like toxin expressed by E. coli O157:H7. The detection limit of the assay is consistent with the consumer protection limits of 1 pg genomic DNA or 1 CFU 25 g(-1) fish meat (with enrichment) allowing the test to be considered as a substitute to standard plate culture methods. The study highlights a novel duplex qPCR for Listeria monocytogenes and Escherichia coli O157:H7 that could be used as an alternative to plate-based ISO or singleplex PCR methods while minimizing the costs. The assay uses rapid DNA extraction methods and locked nucleic acid probes. Sensitivity and specificity are 100 and 98·95% respectively. The potential for quantitative rage of the assay is 10(8) -10(1) CFU ml(-1) . © 2015 The Society for Applied Microbiology.

  1. Synthetic oligonucleotide antigens modified with locked nucleic acids detect disease specific antibodies

    PubMed Central

    Samuelsen, Simone V.; Solov’yov, Ilia A.; Balboni, Imelda M.; Mellins, Elizabeth; Nielsen, Christoffer Tandrup; Heegaard, Niels H. H.; Astakhova, Kira

    2016-01-01

    New techniques to detect and quantify antibodies to nucleic acids would provide a significant advance over current methods, which often lack specificity. We investigate the potential of novel antigens containing locked nucleic acids (LNAs) as targets for antibodies. Particularly, employing molecular dynamics we predict optimal nucleotide composition for targeting DNA-binding antibodies. As a proof of concept, we address a problem of detecting anti-DNA antibodies that are characteristic of systemic lupus erythematosus, a chronic autoimmune disease with multiple manifestations. We test the best oligonucleotide binders in surface plasmon resonance studies to analyze binding and kinetic aspects of interactions between antigens and target DNA. These DNA and LNA/DNA sequences showed improved binding in enzyme-linked immunosorbent assay using human samples of pediatric lupus patients. Our results suggest that the novel method is a promising tool to create antigens for research and point-of-care monitoring of anti-DNA antibodies. PMID:27775006

  2. Synthetic oligonucleotide antigens modified with locked nucleic acids detect disease specific antibodies

    NASA Astrophysics Data System (ADS)

    Samuelsen, Simone V.; Solov’Yov, Ilia A.; Balboni, Imelda M.; Mellins, Elizabeth; Nielsen, Christoffer Tandrup; Heegaard, Niels H. H.; Astakhova, Kira

    2016-10-01

    New techniques to detect and quantify antibodies to nucleic acids would provide a significant advance over current methods, which often lack specificity. We investigate the potential of novel antigens containing locked nucleic acids (LNAs) as targets for antibodies. Particularly, employing molecular dynamics we predict optimal nucleotide composition for targeting DNA-binding antibodies. As a proof of concept, we address a problem of detecting anti-DNA antibodies that are characteristic of systemic lupus erythematosus, a chronic autoimmune disease with multiple manifestations. We test the best oligonucleotide binders in surface plasmon resonance studies to analyze binding and kinetic aspects of interactions between antigens and target DNA. These DNA and LNA/DNA sequences showed improved binding in enzyme-linked immunosorbent assay using human samples of pediatric lupus patients. Our results suggest that the novel method is a promising tool to create antigens for research and point-of-care monitoring of anti-DNA antibodies.

  3. Lactam nonanic acid, a new substance from Cleome viscosa with allelopathic and antimicrobial properties.

    PubMed

    Jana, Anirban; Biswas, Suparna Mandal

    2011-03-01

    Cleome viscosa L. (Capparidaceae) is well known for its medicinal properties. Lactam nonanoic acid (LNA) [2-amino-9-(4-oxoazetidin-2-yl)-nonanoic acid; C12H22N2O3, mol. wt. 242] has been isolated and purified from the root exudates of Cleome viscosa. The aqueous solution of this pure compound has been tested on bacteria (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) and fungi (Aspergillus fumigatus, A. niger and A. tamarii). At a dosage of 500 ppm and above, P. aeruginosa and S. aureus were totally inhibited while E. coli remained unaffected. On the other hand, growth of A. niger and A. tamarii was stimulated while there was no effect on A. fumigatus. This pure compound showed concentration-dependent inhibitory activity on rice, gram and mustard seeds.

  4. Experimental Warming Decreases the Average Size and Nucleic Acid Content of Marine Bacterial Communities

    PubMed Central

    Huete-Stauffer, Tamara M.; Arandia-Gorostidi, Nestor; Alonso-Sáez, Laura; Morán, Xosé Anxelu G.

    2016-01-01

    Organism size reduction with increasing temperature has been suggested as a universal response to global warming. Since genome size is usually correlated to cell size, reduction of genome size in unicells could be a parallel outcome of warming at ecological and evolutionary time scales. In this study, the short-term response of cell size and nucleic acid content of coastal marine prokaryotic communities to temperature was studied over a full annual cycle at a NE Atlantic temperate site. We used flow cytometry and experimental warming incubations, spanning a 6°C range, to analyze the hypothesized reduction with temperature in the size of the widespread flow cytometric bacterial groups of high and low nucleic acid content (HNA and LNA bacteria, respectively). Our results showed decreases in size in response to experimental warming, which were more marked in 0.8 μm pre-filtered treatment rather than in the whole community treatment, thus excluding the role of protistan grazers in our findings. Interestingly, a significant effect of temperature on reducing the average nucleic acid content (NAC) of prokaryotic cells in the communities was also observed. Cell size and nucleic acid decrease with temperature were correlated, showing a common mean decrease of 0.4% per °C. The usually larger HNA bacteria consistently showed a greater reduction in cell and NAC compared with their LNA counterparts, especially during the spring phytoplankton bloom period associated to maximum bacterial growth rates in response to nutrient availability. Our results show that the already smallest planktonic microbes, yet with key roles in global biogeochemical cycling, are likely undergoing important structural shrinkage in response to rising temperatures. PMID:27242747

  5. Experimental Warming Decreases the Average Size and Nucleic Acid Content of Marine Bacterial Communities.

    PubMed

    Huete-Stauffer, Tamara M; Arandia-Gorostidi, Nestor; Alonso-Sáez, Laura; Morán, Xosé Anxelu G

    2016-01-01

    Organism size reduction with increasing temperature has been suggested as a universal response to global warming. Since genome size is usually correlated to cell size, reduction of genome size in unicells could be a parallel outcome of warming at ecological and evolutionary time scales. In this study, the short-term response of cell size and nucleic acid content of coastal marine prokaryotic communities to temperature was studied over a full annual cycle at a NE Atlantic temperate site. We used flow cytometry and experimental warming incubations, spanning a 6°C range, to analyze the hypothesized reduction with temperature in the size of the widespread flow cytometric bacterial groups of high and low nucleic acid content (HNA and LNA bacteria, respectively). Our results showed decreases in size in response to experimental warming, which were more marked in 0.8 μm pre-filtered treatment rather than in the whole community treatment, thus excluding the role of protistan grazers in our findings. Interestingly, a significant effect of temperature on reducing the average nucleic acid content (NAC) of prokaryotic cells in the communities was also observed. Cell size and nucleic acid decrease with temperature were correlated, showing a common mean decrease of 0.4% per °C. The usually larger HNA bacteria consistently showed a greater reduction in cell and NAC compared with their LNA counterparts, especially during the spring phytoplankton bloom period associated to maximum bacterial growth rates in response to nutrient availability. Our results show that the already smallest planktonic microbes, yet with key roles in global biogeochemical cycling, are likely undergoing important structural shrinkage in response to rising temperatures.

  6. Simultaneous Detection and Differentiation of Human Rhino- and Enteroviruses in Clinical Specimens by Real-Time PCR with Locked Nucleic Acid Probes

    PubMed Central

    Österback, Riikka; Tevaluoto, Tuire; Ylinen, Tiina; Peltola, Ville; Susi, Petri; Hyypiä, Timo

    2013-01-01

    Human rhinoviruses (HRVs) and human enteroviruses (HEVs) are significant respiratory pathogens. While HRV infections are restricted to the respiratory tract, HEV infections may spread to secondary target organs. The method of choice for sensitive specific detection of these viruses is reverse transcription (RT)-PCR with primers targeting the conserved 5′ noncoding region of the viral RNA. On the other hand, sequence similarities between HRVs and HEVs complicate their differential detection. In this study, we describe the use of locked nucleic acid (LNA) analogues in short double-dye probes which contained only two selectively HRV- or HEV-specific bases. The double-stranded DNA dye BOXTO (4-[6-(benzoxazole-2-yl-(3-methyl-)-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]-1-methyl-quinolinium chloride) was used with the LNA probes in a tricolor real-time PCR assay to allow specific detection of HRVs (probes labeled with 6-carboxyfluorescein [FAM] [green]) and HEVs (Cy5 [red]) with additional melting curve analysis (BOXTO [yellow]). The functionality of the probes was validated in PCR and RT-PCR assays using plasmids containing viral cDNA, quantified viral RNA transcripts, cultivated rhino- and enterovirus prototypes, and clinical specimens. Of 100 HRV and 63 HEV prototypes, the probes correctly identified all HEVs except one that produced only a BOXTO signal. Among 118 clinical specimens with sequencing results, concordant results were obtained for 116 specimens. Two specimens were reactive with both probes, but sequencing yielded only a single virus. Real-time PCR with LNA probes allowed sensitive group-specific identification of HRVs and HEVs and would enable relative copy number determination. The assay is suitable for rapid and accurate differential detection of HRVs and HEVs in a diagnostic laboratory setting. PMID:24048533

  7. Simultaneous detection and differentiation of human rhino- and enteroviruses in clinical specimens by real-time PCR with locked nucleic Acid probes.

    PubMed

    Osterback, Riikka; Tevaluoto, Tuire; Ylinen, Tiina; Peltola, Ville; Susi, Petri; Hyypiä, Timo; Waris, Matti

    2013-12-01

    Human rhinoviruses (HRVs) and human enteroviruses (HEVs) are significant respiratory pathogens. While HRV infections are restricted to the respiratory tract, HEV infections may spread to secondary target organs. The method of choice for sensitive specific detection of these viruses is reverse transcription (RT)-PCR with primers targeting the conserved 5' noncoding region of the viral RNA. On the other hand, sequence similarities between HRVs and HEVs complicate their differential detection. In this study, we describe the use of locked nucleic acid (LNA) analogues in short double-dye probes which contained only two selectively HRV- or HEV-specific bases. The double-stranded DNA dye BOXTO (4-[6-(benzoxazole-2-yl-(3-methyl-)-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]-1-methyl-quinolinium chloride) was used with the LNA probes in a tricolor real-time PCR assay to allow specific detection of HRVs (probes labeled with 6-carboxyfluorescein [FAM] [green]) and HEVs (Cy5 [red]) with additional melting curve analysis (BOXTO [yellow]). The functionality of the probes was validated in PCR and RT-PCR assays using plasmids containing viral cDNA, quantified viral RNA transcripts, cultivated rhino- and enterovirus prototypes, and clinical specimens. Of 100 HRV and 63 HEV prototypes, the probes correctly identified all HEVs except one that produced only a BOXTO signal. Among 118 clinical specimens with sequencing results, concordant results were obtained for 116 specimens. Two specimens were reactive with both probes, but sequencing yielded only a single virus. Real-time PCR with LNA probes allowed sensitive group-specific identification of HRVs and HEVs and would enable relative copy number determination. The assay is suitable for rapid and accurate differential detection of HRVs and HEVs in a diagnostic laboratory setting.

  8. Quick chip assay using locked nucleic acid modified epithelial cell adhesion molecule and nucleolin aptamers for the capture of circulating tumor cells

    PubMed Central

    Maremanda, Nihal G.; Roy, Kislay; Kanwar, Rupinder K.; Shyamsundar, Vidyarani; Ramshankar, Vijayalakshmi; Krishnamurthy, Arvind; Krishnakumar, Subramanian; Kanwar, Jagat R.

    2015-01-01

    The role of circulating tumor cells (CTCs) in disease diagnosis, prognosis, monitoring of the therapeutic efficacy, and clinical decision making is immense and has attracted tremendous focus in the last decade. We designed and fabricated simple, flat channel microfluidic devices polydimethylsiloxane (PDMS based) functionalized with locked nucleic acid (LNA) modified aptamers (targeting epithelial cell adhesion molecule (EpCAM) and nucleolin expression) for quick and efficient capture of CTCs and cancer cells. With optimized flow rates (10 μl/min), it was revealed that the aptamer modified devices offered reusability for up to six times while retaining optimal capture efficiency (>90%) and specificity. High capture sensitivity (92%) and specificity (100%) was observed in whole blood samples spiked with Caco-2 cells (10–100 cells/ml). Analysis of blood samples obtained from 25 head and neck cancer patients on the EpCAM LNA aptamer functionalized chip revealed that an average count of 5 ± 3 CTCs/ml of blood were captured from 22/25 samples (88%). EpCAM intracellular domain (EpICD) immunohistochemistry on 9 oral squamous cell carcinomas showed the EpICD positivity in the tumor cells, confirming the EpCAM expression in CTCs from head and neck cancers. These microfluidic devices also maintained viability for in vitro culture and characterization. Use of LNA modified aptamers provided added benefits in terms of cost effectiveness due to increased reusability and sustainability of the devices. Our results present a robust, quick, and efficient CTC capture platform with the use of simple PDMS based devices that are easy to fabricate at low cost and have an immense potential in cancer diagnosis, prognosis, and therapeutic planning. PMID:26487896

  9. Intercalator conjugates of pyrimidine locked nucleic acid-modified triplex-forming oligonucleotides: improving DNA binding properties and reaching cellular activities

    PubMed Central

    Brunet, Erika; Corgnali, Maddalena; Perrouault, Loïc; Roig, Victoria; Asseline, Ulysse; Sørensen, Mads D.; Babu, B. Ravindra; Wengel, Jesper; Giovannangeli, Carine

    2005-01-01

    Triplex-forming oligonucleotides (TFOs) are powerful tools to interfere sequence-specifically with DNA-associated biological functions. (A/T,G)-containing TFOs are more commonly used in cells than (T,C)-containing TFOs, especially C-rich sequences; indeed the low intracellular stability of the non-covalent pyrimidine triplexes make the latter less active. In this work we studied the possibility to enhance DNA binding of (T,C)-containing TFOs, aiming to reach cellular activities; to this end, we used locked nucleic acid-modified TFOs (TFO/LNAs) in association with 5′-conjugation of an intercalating agent, an acridine derivative. In vitro a stable triplex was formed with the TFO-acridine conjugate: by SPR measurements at 37°C and neutral pH, the dissociation equilibrium constant was found in the nanomolar range and the triplex half-life ∼10 h (50-fold longer compared with the unconjugated TFO/LNA). Moreover to further understand DNA binding of (T,C)-containing TFO/LNAs, hybridization studies were performed at different pH values: triplex stabilization associated with pH decrease was mainly due to a slower dissociation process. Finally, biological activity of pyrimidine TFO/LNAs was evaluated in a cellular context: it occurred at concentrations ∼0.1 μM for acridine-conjugated TFO/LNA (or ∼2 μM for the unconjugated TFO/LNA) whereas the corresponding phosphodiester TFO was inactive, and it was demonstrated to be triplex-mediated. PMID:16049028

  10. Detection and Typing of Herpes Simplex Virus (HSV) in Mucocutaneous Samples by TaqMan PCR Targeting a gB Segment Homologous for HSV Types 1 and 2

    PubMed Central

    Namvar, Lilly; Olofsson, Sigvard; Bergström, Tomas; Lindh, Magnus

    2005-01-01

    Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) are major causes of mucocutaneous lesions and severe infections of the central nervous system. Here a new semiautomated method for detecting and typing of HSV was used to analyze 479 mucocutaneous swab samples. After DNA extraction using a Magnapure LC robot, a 118-bp segment of the gB region was amplified by real-time PCR utilizing type-specific TaqMan probes to identify HSV-1 or HSV-2. HSV detection in a single well using probes labeled with carboxyfluorescein (FAM) for HSV-1 and JOE (6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein) for HSV-2 had a sensitivity similar to that seen in separate reactions. All but one of 217 samples (99.5%) that had been positive by virus culture were positive by TaqMan PCR, with a correct identification of type in all cases. Out of 262 samples negative by virus culture, 48 (18.3%) were positive by TaqMan PCR, with higher Ct values compared with culture positive samples (P < 0.0001). Overall, the Ct values for HSV-1 were lower than for HSV-2 (mean, 25.5 versus 27.9), but to some extent this could be due to weaker fluorescence by JOE. Lower Ct values for HSV-1 were seen also in the 202 genital samples (79 HSV-1, 122 HSV-2, 1 HSV-1 and HSV-2), indicating that HSV-1 replicates as well as HSV-2 in the genital area. HSV-1 constituted 40% of genital infections and was associated with lower mean age (29.2 versus 36.4 years), probably reflecting the fact that recurrent genital HSV-1 infections are rare. PMID:15872222

  11. A comparison of two West Nile virus detection assays (TaqMan reverse transcriptase polymerase chain reaction and VecTest antigen assay) during three consecutive outbreaks in northern Illinois.

    PubMed

    Lampman, Richard L; Krasavin, Nina M; Szyska, Michael; Novak, Robert J

    2006-03-01

    Mosquitoes identified as female Culex (Culex) species, primarily mixtures or uniform batches of Culex pipiens and Culex restuans, were collected daily from gravid traps by 2 mosquito abatement districts (MADs) in Cook County, Illinois. From 2002 through 2004, batches (pools) of mosquitoes were tested by the MADs for West Nile virus (WNV) by using VecTest WNV antigen assays and the same samples were retested, usually within 1-2 wk, for WNV RNA by the TaqMan reverse transcriptase polymerase chain reaction (RT-PCR). There were 952 TaqMan-positive pools out of 3,953 pools over the 3 years, and about one half of that number were VecTest-positive. The difference between the 2 detection assays varied between and within years. The VecTest assays detected about 57% and 69% of the TaqMan RT-PCR-positive pools from Des Plaines Valley MAD and Northwest MAD in 2002, but only about 40% and 46% in 2003, and 36% and 55% in 2004, respectively. Based on a subset of the 2004 data, a linear relationship was found between VecTest detection of WNV and TaqMan cycle threshold between 18 and 28 cycles. A temporal decrease in the difference between the 2 assays was observed in 2003 and 2004, which we conjecture is due, at least partially, to a seasonal decline in the proportion of recently infected mosquitoes. This trend was not observed in 2002 because infection rates indicated a high likelihood of more than 1 infected mosquito per pool at the peak of transmission. Unlike a previous study, the 95% confidence intervals of infection rates based on the 2 detection methods did not always overlap. The highest infection rates occurred in 2002 when mean monthly temperatures were above average.

  12. Rapid genotyping of 25 autosomal STRs in a Japanese population using fluorescent universal primers containing locked nucleic acids.

    PubMed

    Asari, Masaru; Okuda, Katsuhiro; Yajima, Daisuke; Maseda, Chikatoshi; Hoshina, Chisato; Omura, Tomohiro; Shiono, Hiroshi; Matsubara, Kazuo; Shimizu, Keiko

    2015-04-01

    Amplification of fluorescently labeled products is one of the most popular methods for genotyping genetic variations. Two-step amplification using fluorescent universal primers simultaneously produces multiple targeted fragments labeled with fluorescent dyes, and this strategy is applicable to large-scale, cost-effective genotyping. In this study, we developed a fast PCR-based, multiple short tandem repeat (STR) genotyping method using fluorescent universal primers containing locked nucleic acids (LNAs). Four amplification reactions, each assaying six or seven markers and using 0.5-1.0 ng of genomic DNA, produced obvious Fam-labeled peaks in all 26 loci tested (25 autosomal STRs and amelogenin). The overall amplification time was 37 min. Moreover, fluorescent signals for the 25 STRs obtained from LNA-containing primers were 1.5-9.0 fold higher compared to those from non-LNA primers. Using genomic DNA from 120 Japanese individuals, 16 out of the 25 STRs had observed heterozygosity greater than 0.7. Some of these 25 STRs also had high discriminatory power, similar to that of the 13 core STRs in the Combined DNA Index System dataset. The probability of incorrectly assigning a match based on the accumulated matching probability for these 25 STRs is 1.2 × 10(-22), and their combined use can provide robust information for Japanese forensics. Copyright © 2015 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

  13. Sequence heterogeneity in the equi merozoite antigen gene (ema-1) of Theileria equi and development of an ema-1-specific TaqMan MGB assay for the detection of T. equi.

    PubMed

    Bhoora, Raksha; Quan, Melvyn; Matjila, Paul T; Zweygarth, Erich; Guthrie, Alan J; Collins, Nicola E

    2010-08-27

    Although a quantitative real-time PCR assay (qPCR) assay for the detection of Theileria equi has been developed and evaluated, it is possible that additional, as yet undetected 18S rRNA gene sequence variants may exist. A qPCR assay targeting a different gene, used in conjunction with the T. equi 18S rRNA qPCR assay, could assist in the detection of all T. equi genotypes in field samples. A T. equi ema-1-specific qPCR (Ueti et al., 2003) was tested on 107 South African field samples, 90 of which tested positive for T. equi antibody using the immuno-fluorescent antibody test (IFAT). The qPCR assay performed poorly, as T. equi was detected in only 67 of the 90 IFAT-positive field samples at quantification cycle (C(q)) values ranging from 27 to 39.95. Furthermore, a high C(q) value of 36.18 was obtained from DNA extracted from a South African in vitro-cultured T. equi WL isolate [1.38% parasitized erythrocytes (PE)] when a low C(q) value (indicative of a high T. equi DNA concentration) was expected. Approximately 600 bp of the ema-1 gene from 38 South African samples were sequenced and BLASTN analysis confirmed all sequences to be merozoite surface protein genes, with an identity of 87.1-100% to previously published T. equi ema-1 gene sequences. Alignment of the sequences revealed extensive sequence variations in the target regions of the primers and probes (Ueti et al., 2003), explaining the poor performance of the qPCR assay. Based on these observations, we developed a new TaqMan minor-groove binder (MGB) probe-based qPCR assay, targeting a more conserved region of the ema-1 gene. This assay was shown to be efficient and specific, and the detection limit, defined as the concentration at which 95% of T. equi-positive samples are detected, was determined to be 1.4 x 10(-4)% PE. The two ema-1 assays were compared by testing 41 South African field samples in parallel. The results suggested that the new assay was more sensitive than the original assay, as T. equi was

  14. SYBR Green I and TaqMan quantitative real-time polymerase chain reaction methods for the determination of amplification of Plasmodium falciparum multidrug resistance-1 gene (pfmdr1).

    PubMed

    Muhamad, Poonuch; Chaijaroenkul, Wanna; Congpuong, Kanungnit; Na-Bangchang, Kesara

    2011-10-01

    The pfmdr1 gene, which encodes P-glycoprotein homolog 1, has been shown to be a reliable marker of resistance for Plasmodium falciparum related to artesunate and mefloquine combination therapy. The aims of this study are to investigate the copy number of pfmdr1 in P. falciparum isolates collected from the 4 malaria-endemic areas of Thailand (Kanchanaburi, Mae Hongson, Ranong, and Tak) along the Thailand-Myanmar (Burma) border (Thai-Myanmar border) by using SYBR Green I and the standard method TaqMan real-time polymerase chain reaction (RT-PCR) and to compare the efficiency (sensitivity and specificity) of SYBR Green I with TaqMan RT-quantitative (q)PCR methods in determining pfmdr1 gene copy number. Ninety-six blood samples were collected onto filter paper from patients with uncomplicated falciparum malaria who attended malaria clinics in the Kanchanaburi (n  =  45), Mae Hongson (n  =  18), Ranong (n  =  11), and Tak (n  =  22) provinces in Thailand. Parasite genomic DNA was extracted from dried blood spots by using QIAcube™ automated sample preparation. Pfmdr1 gene copy number was determined by TaqMan (63 samples) and SYBR Green I (96 samples) real-time PCR. Seventy-one (74.0%), 14 (14.6%), 10 (10.4%), and 1 (1%) isolates carried 1, 2, 3, and 4 pfmdr1 gene copies, respectively. Forty-three of 48 (89.6%), 6 of 11 (54.5%), and 3 of 4 (75.0%) samples, respectively, showed agreement with results of 1, 2, and 3 pfmdr1 gene copies as determined by both methods. The efficiency of SYBR Green I in identifying pfmdr1 gene copy number was found to be significantly correlated with that of TaqMan. Considering its simplicity and relatively low cost, SYBR Green I RT-qPCR is therefore a promising alternative technique for the determination of pfmdr1 copy number.

  15. Development and evaluation of a real-time Taqman RT-PCR assay for the detection of infectious bronchitis virus from infected chickens.

    PubMed

    Callison, Scott A; Hilt, Deborah A; Boynton, Tye O; Sample, Brenda F; Robison, Robert; Swayne, David E; Jackwood, Mark W

    2006-12-01

    It is important to rapidly differentiate infectious bronchitis virus (IBV) from disease agents like highly pathogenic avian influenza virus and exotic Newcastle disease virus, which can be extremely similar in the early stages of their pathogenesis. In this study, we report the development and testing of a real-time RT-PCR assay using a Taqman-labeled probe for early and rapid detection of IBV. The assay amplifies a 143-bp product in the 5'-UTR of the IBV genome and has a limit of detection and quantification of 100 template copies per reaction. All 15 strains of IBV tested as well as two Turkey coronavirus strains were amplified, whereas none of the other pathogens examined, tested positive. Evaluation of the assay was completed with 1329 tracheal swab samples. A total of 680 samples collected from IBV antibody negative birds were negative for IBV by the real-time RT-PCR assay. We tested 229 tracheal swabs submitted to two different diagnostic laboratories and found 79.04% of the tracheal swabs positive for IBV by real-time RT-PCR, whereas only 27.51% of the samples were positive by virus isolation, which is the reference standard test. We also collected a total of 120 tracheal swabs at six different time points from birds experimentally infected with different dosages of IBV and found that, independent of the dose given, the viral load in the trachea plateau at 5 days post-inoculation. In addition, an inverse relationship between the dose of virus given and the viral load at 14 days post-inoculation was observed. Finally, we tested 300 total tracheal swab samples, from a flock of commercial broilers spray vaccinated for IBV in the field. The percentage of birds infected with the IBV vaccine at 3, 7, and 14 days post-vaccination was 58%, 65%, and 83%, respectively, indicating that only slightly more than half the birds were initially infected then the vaccine was subsequently transmitted to other birds in the flock. This observation is significant because

  16. Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

    PubMed Central

    Cura, Carolina I.; Duffy, Tomas; Lucero, Raúl H.; Bisio, Margarita; Péneau, Julie; Jimenez-Coello, Matilde; Calabuig, Eva; Gimenez, María J.; Valencia Ayala, Edward; Kjos, Sonia A.; Santalla, José; Mahaney, Susan M.; Cayo, Nelly M.; Nagel, Claudia; Barcán, Laura; Málaga Machaca, Edith S.; Acosta Viana, Karla Y.; Brutus, Laurent; Ocampo, Susana B.; Aznar, Christine; Cuba Cuba, Cesar A.; Gürtler, Ricardo E.; Ramsey, Janine M.; Ribeiro, Isabela; VandeBerg, John L.; Yadon, Zaida E.; Osuna, Antonio; Schijman, Alejandro G.

    2015-01-01

    Background Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). Methods/Principal Findings The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. Conclusions/Significance Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production. PMID:25993316

  17. Design of a sandwich-mode amperometric biosensor for detection of PML/RARα fusion gene using locked nucleic acids on gold electrode.

    PubMed

    Wang, Kun; Sun, Zhouliang; Feng, Meijuan; Liu, Ailin; Yang, Shuyu; Chen, Yuanzhong; Lin, Xinhua

    2011-02-15

    In this study, a novel DNA electrochemical probe (locked nucleic acid, LNA) was designed and involved in constructing an electrochemical DNA biosensor for detection of promyelocytic leukemia/retinoic acid receptor alpha (PML/RARα) fusion gene in acute promyelocytic leukemia for the first time. This biosensor was based on a 'sandwich' sensing mode, which involved a pair of LNA probes (capture probe immobilized at electrode surface and biotinyl reporter probe as an affinity tag for streptavidin-horseradish peroxidase (streptavidin-HRP). Since biotin can be connected with streptavidin-HRP, this biosensor offered an enzymatically amplified electrochemical current signal for the detection of target DNA. In the simple hybridization system, DNA fragment with its complementary DNA fragment was evidenced by amperometric detection, with a detection limit of 74 fM and a linear response range of 0.1-10 pM for synthetic PML/RARα fusion gene in acute promyelocytic leukemia (APL). Otherwise, the biosensor showed an excellent specificity to distinguish the complementary sequence and different mismatch sequences. The new pattern also exhibited high sensitivity and selectivity in mixed hybridization system. Copyright © 2010 Elsevier B.V. All rights reserved.

  18. TaqMan PCR Simulator

    SciTech Connect

    Dyer, M. D.; Gardner, S. N.; Smith, Ja R.; Vitalis, E.; Torres, C.; Kuczmarski, T. A.; Slezak, T.

    2007-05-01

    TaqSim simulates various types of PRC reactions, including multiplex reactions. Given a set of primers and dearch databases, TaqSim identifies amplicons that match user defined criteria and can generate output files in a number of formats allowing it to serve as a front-end or back-end for other software.

  19. Linoleate isomerase activity occurs in lactic acid bacteria strains and is affected by pH and temperature.

    PubMed

    Gorissen, L; Weckx, S; Vlaeminck, B; Raes, K; De Vuyst, L; De Smet, S; Leroy, F

    2011-09-01

    To investigate the ability of lactic acid bacteria (LAB) to convert linoleic acid (LA) and α-linolenic acid (α-LNA) to conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA), respectively. To assess pH and temperature influences on CLA and CLNA production by Lactobacillus sakei LMG 13558. A screening of 48 LAB yielded one Lactobacillus curvatus, five Lactobacillus plantarum and four Lact. sakei strains displaying linoleate isomerase (LAI) activity. CLNA conversion percentages varied largely (1-60%). CLA conversion, occurring in three strains, was lower (2-5%). The LAI gene sequences of the ten LAI-positive strains shared 75-99% identity with the LAI gene sequence of a Lact. plantarum AS1.555. At pH 6.2, CLA and CLNA production by Lact. sakei LMG 13558 was higher at 30°C than at 20 and 25°C. At pH 5.5 (30°C) or 37°C (pH 6.2), LA was not converted and α-LNA only slightly converted. LAB show strain-dependent LAI activity. Production of CLA and CLNA is affected by pH and temperature, as shown for Lact. sakei LMG 13558. Several LAB produce CLA and/or CLNA, as shown for Lact. sakei and Lact. curvatus for the first time. These findings offer potential for the manufacturing of fermented functional foods. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

  20. Comparison of n-3 fatty acid sources in laying hen rations for improvement of whole egg nutritional quality: a review.

    PubMed

    Van Elswyk, M E

    1997-07-01

    The nutritional manipulation of the diets of laying hens to include sources of n-3 fatty acids promotes the deposition of these nutrients into egg yolk, n-3 Fatty acid-rich eggs may provide an exciting alternative food source for enhancing consumer intake of these proposed healthful fatty acids. Care must be taken when designing n-3 fatty acid-rich poultry rations, however, to assure that the resulting egg fatty acid profile is useful for promoting consumer health yet maintaining egg sensory quality. In study 1 laying hens were fed on diets supplemented with graded levels of menhaden oil (MO), rich in both eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), for 4 weeks to determine maximal yolk fatty acid deposition attainable without sensory compromise. Yolk fatty acids were analysed for an additional 4 weeks, post-MO removal, to investigate yolk n-3 fatty acid tenacity. Dietary MO levels between 15 and 30 g/kg yielded the greatest yolk n-3 fatty acid content; however, only eggs from birds fed with 15 g MO/kg were considered acceptable by trained flavour panelists. Evaluation of eggs from hens fed with 15 g MO/ kg during storage verified that the shelf-life of enriched eggs was comparable with that of typical eggs. In study 2, graded levels of whole or ground flaxseed were used for the deposition of linolenic acid (LNA; 18:3n-3) and to determine in vivo production of DHA from dietary LNA for yolk deposition. Flaxseed form influenced yolk n-3 fatty acids only when given at 150 g/kg diet. In vivo production of DHA, while significant, was not enhanced by increasing the level of dietary flaxseed nor by grinding the seed. In the third study, a DHA-rich natural marine alga (MA) was investigated as an n-3 fatty acid supplement. Despite similar DHA profiles, dietary MA was found to be more efficient for yolk DHA deposition than dietary MO. These studies suggest that there are numerous viable n-3 fatty acid supplements for poultry rations. It must

  1. Sensitive and specific detection of phaseolotoxigenic and nontoxigenic strains of Pseudomonas syringae pv. phaseolicola by TaqMan real-time PCR using site-specific recombinase gene sequences.

    PubMed

    Cho, Min Seok; Jeon, Yong Ho; Kang, Man Jung; Ahn, Hong Il; Baek, Hyung-Jin; Na, Young Wang; Choi, Yu Mi; Kim, Tae San; Park, Dong Suk

    2010-09-20

    Pseudomonas syringae pv. phaseolicola, the causative agent of halo blight, is the most important bacterial pathogen of bean. Both nontoxigenic (Tox(-)) and toxigenic (Tox+) strains of this pathogen cause halo blight in beans. However, nontoxigenic strains cannot be detected by currently available molecular and serological tools. In this study, a TaqMan probe and primer set were designed based on the phage integrase family site-specific recombinase of P. s. pv. phaseolicola 1448A because it is known that most site-specific recombinases are structurally and functionally diverse. The specificity of the probe and primers was evaluated using purified DNA from 29 isolates of 3 different pathovars of P. syringae. The probe and primer set were able to detect Tox(-) and Tox+ isolates of P. s. pv. Phaseolicola, but no other phytopathogenic bacteria. The assay was also able to detect at least 5 genome equivalents of cloned amplified target DNA, using purified DNA, or 7 colony forming unit (CFU) per reaction when using calibrated cell suspensions. Thus, the TaqMan real-time PCR-based method can be used for the rapid detection of both types of P. s. pv. Phaseolicola, and will potentially simplify and facilitate the diagnosis and monitoring of this pathogen, and guide plant disease management.

  2. In vitro selection of DNA-based aptamers that exhibit RNA-like conformations using a chimeric oligonucleotide library that contains two different xeno-nucleic acids.

    PubMed

    Hagiwara, Kenta; Fujita, Hiroto; Kasahara, Yuuya; Irisawa, Yuuta; Obika, Satoshi; Kuwahara, Masayasu

    2015-01-01

    We successfully generated chimeric DNA aptamers that contained six nucleoside analogs of 2'-O,4'-C-methylene bridged/locked nucleic acid (2',4'-BNA/LNA) in the primer region and multiple guanosine analogs of 2'-deoxy-2'-fluoro-ribonucleic acid (FNA) in the non-primer region using capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX). Active species enrichment became saturated only after five selection rounds, and we obtained DNA-based xeno-nucleic acid (XNA) aptamers that had high binding affinities for the target human thrombin, with dissociation constant (Kd) values of ≥10 nanomolar. Based on sequence and circular dichroism (CD) analyses, these XNA aptamers exhibited RNA-like conformations, which could cause DNA-based strands to adopt structurally diverse conformations.

  3. Lipid metabolic dose response to dietary alpha-linolenic acid in monk parrot (Myiopsitta monachus).

    PubMed

    Petzinger, Christina; Heatley, J J; Bailey, Christopher A; Bauer, John E

    2014-03-01

    Monk parrots (Myiopsitta monachus) are susceptible to atherosclerosis, a progressive disease characterized by the formation of plaques in the arteries accompanied by underlying chronic inflammation. The family of n-3 fatty acids, especially eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA), have consistently been shown to reduce atherosclerotic risk factors in humans and other mammals. Some avian species have been observed to convert α-linolenic acid (18:3n-3, ALA) to EPA and DHA (Htin et al. in Arch Geflugelk 71:258-266, 2007; Petzinger et al. in J Anim Physiol Anim Nutr, 2013). Therefore, the metabolic effects of including flaxseed oil, as a source of ALA, in the diet at three different levels (low, medium, and high) on the lipid metabolism of Monk parrots was evaluated through measuring plasma total cholesterol (TC), free cholesterol (FC), triacylglycerols (TAG), and phospholipid fatty acids. Feed intake, body weight, and body condition score were also assessed. Thus the dose and possible saturation response of increasing dietary ALA at constant linoleic acid (18:2n-6, LNA) concentration on lipid metabolism in Monk parrots (M. monachus) was evaluated. Calculated esterified cholesterol in addition to plasma TC, FC, and TAG were unaltered by increasing dietary ALA. The high ALA group had elevated levels of plasma phospholipid ALA, EPA, and docosapentaenoic acid (DPAn-3, 22:5n-3). The medium and high ALA groups had suppressed plasma phospholipid 20:2n-6 and adrenic acid (22:4n-6, ADA) compared to the low ALA group. When the present data were combined with data from a previous study (Petzinger et al. in J Anim Physiol Anim Nutr, 2013) a dose response to dietary ALA was observed when LNA was constant. Plasma phospholipid ALA, EPA, DPAn-3, DHA, and total n-3 were positively correlated while 20:2n-6, di-homo-gamma-linoleic acid (20:3n-6Δ7), arachidonic acid (20:4n-6), ADA, and total n-6 were inversely correlated with dietary en% ALA.

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

    PubMed Central

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

    2012-01-01

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

  5. Comparative crystallization and preliminary X-ray diffraction studies of locked nucleic acid and RNA stems of a tenascin C-binding aptamer

    PubMed Central

    Förster, Charlotte; Brauer, Arnd B. E.; Brode, Svenja; Schmidt, Kathrin S.; Perbandt, Markus; Meyer, Arne; Rypniewski, Wojciech; Betzel, Christian; Kurreck, Jens; Fürste, Jens P.; Erdmann, Volker A.

    2006-01-01

    The pharmacokinetic properties of an aptamer against the tumour-marker protein tenascin-C have recently been successfully improved by the introduction of locked nucleic acids (LNAs) into the terminal stem of the aptamer. Since it is believed that this post-SELEX optimization is likely to provide a more general route to enhance the in vitro and in vivo stability of aptamers, elucidation of the structural basis of this improvement was embarked upon. Here, the crystallographic and X-ray diffraction data of the isolated aptamer stem encompassed in a six-base-pair duplex both with and without the LNA modification are presented. The obtained all-LNA crystals belong to space group P41212 or P43212, with unit-cell parameters a = b = 52.80, c = 62.83 Å; the all-RNA crystals belong to space group R32, with unit-cell parameters a = b = 45.21, c = 186.97 Å, γ = 120.00°. PMID:16820689

  6. Possible effect of lysophosphatidic acid on cell proliferation and involvement of lysophosphatidic acid and lysophosphatidic acid receptors in mechanical stretch-induced mitogen-activated protein kinase.

    PubMed

    Kawashima, Yohei; Kushida, Nobuhiro; Kokubun, Shuko; Ogawa, Soichiro; Shiomi, Homare; Ishibashi, Kei; Aikawa, Ken; Ikegami, Kentaro; Nomiya, Masanori; Yamaguchi, Osamu

    2015-08-01

    To determine whether lysophosphatidic acid activates the mitogen-activated protein kinase and increases DNA synthesis in human bladder smooth muscle cells, and to examine the involvement of lysophosphatidic acid and lysophosphatidic acid receptor in mechanical stretch-induced mitogen-activated protein kinase activation in cultured human bladder smooth muscle cells. TaqMan reverse transcription polymerase chain reaction was used to determine the mRNA expression levels of six lysophosphatidic acid receptor subtypes. Mitogen-activated protein kinase activity enhanced by either lysophosphatidic acid or mechanical stretch was measured by western blotting. The effect of lysophosphatidic acid on DNA synthesis was assessed by 5-bromo-2'-deoxy-uridine incorporation assay. Lysophosphatidic acid 1 subtype mRNA was predominantly expressed (96%). Lysophosphatidic acid activated the mitogen-activated protein kinase in a concentration-dependent manner. C-jun NH2 -terminal kinase showed the highest activity among the three subsets of the mitogen-activated protein kinase family members (c-jun NH2 -terminal kinase, extracellular signal-regulated kinases, p38). Lysophosphatidic acid also increased incorporation of 5-bromo-2'-deoxy-uridine. These responses were suppressed by Ki16425 (lysophosphatidic acid receptor antagonist). Mechanical stretch mainly induced c-jun NH2 -terminal kinase activation. This activation was partially inhibited by Ki16425. Lysophosphatidic acid might activate the c-jun NH2 -terminal kinase component of the mitogen-activated protein kinase family and DNA synthesis through lysophosphatidic acid receptors (presumably, through lysophosphatidic acid 1) in human bladder smooth muscle cells. The present study also implicates the involvement of lysophosphatidic acid and lysophosphatidic acid receptors in mechanical stretch-induced c-jun NH2 -terminal kinase activation. Lysophosphatidic acid receptor can be partially activated by mechanical stretching through

  7. Long Chain Fatty Acid Acylated Derivatives of Quercetin-3-O-Glucoside as Antioxidants to Prevent Lipid Oxidation

    PubMed Central

    Warnakulasuriya, Sumudu N.; Ziaullah; Rupasinghe, H.P. Vasantha

    2014-01-01

    Flavonoids have shown promise as natural plant-based antioxidants for protecting lipids from oxidation. It was hypothesized that their applications in lipophilic food systems can be further enhanced by esterification of flavonoids with fatty acids. Quercetin-3-O-glucoside (Q3G) was esterified individually with six selected long chain fatty acids: stearic acid (STA), oleic acid (OLA), linoleic acid (LNA), α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and decosahexaenoic acid (DHA), using Candida antarctica B lipase as the biocatalyst. The antioxidant activity of esterified flavonoids was evaluated using lipid oxidation model systems of poly-unsaturated fatty acids-rich fish oil and human low density lipoprotein (LDL), in vitro. In the oil-in-water emulsion, Q3G esters exhibited 50% to 100% inhibition in primary oxidation and 30% to 75% inhibition in secondary oxidation. In bulk oil, Q3G esters did not provide considerable protection from lipid oxidation; however, Q3G demonstrated more than 50% inhibition in primary oxidation. EPA, DHA and ALA esters of Q3G showed significantly higher inhibition in Cu2+- and peroxyl radical-induced LDL oxidation in comparison to Q3G. PMID:25384198

  8. Role of arachidonic acid lipoxygenase metabolites in acetylcholine-induced relaxations of mouse arteries

    PubMed Central

    Goldman, Daniel H.; Aggarwal, Nitin T.; Chawengsub, Yuttana; Falck, J. R.; Campbell, William B.

    2011-01-01

    Arachidonic acid (AA) metabolites function as EDHFs in arteries of many species. They mediate cyclooxygenase (COX)- and nitric oxide (NO)-independent relaxations to acetylcholine (ACh). However, the role of AA metabolites as relaxing factors in mouse arteries remains incompletely defined. ACh caused concentration-dependent relaxations of the mouse thoracic and abdominal aorta and carotid, femoral, and mesentery arteries (maximal relaxation: 57 ± 4%, 72 ± 4%, 82 ± 3%, 80 ± 3%, and 85 ± 3%, respectively). The NO synthase inhibitor nitro-l-arginine (l-NA; 30 μM) blocked relaxations in the thoracic aorta, and l-NA plus the COX inhibitor indomethacin (10 μM) inhibited relaxations in the abdominal aorta and carotid, femoral, and mesenteric arteries (maximal relaxation: 31 ± 10%, 33 ± 5%, 41 ± 8%, and 73 ± 3%, respectively). In mesenteric arteries, NO- and COX-independent relaxations to ACh were inhibited by the lipoxygenase (LO) inhibitors nordihydroguaiaretic acid (NDGA; 10 μM) and BW-755C (200 μM), the K+ channel inhibitor apamin (1 μM), and 60 mM KCl and eliminated by endothelium removal. They were not altered by the cytochrome P-450 inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (20 μM) or the epoxyeicosatrienoic acid antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10 μM). AA relaxations were attenuated by NDGA or apamin and eliminated by 60 mM KCl. Reverse-phase HPLC analysis revealed arterial [14C]AA metabolites that comigrated with prostaglandins, trihydroxyeicosatrienoic acids (THETAs), hydroxyepoxyeicosatrienoic acids (HEETAs), and hydroxyeicosatetraenoic acids (HETEs). Epoxyeicosatrienoic acids were not observed. Mass spectrometry confirmed the identity of 6-keto-PGF1α, PGE2, 12-HETE, 15-HETE, HEETAs, 11,12,15-THETA, and 11,14,15-THETA. AA metabolism was blocked by NDGA and endothelium removal. 11(R),12(S),15(S)-THETA relaxations (maximal relaxation: 73 ± 3%) were endothelium independent and blocked by 60 mM KCl. Western

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

    PubMed

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

    2015-02-17

    Advances in real-time polymerase chain reaction (PCR), as well as the emergence of digital PCR (dPCR) and useful modified nucleotide chemistries, including locked nucleic acids (LNAs), have created the potential to improve and expand clinical applications of PCR through their ability to better quantify and differentiate amplification products, but fully realizing this potential will require robust methods for designing dual-labeled hydrolysis probes and predicting their hybridization thermodynamics as a function of their sequence, chemistry, and template complementarity. We present here a nearest-neighbor thermodynamic model that accurately predicts the melting thermodynamics of a short oligonucleotide duplexed either to its perfect complement or to a template containing mismatched base pairs. The model may be applied to pure-DNA duplexes or to duplexes for which one strand contains any number and pattern of LNA substitutions. Perturbations to duplex stability arising from mismatched DNA:DNA or LNA:DNA base pairs are treated at the Gibbs energy level to maintain statistical significance in the regressed model parameters. This approach, when combined with the model's accounting of the temperature dependencies of the melting enthalpy and entropy, permits accurate prediction of T(m) values for pure-DNA homoduplexes or LNA-substituted heteroduplexes containing one or two independent mismatched base pairs. Terms accounting for changes in solution conditions and terminal addition of fluorescent dyes and quenchers are then introduced so that the model may be used to accurately predict and thereby tailor the T(m) of a pure-DNA or LNA-substituted hydrolysis probe when duplexed either to its perfect-match template or to a template harboring a noncomplementary base. The model, which builds on classic nearest-neighbor thermodynamics, should therefore be of use to clinicians and biologists who require probes that distinguish and quantify two closely related alleles in either a

  10. Evaluation of performances of VERSANT HCV RNA 1.0 assay (kPCR) and Roche COBAS AmpliPrep/COBAS TaqMan HCV test v2.0 at low level viremia.

    PubMed

    Mazzuti, Laura; Lozzi, Maria Antonietta; Riva, Elisabetta; Maida, Paola; Falasca, Francesca; Antonelli, Guido; Turriziani, Ombretta

    2016-07-01

    We assess the concordance between low level HCV values obtained using the VERSANT HCV RNA 1.0 Assay (kPCR) and COBAS AmpliPrep/COBAS TaqMan HCV Quantitative Test v2.0. The correlation between the values obtained by the two RT-PCR assays for samples with quantifiable HCV RNA levels revealed that viral load measured by kPCR significantly correlated with that of the CAP/CTM (R=0.644, P<0.0001). The results show a good concordance (n=126/144, 87%); discordant results were mainly observed in the assessment of values below the lower limit of detection of the assays. These variations may have an impact on clinical decisions for patients on HCV triple therapy or interferon- free regimens. It is therefore recommended to monitor individual patients with the same test throughout treatment.

  11. Development and validation of a TaqMan real-time PCR assay for the identification and quantification of roe deer (Capreolus capreolus) in food to detect food adulteration.

    PubMed

    Druml, Barbara; Mayer, Walter; Cichna-Markl, Margit; Hochegger, Rupert

    2015-07-01

    In order to protect the consumer from meat adulteration it is necessary to identify and quantify the meat content in foodstuffs. Game meat is particularly susceptible for fraudulent labeling since it is more valuable than meat from domestic animals. The paper presents a TaqMan real-time PCR assay for the quantitative determination of roe deer in meat products. The assay developed does not show cross-reactivity with 23 animal and 43 plant species tested and is therefore specific for roe deer. The amplification efficiency determined by analyzing serially diluted roe deer DNA extracts was found to be 93.9%. For quantifying the roe deer content in % (w/w), a reference system based on the myostatin gene was used. The quantification strategy was validated by determining the roe deer content in model meat mixtures and a model sausage. In addition, the real-time PCR assay was applied to the analysis of commercially available meat products.

  12. Comparison of the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 test v1.0 with v2.0 in HIV-1 viral load quantification.

    PubMed

    Tung, Yi-Ching; Ke, Liang-Yin; Lu, Po-Liang; Lin, Kuei-Hsiang; Lee, Su-Chen; Lin, Yi-Ying; Chou, Li-Chiu; Tsai, Wen-Chan

    2015-04-01

    Roche modified the COBAS AmpliPrep/COBAS TaqMan human immunodeficiency virus type 1 (HIV-1) test version 1.0 (CAP/CTM v1.0), resulting in the COBAS AmpliPrep/COBAS TaqMan HIV-1 test version 2.0 (CAP/CTM v2.0). The aim of this study was to evaluate the performance of the CAP/CTM v2.0 and to compare this performance with that of the CAP/CTM v1.0. The study was conducted in a small local study group in Kaohsiung Medical University Hospital, Kaohsiung, Taiwan. A total of 86 plasma samples from HIV-1-seropositive patients were tested using the two assays. The correlation and concordance of results between the two assays were calculated. The CAP/CTM v2.0 generated higher values than did the CAP/CTM v1.0, and five samples (5.8%) yielded a difference of > 1 log10 copies/mL. In addition, our data show that CAP/CTM v1.0 and CAP/CTM v2.0 yielded relatively consistent values for 23 samples with low viral loads (< 200 copies/mL). Furthermore, when viral loads were in a medium range (2-5 log10 copies/mL), the results of the two assays were more compatible. This study shows a good correlation between CAP/CTM v1.0 and v2.0 in HIV-1 viral load measurement. Further attention must be paid to those cases in which measured viral loads present larger differences between the two assays.

  13. Performance evaluation of the new Roche cobas AmpliPrep/cobas TaqMan HCV test, version 2.0, for detection and quantification of hepatitis C virus RNA.

    PubMed

    Pas, S; Molenkamp, R; Schinkel, J; Rebers, S; Copra, C; Seven-Deniz, S; Thamke, D; de Knegt, R J; Haagmans, B L; Schutten, M

    2013-01-01

    To evaluate the analytical performance and explore the clinical applicability of the new Roche cobas AmpliPrep/cobas TaqMan HCV test, v2.0 (CAP/CTM v2.0), a platform comparison was performed on panels and diagnostic samples with the Roche cobas AmpliPrep/cobas TaqMan HCV test (CAP/CTM v1.0), the Siemens Versant HCV RNA 3.0 branched DNA (bDNA) test, the Abbott m2000 RealTime HCV assay (Realtime assay), and the Siemens Versant HCV transcription-mediated amplification (TMA) test (TMA assay). The analytical performance of the CAP/CTM v2.0 on WHO and Acrometrix panels and clinical specimens of patients infected with HCV genotype 1, 2, 3, 4, 5, or 6 relative to that of the CAP/CTM v1.0 was significantly improved. In a qualitative comparison of the CAP/CTM v2.0 relative to the TMA assay on genotype 1 to 4 samples, the two tests proved to be almost equally sensitive. Response-guided therapy in one of five HCV genotype 4-infected patients previously tested with the CAP/CTM v1.0 would have significantly changed if tested with the CAP/CTM v2.0. In conclusion, the Roche CAP/CTM v2.0 has significantly better performance characteristics than the former CAP/CTM HCV v1.0 and the bDNA assay and performance characteristics comparable to those of the Realtime assay.

  14. Improved COBAS TaqMan hepatitis C virus test (Version 2.0) for use with the High Pure system: enhanced genotype inclusivity and performance characteristics in a multisite study.

    PubMed

    Colucci, G; Ferguson, J; Harkleroad, C; Lee, S; Romo, D; Soviero, S; Thompson, J; Velez, M; Wang, A; Miyahara, Y; Young, S; Sarrazin, C

    2007-11-01

    We have evaluated the COBAS TaqMan hepatitis C virus (HCV) test (version 2.0) for use with the High Pure system (HCVHPS V2), a new, revised real-time reverse transcription-PCR assay developed to improve the genotype quantitation of version 1.0 (HCVHPS V1). Revisions were made in the wash buffer and in the reverse transcription temperature. The genotype inclusivity of HCVHPS V2 was evaluated at three different sites, using HCVHPS V2, HCVHPS V1, and the COBAS AMPLICOR HCV MONITOR test (version 2.0) (CAHCM). The fully automated COBAS Ampliprep/COBAS TaqMan HCV test was also used in one of the participating laboratories. The mean differences in HCV RNA values between HCVHPS V2 and CAHCM and between HCVHPS V2 and HCVHPS V1 ranged from -0.21 to 0.13 log and from 0.24 to 1.27 log, respectively, with >0.5-log differences for genotypes 2, 3, 4, and 5. With a NIBSC panel of HCV genotypes 1 through 6, the measured HCVHPS V2 values were within 0.25 log of the nominal values for all 6 genotypes. When serial dilutions of genotype-specific clinical HCV specimens were tested, the assay showed a limit of detection between 10 and 20 IU/ml and a linear range of 25 IU/ml to 3.91 x 10(8) IU/ml. Clinical and analytical specificities of 100% were demonstrated with 100 HCV-seronegative specimens as well as with 12 non-HCV members of Flaviviridae and 22 additional microorganisms. These data indicate that HCVHPS V2 is a robust and accurate test for the quantitation of all six HCV genotypes and useful in monitoring viral load in all clinical HCV specimens.

  15. Broad-range detection of arboviruses belonging to Simbu serogroup lineage 1 and specific detection of Akabane, Aino and Peaton viruses by newly developed multiple TaqMan assays.

    PubMed

    Shirafuji, Hiroaki; Yazaki, Ryu; Shuto, Yozo; Yanase, Tohru; Kato, Tomoko; Ishikura, Youji; Sakaguchi, Zenjiro; Suzuki, Moemi; Yamakawa, Makoto

    2015-12-01

    TaqMan assays were developed for the broad-range detection of arboviruses belonging to Simbu serogroup lineage 1 in the genus Orthobunyavirus and also for the specific detection of three viruses in the lineage, Akabane, Aino and Peaton viruses (AKAV, AINOV and PEAV, respectively). A primer and probe set was designed for the broad-range detection of Simbu serogroup lineage 1 (Pan-Simbu1 set) mainly targeting AKAV, AINOV, PEAV, Sathuperi and Shamonda viruses (SATV and SHAV), and the forward and reverse primers of the Pan-Simbu1 set were also used for the specific detection of AKAV with another probe (AKAV-specific set). In addition, two more primer and probe sets were designed for AINOV- and PEAV-specific detection, respectively (AINOV- and PEAV-specific sets). All of the four primer and probe sets successfully detected targeted viruses, and thus broad-range and specific detection of all the targeted viruses can be achieved by using two multiplex assays and a single assay in a dual (two-color) assay format when another primer and probe set for a bovine β-actin control is also used. The assays had an analytical sensitivity of 10 copies/tube for AKAV, at least 100 copies/tube for AINOV, 100 copies/tube for PEAV, one copy/tube for SATV and at least 10 copies/tube for SHAV, respectively. Diagnostic sensitivity of the assays was tested with field-collected bovine samples, and the results suggested that the sensitivity was higher than that of a conventional RT-PCR. These data indicate that the newly developed TaqMan assays will be useful tools for the diagnosis and screening of field-collected samples for infections of AKAV and several other arboviruses belonging to the Simbu serogroup lineage 1.

  16. Design and validation of an H5 TaqMan real-time one-step reverse transcription-PCR and confirmatory assays for diagnosis and verification of influenza A virus H5 infections in humans.

    PubMed

    Ellis, Joanna S; Smith, Joanne W; Braham, Sharleen; Lock, Matthew; Barlow, Katrina; Zambon, Maria C

    2007-05-01

    Increasing diversity among influenza H5N1 viruses has resulted in the need for sensitive and specific diagnostic assays, fully validated for the detection of H5 viruses belonging to all hemagglutinin (HA) clades, particularly the recently circulating H5N1 viruses of clade 2. In this report, the development and validation of a real-time, one-step TaqMan reverse transcription-PCR (RT-PCR) assay specific for the detection of influenza A H5 viruses from clades 1, 1', 2, and 3 is described. The real-time assay for H5 virus was shown to be highly sensitive, detecting H5 virus levels of <1 PFU from each of the HA clades. Specificity of the H5 RT-PCR for influenza A H5 viruses was demonstrated by using influenza A viruses of different subtypes, clinical samples containing influenza A viruses H1N1, H3N2, and H5N1, influenza B viruses, and other respiratory viruses. The usefulness of the inclusion of a distinguishable assay positive control and of confirmatory assays for the laboratory diagnosis and verification of H5 virus infections was demonstrated. A real-time RT-PCR pyrosequencing assay, a restriction enzyme digestion assay, and direct sequencing of the H5 real-time RT-PCR amplicon were validated for the confirmation of H5 detection by the diagnostic real-time assay. The H5 real-time assay was applied to diagnostic testing for suspected cases of influenza A virus H5 infection in the United Kingdom. Influenza A H5 viruses were not detected in the cases analyzed; however, influenza A H3N2 virus was detected in 57% of the suspected cases of H5. The H5 TaqMan real-time RT-PCR and confirmatory assays will be useful tools for the laboratory surveillance and rapid diagnosis of H5 infections in humans.

  17. TaqMan real-time reverse transcription-PCR assay for universal detection and quantification of avian hepatitis E virus from clinical samples in the presence of a heterologous internal control RNA.

    PubMed

    Troxler, Salome; Marek, Ana; Prokofieva, Irina; Bilic, Ivana; Hess, Michael

    2011-04-01

    Avian hepatitis E virus (HEV) isolates could be separated into at least three genotypes. In this study, the development of the first duplex TaqMan real-time reverse transcription-PCR (RT-PCR) assay for detection and quantification of avian HEV is presented. Primers and probes binding within relatively conserved open reading frame 3 (ORF3) were designed. Tenfold dilution series of in vitro-transcribed avian HEV RNA were used as the standard for quantification. A 712-bp region of the green fluorescent protein gene was transcribed in vitro and used as a heterologous internal control for both RNA isolation and real-time RT-PCR. The duplex real-time RT-PCR for avian HEV had an efficiency of 1.04, a regression squared value of 0.996, and a sensitivity of approximately 3.6 × 10(3) copies per reaction mixture when in vitro-transcribed RNA was used as the template. The presence of in vitro-transcribed heterologous internal control RNA did not affect amplification of avian HEV RNA compared to that achieved by the single assay. The sensitivity of the real-time RT-PCR assay was comparable to that of conventional RT-PCR, and it was shown to be highly specific, as tissues from uninfected chickens, mammalian HEVs, and other viral genomes did not produce positive signals. All tested field samples with virus belonging to different avian HEV genotypes were successfully detected with this new duplex TaqMan real-time RT-PCR assay.

  18. Improved COBAS TaqMan Hepatitis C Virus Test (Version 2.0) for Use with the High Pure System: Enhanced Genotype Inclusivity and Performance Characteristics in a Multisite Study▿

    PubMed Central

    Colucci, G.; Ferguson, J.; Harkleroad, C.; Lee, S.; Romo, D.; Soviero, S.; Thompson, J.; Velez, M.; Wang, A.; Miyahara, Y.; Young, S.; Sarrazin, C.

    2007-01-01

    We have evaluated the COBAS TaqMan hepatitis C virus (HCV) test (version 2.0) for use with the High Pure system (HCVHPS V2), a new, revised real-time reverse transcription-PCR assay developed to improve the genotype quantitation of version 1.0 (HCVHPS V1). Revisions were made in the wash buffer and in the reverse transcription temperature. The genotype inclusivity of HCVHPS V2 was evaluated at three different sites, using HCVHPS V2, HCVHPS V1, and the COBAS AMPLICOR HCV MONITOR test (version 2.0) (CAHCM). The fully automated COBAS Ampliprep/COBAS TaqMan HCV test was also used in one of the participating laboratories. The mean differences in HCV RNA values between HCVHPS V2 and CAHCM and between HCVHPS V2 and HCVHPS V1 ranged from −0.21 to 0.13 log and from 0.24 to 1.27 log, respectively, with >0.5-log differences for genotypes 2, 3, 4, and 5. With a NIBSC panel of HCV genotypes 1 through 6, the measured HCVHPS V2 values were within 0.25 log of the nominal values for all 6 genotypes. When serial dilutions of genotype-specific clinical HCV specimens were tested, the assay showed a limit of detection between 10 and 20 IU/ml and a linear range of 25 IU/ml to 3.91 × 108 IU/ml. Clinical and analytical specificities of 100% were demonstrated with 100 HCV-seronegative specimens as well as with 12 non-HCV members of Flaviviridae and 22 additional microorganisms. These data indicate that HCVHPS V2 is a robust and accurate test for the quantitation of all six HCV genotypes and useful in monitoring viral load in all clinical HCV specimens. PMID:17898157

  19. Development and evaluation of a real-time PCR assay for quantification of Giardia and Cryptosporidium in sewage samples.

    PubMed

    Alonso, José L; Amorós, Inmaculada; Cañigral, Irene

    2011-02-01

    Cryptosporidium and Giardia are major causes of diarrheal disease in humans worldwide and are major causes of protozoan waterborne diseases. Two DNA TaqMan PCR-based Giardia and Cryptosporidium methods targeting a 74-bp sequence of the β-giardin Giardia gene and a 151-bp sequence of the COWP Cryptosporidium gene, respectively, were used as models to compare two different LNA/DNA TaqMan probes to improve the detection limit in a real-time PCR assay. The LNA probes were the most sensitive resulting in 0.96 to 1.57 lower C t values than a DNA Giardia TaqMan probe and 0.56 to 2.21 lower than a DNA Cryptosporidium TaqMan probe. Evaluation of TaqMan Giardia and Cryptosporidium probes with LNA substitutions resulted in real-time PCR curves with an earlier C t values than conventional DNA TaqMan probes. In conclusion, the LNA probes could be useful for more sensitive detection limits.

  20. Apoptosis- and differentiation-inducing activities of jacaric acid, a conjugated linolenic acid isomer, on human eosinophilic leukemia EoL-1 cells.

    PubMed

    Liu, Wai-Nam; Leung, Kwok-Nam

    2014-11-01

    Conjugated linolenic acids (CLNAs) are a group of naturally occurring positional and geometrical isomers of the C18 polyunsaturated essential fatty acid, linolenic acid (LNA), with three conjugated double bonds (C18:3). Although previous research has demonstrated the growth-inhibitory effects of CLNA on a wide variety of cancer cell lines in vitro, their action mechanisms and therapeutic potential on human myeloid leukemia cells remain poorly understood. In the present study, we found that jacaric acid (8Z,10E,12Z-octadecatrienoic acid), a CLNA isomer which is present in jacaranda seed oil, inhibited the in vitro growth of human eosinophilic leukemia EoL-1 cells in a time- and concentration-dependent manner. Mechanistic studies showed that jacaric acid triggered cell cycle arrest of EoL-1 cells at the G0/G1 phase and induced apoptosis of the EoL-1 cells, as measured by the Cell Death Detection ELISAPLUS kit, Annexin V assay and JC-1 dye staining. Notably, the jacaric acid-treated EoL-1 cells also underwent differentiation as revealed by morphological and phenotypic analysis. Collectively, our results demonstrated the capability of jacaric acid to inhibit the growth of EoL-1 cells in vitro through triggering cell cycle arrest and by inducing apoptosis and differentiation of the leukemia cells. Therefore, jacaric acid might be developed as a potential candidate for the treatment of certain forms of myeloid leukemia with minimal toxicity and few side effects.

  1. Core-shell H-ZSM-5/silicalite-1 composites: Brønsted acidity and catalyst deactivation at the individual particle level.

    PubMed

    Mores, Davide; Stavitski, Eli; Verkleij, Suzanna P; Lombard, Antoinette; Cabiac, Amandine; Rouleau, Loïc; Patarin, Joël; Simon-Masseron, Angélique; Weckhuysen, Bert M

    2011-09-21

    A combination of in situ UV-Vis and confocal fluorescence micro-spectroscopy is applied to investigate the influence of an external silicalite-1 shell on the Brønsted acidity and coke formation process of individual H-ZSM-5 zeolite crystals. Three probe reactions were used: oligomerization of styrene, methanol-to-olefin (MTO) conversion and aromatization of light naphtha (LNA) derivatives. Oligomerization of styrene leads to the formation of optically active carbocationic oligomers. Different styrene substitutions indicate the conversion ability of the catalyst acid core, a preferred alignment of the oligomers within the straight zeolite channels and a Brønsted acidity gradient throughout the zeolite crystal. Both the MTO conversion and the LNA process lead to limited carbonaceous deposition within the external silicalite-1 layer. This outer shell furthermore prevents the growth of extended coke species at the zeolite external surface. During MTO, the formation of carbonaceous compounds initiates at the center of the H-ZSM-5 zeolite core and expands towards the zeolite exterior. This coke build-up starts with a 420 nm UV-Vis absorption band, assigned to methyl-substituted aromatic carbocations, and a second band around 550 nm, which is indicative of their growth towards larger conjugated systems. Aromatization of linear and branched C5 paraffins causes negligible darkening of the zeolite crystals though it forms fluorescent coke deposits and their precursors within the H-ZSM-5 catalyst. Olefin homologues on the contrary cause pronounced darkening of the zeolite composite. Methyl-branching of these reactants slows down the coke formation rate and produces carbonaceous species that are more restricted in their molecular size.

  2. Sensitive and specific detection of potentially allergenic almond (Prunus dulcis) in complex food matrices by Taqman(®) real-time polymerase chain reaction in comparison to commercially available protein-based enzyme-linked immunosorbent assay.

    PubMed

    Röder, Martin; Vieths, Stefan; Holzhauser, Thomas

    2011-01-24

    Currently, causative immunotherapies are lacking in food allergy. The only option to prevent allergic reactions in susceptible individuals is to strictly avoid the offending food. Thus, reliable labelling of allergenic constituents is of major importance, but can only be achieved if appropriate specific and sensitive detection techniques for foods with allergenic potential are available. Almond is an allergenic food that requires mandatory labelling on prepackaged foods and belongs to the genus Prunus. Species of this genus are phylogenetically closely related. We observed commercially available almond specific ELISA being highly cross-reactive with other foods of the Prunoideae family, resulting in a false-positive detection of up to 500,000 mg kg(-1) almond. Previously published PCR methods were reported to be cross-reactive with false positive results >1200 mg kg(-1). We describe the development of a novel almond specific real-time PCR, based on mutated mismatch primers and sequence specific Taqman(®) probe detection, in comparison with two quantitative commercially available ELISA. PCR sensitivity was investigated with chocolate, chocolate coating and cookies spiked between 5 and 100,000 mg kg(-1) almond. In all matrices almond was reproducibly detected by real-time PCR at the lowest spike level of 5 mg kg(-1). Further, between 100 and 100,000 mg kg(-1) spiked almond, the method featured good correlation between quantified copy numbers and the amount of spiked almond. Within this range a similar relation between detectable signal and amount of almond was observed for both PCR and ELISA. In contrast to ELISA the Taqman(®) real-time PCR method was highly specific in 59 food items with negligible cross-reactivity for a very limited number of Prunoideae foods. The real-time PCR analysis of 24 retail samples was in concordance with ELISA results: 21% (n=5) contained undeclared almond. This is the first completely disclosed real-time PCR method for a specific and

  3. Design and Validation of an H5 TaqMan Real-Time One-Step Reverse Transcription-PCR and Confirmatory Assays for Diagnosis and Verification of Influenza A Virus H5 Infections in Humans▿

    PubMed Central

    Ellis, Joanna S.; Smith, Joanne W.; Braham, Sharleen; Lock, Matthew; Barlow, Katrina; Zambon, Maria C.

    2007-01-01

    Increasing diversity among influenza H5N1 viruses has resulted in the need for sensitive and specific diagnostic assays, fully validated for the detection of H5 viruses belonging to all hemagglutinin (HA) clades, particularly the recently circulating H5N1 viruses of clade 2. In this report, the development and validation of a real-time, one-step TaqMan reverse transcription-PCR (RT-PCR) assay specific for the detection of influenza A H5 viruses from clades 1, 1′, 2, and 3 is described. The real-time assay for H5 virus was shown to be highly sensitive, detecting H5 virus levels of <1 PFU from each of the HA clades. Specificity of the H5 RT-PCR for influenza A H5 viruses was demonstrated by using influenza A viruses of different subtypes, clinical samples containing influenza A viruses H1N1, H3N2, and H5N1, influenza B viruses, and other respiratory viruses. The usefulness of the inclusion of a distinguishable assay positive control and of confirmatory assays for the laboratory diagnosis and verification of H5 virus infections was demonstrated. A real-time RT-PCR pyrosequencing assay, a restriction enzyme digestion assay, and direct sequencing of the H5 real-time RT-PCR amplicon were validated for the confirmation of H5 detection by the diagnostic real-time assay. The H5 real-time assay was applied to diagnostic testing for suspected cases of influenza A virus H5 infection in the United Kingdom. Influenza A H5 viruses were not detected in the cases analyzed; however, influenza A H3N2 virus was detected in 57% of the suspected cases of H5. The H5 TaqMan real-time RT-PCR and confirmatory assays will be useful tools for the laboratory surveillance and rapid diagnosis of H5 infections in humans. PMID:17473050

  4. Direct detection of circulating free DNA extracted from serum samples of breast cancer using locked nucleic acid molecular beacon.

    PubMed

    Gui, Zhen; Wang, Quanbo; Li, Jinchang; Zhu, Mingchen; Yu, Lili; Xun, Tang; Yan, Feng; Ju, Huangxian

    2016-07-01

    As an emerging noninvasive blood biomarker, circulating free DNA (cfDNA) can be utilized to assess diagnosis, progression and evaluate prognosis of cancer. However, cfDNAs are not "naked", they can be part of complexes, or are bound to the surface of the cells via proteins, which make the detection more challenging. Here, a simple method for the detection of Ubiquitin-like with PHD and ring finger domains 1 (UHRF1) DNA exacted from serum of breast cancer (BC) has been developed using a novel locked nucleic acid molecular beacon (LNA-MB). In order to enhance the stability and detection efficiency of the probe in biofluids, we design a shared-stem molecular beacon containing a 27-mer loop and a 4-mer stem with DNA/LNA alternating bases. The fluorescence is released in the presence of target. The detection procedure is simple and can be completed within 1h. This method shows a sensitive response to UHRF1 DNA with a dynamic range of 3 orders of magnitude. The limit of detection is 11nM (S/N=3) with excellent selectivity. It can discriminate UHRF1 DNA from three-base mismatched DNA with a high specificity. More importantly, this method can distinguish the expression of serum UHRF1 DNA among 5 breast cancer patients and 5 healthy controls. The mentioned superiority may suggest that this assay can be served as a promising noninvasive detection tool for early BC diagnosis and monitoring. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Single Nucleotide rs760370 Polymorphism at the Main Ribavirin Transporter Gene Detection by PCR-RFLP Assay Compared with the TaqMan Assay and Its Relation to Sustained Virological Response in Chronic HCV Patients Treated with Pegylated Interferon-Ribavirin Therapy.

    PubMed

    Fouad, Rabab; Zachariah, Khaled; Khairy, Marwa; Khorshied, Mervat; Ezzat, Wafaa; Sheta, Marwa M; Heiba, Ahmed

    2017-02-01

    Ribavirin clearly plays a role in chronic hepatitis C treatment response. The equilibrative nucleoside transporter-1 codified by SLC29A1 gene has been associated with ribavirin uptake into hepatocytes and erythrocytes. rs760370A>G single nucleotide polymorphism (SNP) at the SLC29A1 gene may have a role in ribavirin-based regimen treatment response. Accuracy of the polymerase-chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay compared with the TaqMan assay for the detection of the SNP rs760370 at the main ribavirin transporter gene and its relation to sustained virological response in chronic hepatitis C virus (HCV) patients treated with pegylated interferon-ribavirin therapy. The study included 100 chronic HCV patients who were treated with pegylated interferon-ribavirin therapy. The patients were categorized according to the treatment response into responders (50 patients) and null responders (50 patients). rs760370 SNP was measured using TaqMan 5-nuclease assay and by the newly developed PCR-based RFLP assay. The overall accuracy of the newly developed PCR-RFLP assay compared with the TaqMan assay for rs760370 polymorphism detection was 100%. Allelic frequencies at rs760370 were as follows: A/A genotype (28%), A/G genotype (58%), and G/G genotype (14%). Treatment response was not significantly related with rs760370 polymorphism (P = 0.5). Ribavirin-induced anemia was good predictor of sustained virological response (P = 0.001), but was not related to rs760370 polymorphism (P = 0.92). PCR-RFLP assay is an accurate, cost-effective method in the detection of rs760370 compared with TaqMan assay. rs760370 SNP cannot serve as predictor of response in chronic HCV patients treated with interferon ribavirin therapy.

  6. Field evaluation of Abbott Real Time HIV-1 Qualitative test for early infant diagnosis using dried blood spots samples in comparison to Roche COBAS Ampliprep/COBAS TaqMan HIV-1 Qual test in Kenya.

    PubMed

    Chang, Joy; Omuomo, Kenneth; Anyango, Emily; Kingwara, Leonard; Basiye, Frank; Morwabe, Alex; Shanmugam, Vedapuri; Nguyen, Shon; Sabatier, Jennifer; Zeh, Clement; Ellenberger, Dennis

    2014-08-01

    Timely diagnosis and treatment of infants infected with HIV are critical for reducing infant mortality. High-throughput automated diagnostic tests like Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Qual Test (Roche CAPCTM Qual) and the Abbott Real Time HIV-1 Qualitative (Abbott Qualitative) can be used to rapidly expand early infant diagnosis testing services. In this study, the performance characteristics of the Abbott Qualitative were evaluated using two hundred dried blood spots (DBS) samples (100 HIV-1 positive and 100 HIV-1 negative) collected from infants attending the antenatal facilities in Kisumu, Kenya. The Abbott Qualitative results were compared to the diagnostic testing completed using the Roche CAPCTM Qual in Kenya. The sensitivity and specificity of the Abbott Qualitative were 99.0% (95% CI: 95.0-100.0) and 100.0% (95% CI: 96.0-100.0), respectively, and the overall reproducibility was 98.0% (95% CI: 86.0-100.0). The limits of detection for the Abbott Qualitative and Roche CAPCTM Qual were 56.5 and 6.9copies/mL at 95% CIs (p=0.005), respectively. The study findings demonstrate that the Abbott Qualitative test is a practical option for timely diagnosis of HIV in infants.

  7. New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters

    PubMed Central

    Fernandes-Monteiro, Alice G; Trindade, Gisela F; Yamamura, Anna MY; Moreira, Otacilio C; de Paula, Vanessa S; Duarte, Ana Cláudia M; Britto, Constança; Lima, Sheila Maria B

    2015-01-01

    The development and production of viral vaccines, in general, involve several steps that need the monitoring of viral load throughout the entire process. Applying a 2-step quantitative reverse transcription real time PCR assay (RT-qPCR), viral load can be measured and monitored in a few hours. In this context, the development, standardization and validation of a RT-qPCR test to quickly and efficiently quantify yellow fever virus (YFV) in all stages of vaccine production are extremely important. To serve this purpose we used a plasmid construction containing the NS5 region from 17DD YFV to generate the standard curve and to evaluate parameters such as linearity, precision and specificity against other flavivirus. Furthermore, we defined the limits of detection as 25 copies/reaction, and quantification as 100 copies/reaction for the test. To ensure the quality of the method, reference controls were established in order to avoid false negative results. The qRT-PCR technique based on the use of TaqMan probes herein standardized proved to be effective for determining yellow fever viral load both in vivo and in vitro, thus becoming a very important tool to assure the quality control for vaccine production and evaluation of viremia after vaccination or YF disease. PMID:26011746

  8. New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters.

    PubMed

    Fernandes-Monteiro, Alice G; Trindade, Gisela F; Yamamura, Anna M Y; Moreira, Otacilio C; de Paula, Vanessa S; Duarte, Ana Cláudia M; Britto, Constança; Lima, Sheila Maria B

    2015-01-01

    The development and production of viral vaccines, in general, involve several steps that need the monitoring of viral load throughout the entire process. Applying a 2-step quantitative reverse transcription real time PCR assay (RT-qPCR), viral load can be measured and monitored in a few hours. In this context, the development, standardization and validation of a RT-qPCR test to quickly and efficiently quantify yellow fever virus (YFV) in all stages of vaccine production are extremely important. To serve this purpose we used a plasmid construction containing the NS5 region from 17DD YFV to generate the standard curve and to evaluate parameters such as linearity, precision and specificity against other flavivirus. Furthermore, we defined the limits of detection as 25 copies/reaction, and quantification as 100 copies/reaction for the test. To ensure the quality of the method, reference controls were established in order to avoid false negative results. The qRT-PCR technique based on the use of TaqMan probes herein standardized proved to be effective for determining yellow fever viral load both in vivo and in vitro, thus becoming a very important tool to assure the quality control for vaccine production and evaluation of viremia after vaccination or YF disease.

  9. Sensitivity and specificity of Cobas TaqMan MTB real-time polymerase chain reaction for culture-proven Mycobacterium tuberculosis: meta-analysis of 26999 specimens from 17 Studies.

    PubMed

    Horita, Nobuyuki; Yamamoto, Masaki; Sato, Takashi; Tsukahara, Toshinori; Nagakura, Hideyuki; Tashiro, Ken; Shibata, Yuji; Watanabe, Hiroki; Nagai, Kenjiro; Nakashima, Kentaro; Ushio, Ryota; Ikeda, Misako; Sakamaki, Kentaro; Yoshiyama, Takashi; Kaneko, Takeshi

    2015-12-09

    Since 2010, studies on the diagnostic accuracy of COBAS TaqMan MTB (CTM) have been frequently reported with an unignorable discrepancy. The key inclusion criterion for this systematic review was original studies that could provide sufficient data for calculating the sensitivity and the specificity of CTM for M tuberculosis (TB) or M tuberculosis complex. The reference test was Mycobacterium culture. We used bivariate model for meta-analyses. Of the 201 candidate articles, we finally identified 17 eligible articles.Concerning the respiratory specimens, 1900 culture positive specimens and 20983 culture negative specimens from 15 studies were assessed. This provided the summary estimate sensitivity of 0.808 (95% CI 0.758-0.850) and the summary estimate specificity of 0.990 (95% CI 0.981-0.994). The area under curve was 0.956. The diagnostic odds ratio was 459 (95% CI 261-805, I(2) 26%). For the smear positive respiratory specimens, the sensitivity was 0.952 (95% CI 0.926-0.969) and the specificity was 0.916 (95% CI 0.797-0.968). For the smear negative respiratory specimens, the sensitivity and the specificity were 0.600 (95% CI 0.459-0.726) and 0.989 (95% CI 0.981-0.993), respectively. The diagnostic accuracy was poorer for the non-respiratory specimens, than for the respiratory specimens, but was acceptable. We believe that the information obtained from this study will aid physicians' decision making.

  10. The development of a real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay using TaqMan technology for the pan detection of bluetongue virus (BTV).

    PubMed

    Mulholland, Catherine; McMenamy, Michael J; Hoffmann, Bernd; Earley, Bernadette; Markey, Bryan; Cassidy, Joseph; Allan, Gordon; Welsh, Michael D; McKillen, John

    2017-03-23

    Bluetongue virus (BTV) is an infectious, non-contagious viral disease of domestic and wild ruminants that is transmitted by adult females of certain Culicoides species. Since 2006, several serotypes including BTV-1, 2, 4, 6, 8, 9 and 16, have spread from the Mediterranean basin into Northern Europe for the first time. BTV-8 in particular, caused a major epidemic in northern Europe. As a result, it is evident that most European countries are at risk of BTV infection. The objective of this study was to develop and validate a real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay based on TaqMan technology for the detection of representative strains of all BTV serotypes. Primers and probes were based on genome segment 10 of the virus, the NS3 gene. The assay was tested for sensitivity, and specificity. The analytical sensitivity of the rRT-PCR assay was 200 copies of RNA per reaction. The assay did not amplify the closely related orbivirus epizootic hemorrhagic disease virus (EHDV) but successfully detected all BTV reference strains including clinical samples from animals experimentally infected with BTV-8. This real time RT-PCR assay offers a sensitive, specific and rapid alternative assay for the pan detection of BTV that could be used as part of a panel of diagnostic assays for the detection of all serotypes of BTV.

  11. Detection of Mycoplasma mycoides subsp. mycoides SC in bronchoalveolar lavage fluids of cows based on a TaqMan real-time PCR discriminating wild type strains from an lppQ− mutant vaccine strain used for DIVA-strategies

    PubMed Central

    Vilei, Edy M.; Frey, Joachim

    2010-01-01

    Contagious bovine pleuropneumonia (CBPP) is the most serious cattle disease in Africa, caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC). CBPP control strategies currently rely on vaccination with a vaccine based on live attenuated strains of the organism. Recently, an lppQ− mutant of the existing vaccine strain T1/44 has been developed (Janis et al., 2008). This T1lppQ− mutant strain is devoid of lipoprotein LppQ, a potential virulence attribute of M. mycoides subsp. mycoides SC. It is designated as a potential live DIVA (Differentiating Infected from Vaccinated Animals) vaccine strain allowing both serological and etiological differentiation. The present paper reports on the validation of a control strategy for CBPP in cattle, whereby a TaqMan real-time PCR based on the lppQ gene has been developed for the direct detection of M. mycoides subsp. mycoides SC in ex vivo bronchoalveolar lavage fluids of cows and for the discrimination of wild type strains from the lppQ− mutant vaccine strain. PMID:20381545

  12. Analysis of TaqMan Array Cards Data by an Assumption-Free Improvement of the maxRatio Algorithm Is More Accurate than the Cycle-Threshold Method

    PubMed Central

    Shain, Eric; Drumright, Lydia; Lillestøl, Reidun; Somasunderam, Donald; Curran, Martin D.

    2016-01-01

    Quantitative PCR diagnostic platforms are moving towards increased sample throughput, with instruments capable of carrying out thousands of reactions at once already in use. The need for a computational tool to reliably assist in the validation of the results is therefore compelling. In the present study, 328 residual clinical samples provided by the Public Health England at Addenbrooke's Hospital (Cambridge, UK) were processed by TaqMan Array Card assay, generating 15 744 reactions from 54 targets. The amplification data were analysed by the conventional cycle-threshold (CT) method and an improvement of the maxRatio (MR) algorithm developed to filter out the reactions with irregular amplification profiles. The reactions were also independently validated by three raters and a consensus was generated from their classification. The inter-rater agreement by Fleiss' kappa was 0.885; the agreement between either CT or MR with the raters gave Fleiss' kappa 0.884 and 0.902, respectively. Based on the consensus classification, the CT and MR methods achieved an assay accuracy of 0.979 and 0.987, respectively. These results suggested that the assumption-free MR algorithm was more reliable than the CT method, with clear advantages for the diagnostic settings. PMID:27828987

  13. Field evaluation of Abbott Real Time HIV-1 Qualitative test for early infant diagnosis using dried blood spots samples in comparison to Roche COBAS Ampliprep/COBAS TaqMan HIV-1 Qual Test in Kenya

    PubMed Central

    Chang, Joy; Omuomo, Kenneth; Anyango, Emily; Kingwara, Leonard; Basiye, Frank; Morwabe, Alex; Shanmugam, Vedapuri; Nguyen, Shon; Sabatier, Jennifer; Zeh, Clement; Ellenberger, Dennis

    2016-01-01

    Timely diagnosis and treatment of infants infected with HIV are critical for reducing infant mortality. High-throughput automated diagnostic tests like Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Qual Test (Roche CAPCTM Qual) and the Abbott Real Time HIV-1 Qualitative (Abbott Qualitative) can be used to rapidly expand early infant diagnosis testing services. In this study, the performance characteristics of the Abbott Qualitative were evaluated using two hundred dried blood spots (DBS) samples (100 HIV-1 positive and 100 HIV-1 negative) collected from infants attending the antenatal facilities in Kisumu, Kenya. The Abbott Qualitative results were compared to the diagnostic testing completed using the Roche CAPCTM Qual in Kenya. The sensitivity and specificity of the Abbott Qualitative were 99.0% (95% CI: 95.0–100.0) and 100.0% (95% CI: 96.0–100.0), respectively, and the overall reproducibility was 98.0% (95% CI: 86.0–100.0). The limits of detection for the Abbott Qualitative and Roche CAPCTM Qual were 56.5 and 6.9 copies/mL at 95% CIs (p = 0.005), respectively. The study findings demonstrate that the Abbott Qualitative test is a practical option for timely diagnosis of HIV in infants. PMID:24726703

  14. An in-house real-time polymerase chain reaction: standardisation and comparison with the Cobas Amplicor HBV monitor and Cobas AmpliPrep/Cobas TaqMan HBV tests for the quantification of hepatitis B virus DNA

    PubMed Central

    Santos, Ana Paula de Torres; Levi, José Eduardo; Lemos, Marcilio Figueiredo; Calux, Samira Julien; Oba, Isabel Takano; Moreira, Regina Célia

    2016-01-01

    This study aimed to standardise an in-house real-time polymerase chain reaction (rtPCR) to allow quantification of hepatitis B virus (HBV) DNA in serum or plasma samples, and to compare this method with two commercial assays, the Cobas Amplicor HBV monitor and the Cobas AmpliPrep/Cobas TaqMan HBV test. Samples from 397 patients from the state of São Paulo were analysed by all three methods. Fifty-two samples were from patients who were human immunodeficiency virus and hepatitis C virus positive, but HBV negative. Genotypes were characterised, and the viral load was measure in each sample. The in-house rtPCR showed an excellent success rate compared with commercial tests; inter-assay and intra-assay coefficients correlated with commercial tests (r = 0.96 and r = 0.913, p < 0.001) and the in-house test showed no genotype-dependent differences in detection and quantification rates. The in-house assay tested in this study could be used for screening and quantifying HBV DNA in order to monitor patients during therapy. PMID:26872342

  15. Application of TaqMan fluorescent probe-based quantitative real-time PCR assay for the environmental survey of Legionella spp. and Legionella pneumophila in drinking water reservoirs in Taiwan.

    PubMed

    Kao, Po-Min; Hsu, Bing-Mu; Hsu, Tsui-Kang; Ji, Wen-Tsai; Huang, Po-Hsiang; Hsueh, Chih-Jen; Chiang, Chuen-Sheue; Huang, Shih-Wei; Huang, Yu-Li

    2014-08-15

    In this study, TaqMan fluorescent quantitative real-time PCR was performed to quantify Legionella species in reservoirs. Water samples were collected from 19 main reservoirs in Taiwan, and 12 (63.2%) were found to contain Legionella spp. The identified species included uncultured Legionella spp., L. pneumophila, L. jordanis, and L. drancourtii. The concentrations of Legionella spp. and L. pneumophila in the water samples were in the range of 1.8×10(2)-2.6×10(3) and 1.6×10(2)-2.4×10(2) cells/L, respectively. The presence and absence of Legionella spp. in the reservoir differed significantly in pH values. These results highlight the importance that L. pneumophila, L. jordanis, and L. drancourtii are potential pathogens in the reservoirs. The presence of L. pneumophila in reservoirs may be a potential public health concern that must be further examined. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Specific TaqMan allelic discrimination assay for rs1477196 and rs9939609 single nucleotide polymorphisms of FTO gene demonstrated that there is no association between these SNPs and risk of breast cancer in Iranian women.

    PubMed

    Mojaver, Mahboobeh; Mokarian, Fariborz; Kazemi, Mohammad; Salehi, Mansoor

    2015-01-01

    Breast cancer (BC), is the most common cancer in women, that is the major cause of cancer-related morbidity and mortality in women. Obesity is considered as a major risk factor for BC that increases both the rate and intensity of the disease. Polymorphisms in FTO gene, a known obesity related gene, is shown to be associated with obesity-related traits as well. The aim of this study was to evaluate the association between previously reported single nucleotide polymorphisms (SNPs) of intron 1of FTO gene, rs1477196 and rs9939609 and risk of BC in a subset of Iranian BC patients. We genotyped 99 cases and 100 controls for the two SNPs of rs9939609 and rs1477196 by TaqMan allelic discrimination assay. For each sample in an allelic discrimination assay, a unique pair of fluorescent dye probe is used. One fluorescent dye probe has a perfect match with the wild type allele and the other fluorescent dye probe is perfectly matched to the mutated allele. Our research has shown that the observed differences between case and control groups in the studied SNPs of FTO gene are not statistically significant (P > 0.05). Our findings suggest that there is no association between rs9939609 and rs1477196 polymorphisms in FTO gene and increase in risk of BC in the studied Iranian population. These results were inconsistent with that of previously reported case-control studies with BC that means presence of these polymorphisms depends on ethnic group.

  17. [Investigation of viral nucleic acids in middle-ear effusion specimens from children with acute otitis media].

    PubMed

    Abu Sitteh, Muhammed H; Sener, Kenan; Yapar, Mehmet; Kiliç, Abdullah; Güney, Cakir; Kubar, Ayhan

    2008-07-01

    Acute otitis media with effusion (OME) is one of the major causes of antibiotic use, indication for operation and hearing loss in children. In two third of the cases the etiologic agents are bacteria. Nonetheless, increasing numbers of reports have implicated viruses as etiologic agents that may have some effect on prognosis of OME. The aim of this study was to investigate the presence of nucleic acids of respiratory syncytial virus (RSV) type A and B, influenza type A virus, adenovirus, cytomegalovirus (CMV), herpes simplex virus type-1 (HSV-1), and enteroviruses in the middle ear effusion specimens from children with otitis media by TaqMan real-time PCR. As a result, 18 of 30 (60%) OME samples were found positive in terms of viral nucleic acids by real-time PCR. RSV-A was detected in nine samples (30%), CMV in 3 (10%) samples and HSV-1 in 1 (3.3%) sample. In five of the samples two viruses were detected in the same sample (three were positive for adenovirus and RSV-A, and two were positive for CMV and RSV-A). Our data have supported the importance of viruses as etiologic agents of OME. Additionally, it was thought that TaqMan real-time PCR may be used as a reliable and rapid method for the detection of viruses in the middle ear effusion samples.

  18. Glucose, amino acids and fatty acids directly regulate ghrelin and NUCB2/nesfatin-1 in the intestine and hepatopancreas of goldfish (Carassius auratus) in vitro.

    PubMed

    Bertucci, Juan Ignacio; Blanco, Ayelén Melisa; Canosa, Luis Fabián; Unniappan, Suraj

    2017-04-01

    Ghrelin and nesfatin-1 are two peptidyl hormones primarily involved in food intake regulation. We previously reported that the amount of dietary carbohydrates, protein and lipids modulates the expression of these peptides in goldfish in vivo. In the present work, we aimed to characterize the effects of single nutrients on ghrelin and nesfatin-1 in the intestine and hepatopancreas. First, immunolocalization of ghrelin and NUCB2/nesfatin-1 in goldfish hepatopancreas cells was studied by immunohistochemistry. Second, the effects of 2 and 4hour-long exposures of cultured intestine and hepatopancreas sections to glucose, l-tryptophan, oleic acid, linolenic acid (LNA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on ghrelin and nesfatin-1 gene and protein expression were studied. Co-localization of ghrelin and NUCB2/nesfatin-1 in the cytoplasm of goldfish hepatocytes was found. Exposure to glucose led to an upregulation of preproghrelin and a downregulation of nucb2/nesfatin-1 in the intestine. l-Tryptophan mainly decreased the expression of both peptides in the intestine and hepatopancreas. Fatty acids, in general, downregulated NUCB2/nesfatin-1 in the intestine, but only the longer and highly unsaturated fatty acids inhibited preproghrelin. EPA exposure led to a decrease in preproghrelin, and an increase in nucb2/nesfatin-1 expression in hepatopancreas after 2h. These results show that macronutrients exert a dose- and time-dependent, direct regulation of ghrelin and nesfatin-1 in the intestine and hepatopancreas, and suggest a role for these hormones in the digestive process and nutrient metabolism.

  19. Purslane Weed (Portulaca oleracea): A Prospective Plant Source of Nutrition, Omega-3 Fatty Acid, and Antioxidant Attributes

    PubMed Central

    Uddin, Md. Kamal; Juraimi, Abdul Shukor; Hossain, Md Sabir; Nahar, Most. Altaf Un; Ali, Md. Eaqub; Rahman, M. M.

    2014-01-01

    Purslane (Portulaca oleracea L.) is an important plant naturally found as a weed in field crops and lawns. Purslane is widely distributed around the globe and is popular as a potherb in many areas of Europe, Asia, and the Mediterranean region. This plant possesses mucilaginous substances which are of medicinal importance. It is a rich source of potassium (494 mg/100 g) followed by magnesium (68 mg/100 g) and calcium (65 mg/100 g) and possesses the potential to be used as vegetable source of omega-3 fatty acid. It is very good source of alpha-linolenic acid (ALA) and gamma-linolenic acid (LNA, 18 : 3 w3) (4 mg/g fresh weight) of any green leafy vegetable. It contained the highest amount (22.2 mg and 130 mg per 100 g of fresh and dry weight, resp.) of alpha-tocopherol and ascorbic acid (26.6 mg and 506 mg per 100 g of fresh and dry weight, resp.). The oxalate content of purslane leaves was reported as 671–869 mg/100 g fresh weight. The antioxidant content and nutritional value of purslane are important for human consumption. It revealed tremendous nutritional potential and has indicated the potential use of this herb for the future. PMID:24683365

  20. Formula feeding potentiates docosahexaenoic and arachidonic acid biosynthesis in term and preterm baboon neonates.

    PubMed

    Sarkadi-Nagy, Eszter; Wijendran, Vasuki; Diau, Guan Yeu; Chao, Angela Chueh; Hsieh, Andrea T; Turpeinen, Anu; Lawrence, Peter; Nathanielsz, Peter W; Brenna, J Thomas

    2004-01-01

    Infant formulas supplemented with docosahexaenoic acid (DHA) and arachidonic acid (ARA) are now available in the United States; however, little is known about the factors that affect biosynthesis. Baboon neonates were assigned to one of four treatments: term, breast-fed; term, formula-fed; preterm (155 of 182 days gestation), formula-fed; and preterm, formula+DHA/ARA-fed. Standard formula had no DHA/ARA; supplemented formula had 0.61%wt DHA (0.3% of calories) and 1.21%wt ARA (0.6% of calories), and baboon breast milk contained 0.68 +/- 0.22%wt DHA and 0.62 +/- 0.12%wt ARA. At 14 days adjusted age, neonates received a combined oral dose of [U-13C]alpha-linolenic acid (LNA*) and [U-13C]linoleic acid (LA*), and tissues were analyzed 14 days after dose. Brain accretion of linolenic acid-derived DHA was approximately 3-fold greater for the formula groups than for the breast-fed group, and dietary DHA partially attenuated excess DHA synthesis among preterms. A similar, significant pattern was found in other organs. Brain linoleic acid-derived ARA accretion was significantly greater in the unsupplemented term group but not in the preterm groups compared with the breast-fed group. These data show that formula potentiates the biosynthesis/accretion of DHA/ARA in term and preterm neonates compared with breast-fed neonates and that the inclusion of DHA/ARA in preterm formula partially restores DHA/ARA biosynthesis to lower, breast-fed levels. Current formula DHA concentrations are inadequate to normalize long-chain polyunsaturated fatty acids synthesis to that of breast-fed levels.

  1. Performance of the New Aptima HCV Quant Dx Assay in Comparison to the Cobas TaqMan HCV2 Test for Use with the High Pure System in Detection and Quantification of Hepatitis C Virus RNA in Plasma or Serum.

    PubMed

    Schalasta, Gunnar; Speicher, Andrea; Börner, Anna; Enders, Martin

    2016-04-01

    Quantitating the level of hepatitis C virus (HCV) RNA is the standard of care for monitoring HCV-infected patients during treatment. The performances of commercially available assays differ for precision, limit of detection, and limit of quantitation (LOQ). Here, we compare the performance of the Hologic Aptima HCV Quant Dx assay (Aptima) to that of the Roche Cobas TaqMan HCV test, version 2.0, using the High Pure system (HPS/CTM), considered a reference assay since it has been used in trials defining clinical decision points in patient care. The assays' performance characteristics were assessed using HCV RNA reference panels and plasma/serum from chronically HCV-infected patients. The agreement between the assays for the 3 reference panels was good, with a difference in quantitation values of <0.5 log. High concordance was demonstrated between the assays for 245 clinical samples (kappa = 0.80; 95% confidence interval [CI], 0.720 to 0.881); however, Aptima detected and/or quantitated 20 samples that HPS/CTM did not detect, while Aptima did not detect 1 sample that was quantitated by HPS/CTM. For the 165 samples quantitated by both assays, the values were highly correlated (R= 0.98;P< 0.0001). The linearity of quantitation from concentrations of 1.4 to 6 log was excellent for both assays for all HCV genotypes (GT) tested (GT 1a, 1b, 2b, and 3a) (R(2)> 0.99). The assays had similar levels of total and intra-assay variability across all genotypes at concentrations from 1,000 to 25 IU/ml. Aptima had a greater analytical sensitivity, quantitating more than 50% of replicates at 25-IU/ml target. Aptima showed performance characteristics comparable to those of HPS/CTM and increased sensitivity, making it suitable for use as a clinical diagnostic tool on the fully automated Panther platform.

  2. Abbott RealTime Hepatitis C Virus (HCV) and Roche Cobas AmpliPrep/Cobas TaqMan HCV Assays for Prediction of Sustained Virological Response to Pegylated Interferon and Ribavirin in Chronic Hepatitis C Patients ▿

    PubMed Central

    Matsuura, Kentaro; Tanaka, Yasuhito; Hasegawa, Izumi; Ohno, Tomoyoshi; Tokuda, Hiroshi; Kurbanov, Fuat; Sugauchi, Fuminaka; Nojiri, Shunsuke; Joh, Takashi; Mizokami, Masashi

    2009-01-01

    Two commercial real-time PCR assays are currently available for sensitive hepatitis C virus (HCV) RNA quantification: the Abbott RealTime HCV assay (ART) and Roche Cobas AmpliPrep/Cobas TaqMan HCV assay (CAP/CTM). We assessed whether the two real-time PCR assays were more effective than Roche Cobas Amplicor HCV Monitor test, v.2.0 (CAM) for prediction of the sustained virological response (SVR) to pegylated interferon (PEG-IFN) plus ribavirin (RBV) in chronic hepatitis C. Sixty patients chronically infected with HCV genotype 1b (37 males and 23 females, 53 ± 12 years of age) were treated with PEG-IFNα2b plus RBV for 48 weeks. Stored specimens at nine time points for each patient (at baseline, on treatment, and 24 weeks after treatment) were tested by the two real-time PCR assays and CAM. Twenty-six (43.3%) patients reached SVR. The positive predictive values (PPVs) for SVR of undetectable HCV RNA at week 12 by CAM, ART, and CAP/CTM were 74.3%, 88.0%, and 95.2%, respectively. An undetectable HCV RNA level by CAM, ART, and CAP/CTM correctly predicted SVR at week 4 in 100%, 100%, and 100% of patients, at weeks 5 to 8 in 91.7%, 100%, and 100% of patients, at weeks 9 to 12 in 55.6%, 75%, and 87.5% of patients, and at weeks 13 to 24 in 0%, 26.7%, and 40% of patients, respectively. Of 16 patients who relapsed after treatment, HCV RNA was detectable in 2 patients at the end of treatment by CAP/CTM but undetectable by ART and CAM. HCV RNA tests using ART and CAP/CTM are considered to be more effective at predicting SVR than CAM, and the PPV for SVR was slightly higher in CAP/CTM than in ART. PMID:19091819

  3. Performance of the New Aptima HCV Quant Dx Assay in Comparison to the Cobas TaqMan HCV2 Test for Use with the High Pure System in Detection and Quantification of Hepatitis C Virus RNA in Plasma or Serum

    PubMed Central

    Speicher, Andrea; Börner, Anna; Enders, Martin

    2016-01-01

    Quantitating the level of hepatitis C virus (HCV) RNA is the standard of care for monitoring HCV-infected patients during treatment. The performances of commercially available assays differ for precision, limit of detection, and limit of quantitation (LOQ). Here, we compare the performance of the Hologic Aptima HCV Quant Dx assay (Aptima) to that of the Roche Cobas TaqMan HCV test, version 2.0, using the High Pure system (HPS/CTM), considered a reference assay since it has been used in trials defining clinical decision points in patient care. The assays' performance characteristics were assessed using HCV RNA reference panels and plasma/serum from chronically HCV-infected patients. The agreement between the assays for the 3 reference panels was good, with a difference in quantitation values of <0.5 log. High concordance was demonstrated between the assays for 245 clinical samples (kappa = 0.80; 95% confidence interval [CI], 0.720 to 0.881); however, Aptima detected and/or quantitated 20 samples that HPS/CTM did not detect, while Aptima did not detect 1 sample that was quantitated by HPS/CTM. For the 165 samples quantitated by both assays, the values were highly correlated (R = 0.98; P < 0.0001). The linearity of quantitation from concentrations of 1.4 to 6 log was excellent for both assays for all HCV genotypes (GT) tested (GT 1a, 1b, 2b, and 3a) (R2 > 0.99). The assays had similar levels of total and intra-assay variability across all genotypes at concentrations from 1,000 to 25 IU/ml. Aptima had a greater analytical sensitivity, quantitating more than 50% of replicates at 25-IU/ml target. Aptima showed performance characteristics comparable to those of HPS/CTM and increased sensitivity, making it suitable for use as a clinical diagnostic tool on the fully automated Panther platform. PMID:26865682

  4. Specific TaqMan allelic discrimination assay for rs1477196 and rs9939609 single nucleotide polymorphisms of FTO gene demonstrated that there is no association between these SNPs and risk of breast cancer in Iranian women

    PubMed Central

    Mojaver, Mahboobeh; Mokarian, Fariborz; Kazemi, Mohammad; Salehi, Mansoor

    2015-01-01

    Background: Breast cancer (BC), is the most common cancer in women, that is the major cause of cancer-related morbidity and mortality in women. Obesity is considered as a major risk factor for BC that increases both the rate and intensity of the disease. Polymorphisms in FTO gene, a known obesity related gene, is shown to be associated with obesity-related traits as well. The aim of this study was to evaluate the association between previously reported single nucleotide polymorphisms (SNPs) of intron 1of FTO gene, rs1477196 and rs9939609 and risk of BC in a subset of Iranian BC patients. Materials and Methods: We genotyped 99 cases and 100 controls for the two SNPs of rs9939609 and rs1477196 by TaqMan allelic discrimination assay. For each sample in an allelic discrimination assay, a unique pair of fluorescent dye probe is used. One fluorescent dye probe has a perfect match with the wild type allele and the other fluorescent dye probe is perfectly matched to the mutated allele. Results: Our research has shown that the observed differences between case and control groups in the studied SNPs of FTO gene are not statistically significant (P > 0.05). Conclusions: Our findings suggest that there is no association between rs9939609 and rs1477196 polymorphisms in FTO gene and increase in risk of BC in the studied Iranian population. These results were inconsistent with that of previously reported case–control studies with BC that means presence of these polymorphisms depends on ethnic group. PMID:26322284

  5. Folic Acid

    MedlinePlus

    Folic acid is used to treat or prevent folic acid deficiency. It is a B-complex vitamin needed by ... Folic acid comes in tablets. It usually is taken once a day. Follow the directions on your prescription label ...

  6. Aspartic acid

    MedlinePlus

    ... also called asparaginic acid. Aspartic acid helps every cell in the body work. It plays a role in: Hormone production and release Normal nervous system function Plant sources of aspartic acid include: Legumes such as ...

  7. Fatty acid concentration, proximate composition, and mineral composition in fishbone flour of Nile Tilapia.

    PubMed

    Petenuci, Maria Eugênia; Stevanato, Flávia Braidoti; Visentainer, Jeane Eliete Laguila; Matsushita, Makoto; Garcia, Edivaldo Egea; de Souza, Nilson Evelázio; Visentainer, Jesui Vergilio

    2008-03-01

    Nile tilapia (Oreochormis niloticus) fishbone is a fish part with unknown composition. After elaboration of flour fishbone of tilapia it was analysede. The results in 100 g of flour were: moisture (14.2%), protein (40.8%), total lipids (25.3%), and ash (18.3%), and mineral (in 100 g) was 2715.9 mg (calcium), 1.3 mg (iron), and 1132.7 mg (phosphorus). A total of 22 fatty acids were detected in fishbone flour total lipids (TL), being the major ones in (g) of total lipids: 16:0 (208.5 mg); 18:1n-9 (344.3 mg); and 18:2n-6 (109.6 mg). The concentration of linolenic acid--LNA (18:3n-3); eicopentaenoic acid--EPA (20:5n-3), and docosahexaenoic acid--DHA (22:6n-3) were (29.9 mg), (3.3 mg), and (12.9 mg), respectively. The content to saturated (SFA) were (296.2 mg), monounsaturated (MUFA) 415.0 mg, and polyunsaturated (PUFA) 175.6 mg. The ratio PUFA:MUFA:SFA was 1:2.4:1.7, and the ratio omega-6/omega-3 fatty acids were 2.8. The last is within the recommended values. The results show low concentrations of omega-3 fatty acids in flour. The value caloric and calcium, iron, phosphorus, and protein content the fishbone flour of tilapia may results a valuable alternative food in the human diet.

  8. Acid Rain

    USGS Publications Warehouse

    Bricker, Owen P.; Rice, Karen C.

    1995-01-01

    Although acid rain is fading as a political issue in the United States and funds for research in this area have largely disappeared, the acidity of rain in the Eastern United States has not changed significantly over the last decade, and it continues to be a serious environmental problem. Acid deposition (commonly called acid rain) is a term applied to all forms of atmospheric deposition of acidic substances - rain, snow, fog, acidic dry particulates, aerosols, and acid-forming gases. Water in the atmosphere reacts with certain atmospheric gases to become acidic. For example, water reacts with carbon dioxide in the atmosphere to produce a solution with a pH of about 5.6. Gases that produce acids in the presence of water in the atmosphere include carbon dioxide (which converts to carbonic acid), oxides of sulfur and nitrogen (which convert to sulfuric and nitric acids}, and hydrogen chloride (which converts to hydrochloric acid). These acid-producing gases are released to the atmosphere through natural processes, such as volcanic emissions, lightning, forest fires, and decay of organic matter. Accordingly, precipitation is slightly acidic, with a pH of 5.0 to 5.7 even in undeveloped areas. In industrialized areas, most of the acid-producing gases are released to the atmosphere from burning fossil fuels. Major emitters of acid-producing gases include power plants, industrial operations, and motor vehicles. Acid-producing gases can be transported through the atmosphere for hundreds of miles before being converted to acids and deposited as acid rain. Because acids tend to build up in the atmosphere between storms, the most acidic rain falls at the beginning of a storm, and as the rain continues, the acids "wash out" of the atmosphere.

  9. A new, multiplex, quantitative real-time polymerase chain reaction system for nucleic acid detection and quantification.

    PubMed

    Liang, Fang; Arora, Neetika; Zhang, Kang Liang; Yeh, David Che Cheng; Lai, Richard; Pearson, Darnley; Barnett, Graeme; Whiley, David; Sloots, Theo; Corrie, Simon R; Barnard, Ross T

    2013-01-01

    Quantitative real-time polymerase chain reaction (qPCR) has emerged as a powerful investigative and diagnostic tool with potential to generate accurate and reproducible results. qPCR can be designed to fulfil the four key aspects required for the detection of nucleic acids: simplicity, speed, sensitivity, and specificity. This chapter reports the development of a novel real-time multiplex quantitative PCR technology, dubbed PrimRglo™, with a potential for high-degree multiplexing. It combines the capacity to simultaneously detect many viruses, bacteria, or nucleic acids, in a single reaction tube, with the ability to quantitate viral or bacterial load. The system utilizes oligonucleotide-tagged PCR primers, along with complementary fluorophore-labelled and quencher-labelled oligonucleotides. The analytic sensitivity of PrimRglo technology was compared with the widely used Taqman(®) and SYBR green detection systems.

  10. Defense Priming and Jasmonates: A Role for Free Fatty Acids in Insect Elicitor-Induced Long Distance Signaling

    PubMed Central

    Li, Ting; Cofer, Tristan; Engelberth, Marie; Engelberth, Jurgen

    2016-01-01

    Green leaf volatiles (GLV) prime plants against insect herbivore attack resulting in stronger and faster signaling by jasmonic acid (JA). In maize this response is specifically linked to insect elicitor (IE)-induced signaling processes, which cause JA accumulation not only around the damage site, but also in distant tissues, presumably through the activation of electrical signals. Here, we present additional data further characterizing these distal signaling events in maize. Also, we describe how exposure to GLV increases free fatty acid (fFA) levels in maize seedlings, but also in other plants, and how increased fFA levels affect IE-induced JA accumulation. Increased fFA, in particular α-linolenic acid (LnA), caused a significant increase in JA accumulation after IE treatment, while JA induced by mechanical wounding (MW) alone was not affected. We also identified treatments that significantly decreased certain fFA level including simulated wind and rain. In such treated plants, IE-induced JA accumulation was significantly reduced when compared to un-moved control plants, while MW-induced JA accumulation was not significantly affected. Since only IE-induced JA accumulation was altered by changes in the fFA composition, we conclude that changing levels of fFA affect primarily IE-induced signaling processes rather than serving as a substrate for JA. PMID:27135225

  11. Acid Rain.

    ERIC Educational Resources Information Center

    Openshaw, Peter

    1987-01-01

    Provides some background information on acid deposition. Includes a historical perspective, describes some effects of acid precipitation, and discusses acid rain in the United Kingdom. Contains several experiments that deal with the effects of acid rain on water quality and soil. (TW)

  12. Acid Rain.

    ERIC Educational Resources Information Center

    Openshaw, Peter

    1987-01-01

    Provides some background information on acid deposition. Includes a historical perspective, describes some effects of acid precipitation, and discusses acid rain in the United Kingdom. Contains several experiments that deal with the effects of acid rain on water quality and soil. (TW)

  13. Development and Validation of LNA-Based Quantitative Real-Time PCR Assays for Detection and Identification of the Root-Knot Nematode Meloidogyne enterolobii in Complex DNA Backgrounds.

    PubMed

    Kiewnick, Sebastian; Frey, Jürg E; Braun-Kiewnick, Andrea

    2015-09-01

    Meloidogyne enterolobii is a quarantine root-knot nematode posing a major threat to agricultural production systems worldwide. It attacks many host plants, including important agricultural crops, ornamentals, and trees. M. enterolobii is a highly virulent and pathogenic root-knot nematode species, able to reproduce on plants resistant to other Meloidogyne spp. Significant crop damage has been reported in Asia, South America, Africa, the United States, France, and greenhouses in Switzerland. To identify potential introduction pathways and ensure appropriate phytosanitary measures and management strategies, accurate detection and identification tools are needed. Therefore, two real-time quantitative polymerase chain reaction (PCR) assays based on the second intergenic spacer region of the ribosomal DNA cistron and the cytochrome oxidase c subunit I (COI) gene using locked nucleic acid probes were developed and validated for fast and reliable detection and identification of M. enterolobii. Analytical specificity was confirmed with 16 M. enterolobii populations, 16 populations of eight closely related Meloidogyne spp., and four species from other nematode genera. Optimizing and testing the assays on two real-time PCR platforms revealed an analytical sensitivity of one juvenile in a background of 1,000 nematodes and the intended limit of detection of one juvenile per 100 ml of soil. Both assays performed equally well, with the COI-based assay showing a slightly better performance concerning detection of M. enterolobii target DNA in complex DNA backgrounds.

  14. Development and evaluation of a two-step multiplex TaqMan real-time PCR assay for detection/quantification of different genospecies of Borrelia burgdorferi sensu lato.

    PubMed

    Nunes, Mónica; Parreira, Ricardo; Carreira, Teresa; Inácio, João; Vieira, Maria Luísa

    2017-09-11

    Nowadays, at least four clinically important B. burgdorferi sensu lato (s.l.) genospecies (B. afzelii, B. garinii, B. burgdorferi sensu stricto (s.s.) and B. lusitaniae) circulate in Portugal. Each genospecies has a different tropism that resuls in a diverse array of clinical manifestations. The standard diagnostic procedure used is normally simple, nevertheless, during the "window-period" phase, in which specific antibodies cannot yet be detected, diagnosis becomes difficult, and calls for reliable, sensitive and specific laboratory methods, such as molecular tests. The aim of this study was to develop and evaluate a multiplex TaqMan real-time PCR assay to infer the presence of B. burgdorferi s.l. genospecies in clinical and vector-derived samples. The assay consists of two steps: (i) a first duplex real-time PCR targeting both flaB of B. burgdorferi s.l., and an internal control (18S rDNA for tick samples or the mammal β-actin gene for clinical samples); and (ii) a second tetraplex real-time PCR targeting the flaB gene of B. afzelii, B. garinii, B. burgdorferi s.s. and B. lusitaniae. The first step revealed a high specificity and sensitivity, allowing the detection of as low as 20 genome equivalents (GE) of B. burgdorferi s.l. from isolated cultures, clinical samples and ticks. The second step revealed high specificity, but a slightly lower sensitivity (2×10(2) GE) for detection of B. afzelii, B. garinii, B. burgdorferi s.s. and B. lusitaniae in purified DNA extracts, and particularly when testing cerebrospinal fluid (CSF) samples. Nonetheless, both real-time PCR protocols were developed to be applied at the beginning of the infection, to improve early diagnosis of Lyme borreliosis (LB), where detection of Borrelia should not rely on the use of CSF samples. The assay here described is of special interest for the analysis of both environmental and clinical samples, being advantageous in the former phase screening of Lyme borreliosis, when the efficiency of

  15. Real-Time TaqMan PCR Assay for the Detection of Heat-Labile and Heat-Stable Enterotoxin Genes in a Geographically Diverse Collection of Enterotoxigenic Escherichia coli Strains and Stool Specimens.

    PubMed

    Pattabiraman, Vaishnavi; Parsons, Michele B; Bopp, Cheryl A

    2016-04-01

    Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrhea in children under the age of 5 years in developing countries and are the leading bacterial agent of traveler's diarrhea in persons traveling to these countries. ETEC strains secrete heat-labile (LT) and/or heat-stable (ST) enterotoxins that induce diarrhea by causing water and electrolyte imbalance. We describe the validation of a real-time TaqMan PCR (RT-PCR) assay to detect LT, ST1a, and ST1b enterotoxin genes in E. coli strains and in stool specimens. We validated LT/ST1b duplex and ST1a single-plex RT-PCR assay using a conventional PCR assay as a gold standard with 188 ETEC strains and 42 non-ETEC strains. We validated LT/ST1b duplex and ST1a single-plex RT-PCR assay in stool specimens (n = 106) using traditional culture as the gold standard. RT- PCR assay sensitivities for LT, ST1a, and ST1b detection in strains were 100%, 100%, and 98%; specificities were 95%, 98%, and 99%, and Pearson correlation coefficient r was 0.9954 between RT-PCR assay and the gold standard. In stool specimens, RT-PCR assay sensitivities for LT, ST1a, and ST1b detection were 97%, 100%, and 97%; and specificities were 99%, 94%, and 97%. Pearson correlation coefficient r was 0.9975 between RT-PCR results in stool specimens and the gold standard. Limits of detection of LT, ST1a, and ST1b by RT-PCR assay were 0.1 to1.0 pg/μL and by conventional PCR assay were 100 to1000 pg/μL. The accuracy, rapidity and sensitivity of this RT-PCR assay is promising for ETEC detection in public health/clinical laboratories and for laboratories in need of an independent method to confirm results of other culture independent diagnostic tests.

  16. Differences between quantification of genotype 3 hepatitis C virus RNA by Versions 1.0 and 2.0 of the COBAS AmpliPrep/COBAS TaqMan HCV Test.

    PubMed

    Pierce, Virginia M; Eversley, Jacqueline S; Tran, Thuy K; Rosenberg, Eric S

    2017-06-27

    Differences between the designs of hepatitis C virus (HCV) viral load assays can result in genotype-related variability in RNA quantification. We tested paired aliquots of plasma specimens from HCV-infected individuals using two versions (v1.0 and v2.0) of the Roche COBAS AmpliPrep/COBAS TaqMan HCV Test (CAP/CTM HCV) and noted variability between results for a subset of specimens; we then sought to determine whether discrepant results were more prevalent among specific HCV genotypes. Archived and prospectively-collected plasma samples from 114 unique patients were tested using CAP/CTM HCV v1.0 and v2.0. The HCV genotype result for each patient was determined by retrospectively reviewing laboratory records. All (46/46) specimens with quantifiable viral loads from patients with genotype 1 or 2 infection had CAP/CTM HCV v1.0 and v2.0 results that were within 0.5 log10 IU/mL; in contrast, only 3/11 (27.3%) from patients with HCV genotype 3 (mean difference, 0.56 log10 IU/mL higher with v2.0) and 0/3 (0%) from patients with HCV genotype 4 (mean difference, 0.91 log10 IU/mL higher with v2.0) had results within 0.5 log10 IU/mL. Among specimens with detectable HCV RNA below the lower limit of quantification with v1.0, greater proportions of genotype 3 (4/7, 57.1%) and genotype 4 (3/4, 75.0%) specimens than genotype 1 or 2 specimens (6/30, 20.0%) had v2.0 results within the quantifiable range. In patients infected with HCV genotype 3, sequential CAP/CTM HCV viral load results should be compared with caution and interpreted in the context of the specific assay version used.

  17. Smallpox and pan-orthopox virus detection by real-time 3'-minor groove binder TaqMan assays on the roche LightCycler and the Cepheid smart Cycler platforms.

    PubMed

    Kulesh, David A; Baker, Robert O; Loveless, Bonnie M; Norwood, David; Zwiers, Susan H; Mucker, Eric; Hartmann, Chris; Herrera, Rafael; Miller, David; Christensen, Deanna; Wasieloski, Leonard P; Huggins, John; Jahrling, Peter B

    2004-02-01

    We designed, optimized, and extensively tested several sensitive and specific real-time PCR assays for rapid detection of both smallpox and pan-orthopox virus DNAs. The assays are based on TaqMan 3'-minor groove binder chemistry and were performed on both the rapid-cycling Roche LightCycler and the Cepheid Smart Cycler platforms. The hemagglutinin (HA) J7R, B9R, and B10R genes were used as targets for the variola virus-specific assays, and the HA and DNA polymerase-E9L genes were used as targets for the pan-orthopox virus assays. The five orthopox virus assays were tested against a panel of orthopox virus DNAs (both genomic and cloned) at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). The results indicated that each assay was capable of detecting both the appropriate cloned gene and genomic DNA. The assays showed no cross-reactivity to the 78 DNAs in the USAMRIID bacterial cross-reactivity panel. The limit of detection (LOD) of each assay was determined to be between 12 and 25 copies of target DNA. The assays were also run against a blind panel of DNAs at the Centers for Disease Control and Prevention (CDC) on both the LightCycler and the Smart Cycler. The panel consisted of eight different variola virus isolates, five non-variola virus orthopox virus isolates, two varicella-zoster virus isolates, and one herpes simplex virus isolate. Each sample was tested in triplicate at 2.5 ng, 25 pg, 250 fg, and 2.5 fg, which represent 1.24 x 10(7), 1.24 x 10(5), 1.24 x 10(3), and 1.24 x 10(1) genome equivalents, respectively. The results indicated that each of the five assays was 100% specific (no false positives) when tested against both the USAMRIID panels and the CDC blind panel. With the CDC blind panel, the LightCycler was capable of detecting 96.2% of the orthopox virus DNAs and 93.8% of the variola virus DNAs. The Smart Cycler was capable of detecting 92.3% of the orthopox virus DNAs and between 75 and 93.8% of the variola virus DNAs

  18. Smallpox and pan-Orthopox Virus Detection by Real-Time 3′-Minor Groove Binder TaqMan Assays on the Roche LightCycler and the Cepheid Smart Cycler Platforms

    PubMed Central

    Kulesh, David A.; Baker, Robert O.; Loveless, Bonnie M.; Norwood, David; Zwiers, Susan H.; Mucker, Eric; Hartmann, Chris; Herrera, Rafael; Miller, David; Christensen, Deanna; Wasieloski, Leonard P.; Huggins, John; Jahrling, Peter B.

    2004-01-01

    We designed, optimized, and extensively tested several sensitive and specific real-time PCR assays for rapid detection of both smallpox and pan-orthopox virus DNAs. The assays are based on TaqMan 3′-minor groove binder chemistry and were performed on both the rapid-cycling Roche LightCycler and the Cepheid Smart Cycler platforms. The hemagglutinin (HA) J7R, B9R, and B10R genes were used as targets for the variola virus-specific assays, and the HA and DNA polymerase-E9L genes were used as targets for the pan-orthopox virus assays. The five orthopox virus assays were tested against a panel of orthopox virus DNAs (both genomic and cloned) at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). The results indicated that each assay was capable of detecting both the appropriate cloned gene and genomic DNA. The assays showed no cross-reactivity to the 78 DNAs in the USAMRIID bacterial cross-reactivity panel. The limit of detection (LOD) of each assay was determined to be between 12 and 25 copies of target DNA. The assays were also run against a blind panel of DNAs at the Centers for Disease Control and Prevention (CDC) on both the LightCycler and the Smart Cycler. The panel consisted of eight different variola virus isolates, five non-variola virus orthopox virus isolates, two varicella-zoster virus isolates, and one herpes simplex virus isolate. Each sample was tested in triplicate at 2.5 ng, 25 pg, 250 fg, and 2.5 fg, which represent 1.24 × 107, 1.24 × 105, 1.24 × 103, and 1.24 × 101 genome equivalents, respectively. The results indicated that each of the five assays was 100% specific (no false positives) when tested against both the USAMRIID panels and the CDC blind panel. With the CDC blind panel, the LightCycler was capable of detecting 96.2% of the orthopox virus DNAs and 93.8% of the variola virus DNAs. The Smart Cycler was capable of detecting 92.3% of the orthopox virus DNAs and between 75 and 93.8% of the variola virus DNAs. However

  19. Systematic evaluation of three microRNA profiling platforms: microarray, beads array, and quantitative real-time PCR array.

    PubMed

    Wang, Bin; Howel, Paul; Bruheim, Skjalg; Ju, Jingfang; Owen, Laurie B; Fodstad, Oystein; Xi, Yaguang

    2011-02-11

    A number of gene-profiling methodologies have been applied to microRNA research. The diversity of the platforms and analytical methods makes the comparison and integration of cross-platform microRNA profiling data challenging. In this study, we systematically analyze three representative microRNA profiling platforms: Locked Nucleic Acid (LNA) microarray, beads array, and TaqMan quantitative real-time PCR Low Density Array (TLDA). The microRNA profiles of 40 human osteosarcoma xenograft samples were generated by LNA array, beads array, and TLDA. Results show that each of the three platforms perform similarly regarding intra-platform reproducibility or reproducibility of data within one platform while LNA array and TLDA had the best inter-platform reproducibility or reproducibility of data across platforms. The endogenous controls/probes contained in each platform have been observed for their stability under different treatments/environments; those included in TLDA have the best performance with minimal coefficients of variation. Importantly, we identify that the proper selection of normalization methods is critical for improving the inter-platform reproducibility, which is evidenced by the application of two non-linear normalization methods (loess and quantile) that substantially elevated the sensitivity and specificity of the statistical data assessment. Each platform is relatively stable in terms of its own microRNA profiling intra-reproducibility; however, the inter-platform reproducibility among different platforms is low. More microRNA specific normalization methods are in demand for cross-platform microRNA microarray data integration and comparison, which will improve the reproducibility and consistency between platforms.

  20. Acid rain

    SciTech Connect

    Stensland, G.J.

    1983-11-01

    A series of definitions for the field of acid rain studies are presented. Protocols for acid rain sampling and monitoring are also presented. A procedure for calculatory precipitation pH is discussed. 11 references, 1 table.

  1. Folic acid

    MedlinePlus

    ... of childhood cancer of the white blood cells. Iron deficiency. Taking folic acid with iron supplements is not ... supplements without folic acid for treating and preventing iron deficiency and anemia caused by too little iron in ...

  2. Aminocaproic Acid

    MedlinePlus

    Aminocaproic acid is used to control bleeding that occurs when blood clots are broken down too quickly. This type ... the baby is ready to be born). Aminocaproic acid is also used to control bleeding in the ...

  3. Obeticholic Acid

    MedlinePlus

    Obeticholic acid is used alone or in combination with ursodiol (Actigall, Urso) to treat primary biliary cholangitis (PBC; a ... were not treated successfully with ursodiol alone. Obeticholic acid is in a class of medications called farnesoid ...

  4. Acid mucopolysaccharides

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003368.htm Acid mucopolysaccharides To use the sharing features on this page, please enable JavaScript. Acid mucopolysaccharides is a test that measures the amount ...

  5. Ethacrynic Acid

    MedlinePlus

    Ethacrynic acid, a 'water pill,' is used to treat swelling and fluid retention caused by various medical problems. It ... Ethacrynic acid comes as a tablet to take by mouth. It is usually taken once or twice a day ...

  6. Ascorbic Acid

    MedlinePlus

    Ascorbic acid is used to prevent and treat scurvy, a disease caused by a lack of vitamin C in ... Ascorbic acid comes in extended-release (long-acting) capsules and tablets, lozenges, syrup, chewable tablets, and liquid drops to ...

  7. Aristolochic Acids

    MedlinePlus

    ... Sciences NIH-HHS www.niehs.nih.gov Aristolochic Acids Key Points Report on Carcinogens Status Known to be human carcinogens Aristolochia Clematitis Aristolochic Acids n Known human carcinogens n Found in certain ...

  8. Amino acids

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/002222.htm Amino acids To use the sharing features on this page, please enable JavaScript. Amino acids are organic compounds that combine to form proteins . ...

  9. Mefenamic Acid

    MedlinePlus

    Mefenamic acid is used to relieve mild to moderate pain, including menstrual pain (pain that happens before or during a menstrual period). Mefenamic acid is in a class of medications called NSAIDs. ...

  10. Comparison of Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 2.0 (CAP/CTM v2.0) with other real-time PCR assays in HIV-1 monitoring and follow-up of low-level viral loads.

    PubMed

    Wojewoda, Christina M; Spahlinger, Timothy; Harmon, Marlene Louise; Schnellinger, Brian; Li, Qing; Dejelo, Corazon; Schmotzer, Christine; Zhou, Lan

    2013-01-01

    Viral load monitoring of HIV-1 has become standard of care in HIV-1 positive patients. In this study, we evaluated the performance characteristics of the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 2.0 (CAP/CTM v2.0) in comparison with Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 1.0 (CAP/CTM v1.0) and Abbott RealTime HIV-1 assay (m2000), with special emphasis on the quantitation of clinically controversial low-level viral loads. The performance characteristics of CAP/CTM v2.0 were confirmed by the validation study. All three assays performed comparably, with Abbott m2000 showing slightly decreased sensitivity for detection of viral loads close to the lower limit of quantitation. Follow-up of patients with low-level viral loads revealed that some of those represent single viral blips; however, a significant portion of these patients have intermittent or persistent low-positive viremia. We conclude that CAP/CTM v2.0 is an accurate and reliable assay for HIV-1 viral load monitoring.

  11. Valproic Acid

    MedlinePlus

    Valproic acid is used alone or with other medications to treat certain types of seizures. Valproic acid is also used to treat mania (episodes of ... to relieve headaches that have already begun. Valproic acid is in a class of medications called anticonvulsants. ...

  12. Locked nucleic acid-inhibitor of miR-205 decreases endometrial cancer cells proliferation in vitro and in vivo

    PubMed Central

    Torres, Anna; Kozak, Joanna; Korolczuk, Agnieszka; Rycak, Dominika; Wdowiak, Paulina; Maciejewski, Ryszard; Torres, Kamil

    2016-01-01

    Pathogenesis of endometrial cancer has been connected with alterations of microRNA expression and in particular miR-205 up–regulation was consistently reported in this carcinoma. Presented study aimed to investigate if inhibition of miR-205 expression using LNA-modified-nucleotide would attenuate endometrial cancer cells proliferation in vitro and in vivo. In the course of the study we found that the proliferation of endometrial cancer cells (HEC-1-B, RL-95, KLE, Ishikawa) transfected with LNA-miR-205-inhibitor and evaluated using real time cell monitoring as well as standard cell proliferation assay, was significantly decreased. Next, LNA-miR-205-inhibitor was used to assess the in vivo effects of miR-205 inhibition of endometrial cancer growth. Cby.Cg-Foxn1/cmdb mice bearing endometrial cancer xenografts were intraperitoneally injected with nine dosages of 25mg/kg of miR-205-LNA-inhibitor or scramble control or phosphatase buffered saline and were observed for 32 days. We found that systemic administration of miR-205-LNA-inhibitor was technically possible, and exerted inhibitory effect on endometrial cancer xenograft growth in vivo with only mild toxic effects in treated animals. In conclusion our results suggest that systemic delivery of miR-205-LNA-inhibitor is feasible, devoid of significant toxicity, and could be a promising treatment strategy for endometrial cancer. Therefore it warrants further studies in other animal models. PMID:27655663

  13. Locked nucleic acid-inhibitor of miR-205 decreases endometrial cancer cells proliferation in vitro and in vivo.

    PubMed

    Torres, Anna; Kozak, Joanna; Korolczuk, Agnieszka; Rycak, Dominika; Wdowiak, Paulina; Maciejewski, Ryszard; Torres, Kamil

    2016-11-08

    Pathogenesis of endometrial cancer has been connected with alterations of microRNA expression and in particular miR-205 up-regulation was consistently reported in this carcinoma. Presented study aimed to investigate if inhibition of miR-205 expression using LNA-modified-nucleotide would attenuate endometrial cancer cells proliferation in vitro and in vivo.In the course of the study we found that the proliferation of endometrial cancer cells (HEC-1-B, RL-95, KLE, Ishikawa) transfected with LNA-miR-205-inhibitor and evaluated using real time cell monitoring as well as standard cell proliferation assay, was significantly decreased. Next, LNA-miR-205-inhibitor was used to assess the in vivo effects of miR-205 inhibition of endometrial cancer growth. Cby.Cg-Foxn1/cmdb mice bearing endometrial cancer xenografts were intraperitoneally injected with nine dosages of 25mg/kg of miR-205-LNA-inhibitor or scramble control or phosphatase buffered saline and were observed for 32 days. We found that systemic administration of miR-205-LNA-inhibitor was technically possible, and exerted inhibitory effect on endometrial cancer xenograft growth in vivo with only mild toxic effects in treated animals.In conclusion our results suggest that systemic delivery of miR-205-LNA-inhibitor is feasible, devoid of significant toxicity, and could be a promising treatment strategy for endometrial cancer. Therefore it warrants further studies in other animal models.

  14. Analytical characteristics and comparative evaluation of Aptima HCV quant Dx assay with the Abbott RealTime HCV assay and Roche COBAS AmpliPrep/COBAS TaqMan HCV quantitative test v2.0.

    PubMed

    Worlock, A; Blair, D; Hunsicker, M; Le-Nguyen, T; Motta, C; Nguyen, C; Papachristou, E; Pham, J; Williams, A; Vi, M; Vinluan, B; Hatzakis, A

    2017-04-04

    The Aptima HCV Quant Dx assay (Aptima assay) is a fully automated quantitative assay on the Panther® system. This assay is intended for confirmation of diagnosis and monitoring of HCV RNA in plasma and serum specimens. The purpose of the testing described in this paper was to evaluate the performance of the Aptima assay. The analytical sensitivity, analytical specificity, precision, and linearity of the Aptima assay were assessed. The performance of the Aptima assay was compared to two commercially available HCV assays; the Abbott RealTime HCV assay (Abbott assay, Abbott Labs Illinois, USA) and the Roche COBAS Ampliprep/COBAS Taqman HCV Quantitative Test v2.0 (Roche Assay, Roche Molecular Systems, Pleasanton CA, USA). The 95% Lower Limit of Detection (LoD) of the assay was determined from dilutions of the 2nd HCV WHO International Standard (NIBSC 96/798 genotype 1) and HCV positive clinical specimens in HCV negative human plasma and serum. Probit analysis was performed to generate the 95% predicted detection limits. The Lower Limit of Quantitation (LLoQ) was established for each genotype by diluting clinical specimens and the 2nd HCV WHO International Standard (NIBSC 96/798 genotype 1) in HCV negative human plasma and serum. Specificity was determined using 200 fresh and 536 frozen HCV RNA negative clinical specimens including 370 plasma specimens and 366 serum specimens. Linearity for genotypes 1 to 6 was established by diluting armored RNA or HCV positive clinical specimens in HCV negative serum or plasma from 8.08 log IU/mL to below 1 log IU/mL. Precision was tested using a 10 member panel made by diluting HCV positive clinical specimens or spiking armored RNA into HCV negative plasma and serum. A method comparison was conducted against the Abbott assay using 1058 clinical specimens and against the Roche assay using 608 clinical specimens from HCV infected patients. In addition, agreement between the Roche assay and the Aptima assay using specimens with low

  15. Evaluation of the performance of Abbott m2000 and Roche COBAS Ampliprep/COBAS Taqman assays for HIV-1 viral load determination using dried blood spots and dried plasma spots in Kenya

    PubMed Central

    Ndiege, Kenneth; Inzaule, Seth; Achieng, Rebecca; Williamson, John; Chih-Wei Chang, Joy; Ellenberger, Dennis; Nkengasong, John

    2017-01-01

    Background Routine HIV viral load testing is not widely accessible in most resource-limited settings, including Kenya. To increase access to viral load testing, alternative sample types like dried blood spots (DBS), which overcome the logistic barriers associated with plasma separation and cold chain shipment need to be considered and evaluated. The current study evaluated matched dried blood spots (DBS) and dried plasma spots (DPS) against plasma using the Abbott M 2000 (Abbott) and Roche Cobas Ampliprep/Cobas TaqMan (CAP/CTM) quantitative viral load assays in western Kenya. Methods Matched plasma DBS and DPS were obtained from 200 HIV-1 infected antiretroviral treatment (ART)-experienced patients attending patient support centers in Western Kenya. Standard quantitative assay performance parameters with accompanying 95% confidence intervals (CI) were assessed at the assays lower detection limit (400cps/ml for CAP/CTM and 550cps/ml for Abbott) using SAS version 9.2. Receiver operating curves (ROC) were further used to assess viral-load thresholds with best assay performance (reference assay CAP/CTM plasma). Results Using the Abbott test, the sensitivity and specificity, respectively, for DPS were (97.3%, [95%CI: 93.2–99.2] and 98.1% [95%CI: 89.7–100]) and those for DBS (93.9% [95%CI: 88.8–97.2] and 88.0% [95%CI: 82.2–92.4]). The correlation and agreement using paired plasma and DPS/DBS were strong, with r2 = 90.5 and rc = 68.1. The Bland-Altman relative percent change was 95.3 for DPS, (95%CI: 90.4–97.7) and 73.6 (95%CI: 51.6–86.5) for DBS. Using the CAP/CTM assay, the sensitivity for DBS was significantly higher compared to DPS (100.0% [95% CI: 97.6–100.0] vs. 94.7% [95%CI: 89.8–97.7]), while the specificity for DBS was lower: 4%, [95% CI: 0.4–13.7] compared to DPS: 94.0%, [95% CI: 83.5–98.7]. When compared under different clinical relevant thresholds, the accuracy for the Abbott assay was 95% at the 1000cps/ml cut-off with a sensitivity and

  16. Fatty acids - trans fatty acids

    USDA-ARS?s Scientific Manuscript database

    The data supporting a negative effect of dietary trans fatty acids on cardiovascular disease risk is consistent. The primary dietary sources of trans fatty acids include partially hydrogenated fat and rudiment fat. The adverse effect of trans fatty acids on plasma lipoprotein profiles is consisten...

  17. Matching technique yields optimum LNA performance. [Low Noise Amplifiers

    NASA Technical Reports Server (NTRS)

    Sifri, J. D.

    1986-01-01

    The present article is concerned with a case in which an optimum noise figure and unconditional stability have been designed into a 2.385-GHz low-noise preamplifier via an unusual method for matching the input with a suspended line. The results obtained with several conventional line-matching techniques were not satisfactory. Attention is given to the minimization of thermal noise, the design procedure, requirements for a high-impedance line, a sampling of four matching networks, the noise figure of the single-line matching network as a function of frequency, and the approaches used to achieve unconditional stability.

  18. Individualized supplementation of folic acid according to polymorphisms of methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR) reduced pregnant complications.

    PubMed

    Li, Xiujuan; Jiang, Jing; Xu, Min; Xu, Mei; Yang, Yan; Lu, Wei; Yu, Xuemei; Ma, Jianlin; Pan, Jiakui

    2015-01-01

    This study aimed to detect the genotype distributions and allele frequencies of methylenetetrahydrofolate reductase (MTHFR) C677T, A1298C and methionine synthase reductase (MTRR) A66G polymorphisms of pregnant women in Jiaodong region in China, and to investigate whether folic acid supplementation affect the pregnancy complications. A total of 7,812 pregnant women from the Jiaodong region in Shandong province in China. By using Taqman-MGB, 2,928 pregnant women (case group) were tested for the genotype distributions and allele frequencies of MTHFR C677T, A1298C and MTRR A66G polymorphisms. Folic acid metabolism ability was ranked at four levels and then pregnant women in different rank group were supplemented with different doses of folic acid. Their pregnancy complications were followed up and compared with 4,884 pregnant women without folic acid supplementation (control group) in the same hospital. The allele frequencies of MTHFR C677T were 49.1 and 50.9%; those of MTHFR A1298C were 80.2 and 19.8%, and those of MTRR A66G were 74.1 and 25.9%. After supplemented with folic acid, the complication rates in different age groups were significantly reduced, especially for gestational diabetes mellitus and hypertension. Periconceptional folic acid supplementation and healthcare following gene polymorphism testing may be a powerful measure to decrease congenital malformations. © 2015 S. Karger AG, Basel.

  19. Acid rain

    SciTech Connect

    White, J.C. )

    1988-01-01

    This book presents the proceedings of the third annual conference sponsored by the Acid Rain Information Clearinghouse (ARIC). Topics covered include: Legal aspects of the source-receptor relationship: an energy perspective; Scientific uncertainty, agency inaction, and the courts; and Acid rain: the emerging legal framework.

  20. Acid rain

    SciTech Connect

    Elsworth, S.

    1985-01-01

    This book was written in a concise and readable style for the lay public. It's purpose was to make the public aware of the damage caused by acid rain and to mobilize public opinion to favor the elimination of the causes of acid rain.

  1. Acid clusters

    SciTech Connect

    Keesee, R.G.; Castleman, A.W. Jr.

    1986-04-01

    Molecular clusters can be considered to be the smallest size range of an aerosol particle size distribution. Nucleation from the gas phase to particles or droplets involves the formation of clusters in the initial stages. Consequently, knowledge of the properties and formation of clusters containing acids contribute to an understanding of acid rain. This paper presents an overview of results obtained in the laboratory on the formation and stability of both neutral and ionized acid clusters. With free jet expansion techniques, the authors have produced clusters of aqueous nitric acid, aqueous hydrochloric acid, aqueous sulfuric acid, acetic acid and aqueous sulfur dioxide. For analogy to buffering, the formation of clusters containing ammonia have also been examined. These have included ammonia with aqueous nitric acid, hydrogen sulfide and sulfur dioxide. The basic experiment involves expansion of vapor through a nozzle, collimation of the jet with a skimmer to form a well-directed molecular beam, and detection of clusters via electron impact ionization and mass spectrometry. Some variations include the introduction of a reactive gas into vacuum near the expansion as described elsewhere and the implementation of an electrostatic quadrupolar field to examine the polarity of the neutral clusters.

  2. Acid rain

    SciTech Connect

    Sweet, W.

    1980-06-20

    Acid precipitation includes not only rain but also acidified snow, hail and frost, as well as sulfur and nitrogen dust. The principal source of acid precipitation is pollution emitted by power plants and smelters. Sulfur and nitrogen compounds contained in the emissions combine with moisture to form droplets with a high acid content - sometimes as acidic as vinegar. When sufficiently concentrated, these acids can kill fish and damage material structures. Under certain circumstances they may reduce crop and forest yields and cause or aggravate respiratory diseases in humans. During the summer, especially, pollutants tend to collect over the Great Lakes in high pressure systems. Since winds typically are westerly and rotate clockwise around high pressure systems, the pollutants gradually are dispersed throughout the eastern part of the continent.

  3. Asparagusic acid.

    PubMed

    Mitchell, Stephen C; Waring, Rosemary H

    2014-01-01

    Asparagusic acid (1,2-dithiolane-4-carboxylic acid) is a simple sulphur-containing 5-membered heterocyclic compound that appears unique to asparagus, though other dithiolane derivatives have been identified in non-food species. This molecule, apparently innocuous toxicologically to man, is the most probable culprit responsible for the curious excretion of odorous urine following asparagus ingestion. The presence of the two adjacent sulphur atoms leads to an enhanced chemical reactivity, endowing it with biological properties including the ability to substitute potentially for α-lipoic acid in α-keto-acid oxidation systems. This brief review collects the scattered data available in the literature concerning asparagusic acid and highlights its properties, intermediary metabolism and exploratory applications.