Radioactive probes for human gene localisation by in situ hybridisation

International Nuclear Information System (INIS)

Fennell, S.J.

1980-07-01

Radioactive probes of high specific activity have been used for human gene localisation on metaphase chromosome preparations. Human 5S ribosomal RNA was used as a model system, as a probe for the localisation of human 5S ribosomal genes. 125 I-labelled mouse 5S ribosomal RNA was used to study the 5S ribosomal gene content and arrangement in families with translocations on the long arm of chromosome 1 close to or containing the 5S ribosomal RNA locus, by in situ hybridisation to human metaphase chromosomes from peripheral blood cultures. This confirmed the chromosomal assignment of 5S ribosomal genes to 1q 42-43. In situ hybridisation probes were also prepared from recombinant plasmids containing Xenopus laevis oocyte 5S or 28S/18S gene sequences to give [ 3 H]-labelled cRNA and [ 3 H]-labelled nick-translated plasmid DNA. Studies on the kinetics of hybridisation of plasmid probes with and without ribosomal gene sequences questioned the role of plasmid DNA for amplification of signal during gene localisation. Gene localisation was obtained with nick-translated plasmid DNA containing the 28S/18S ribosomal DNA insert after short exposure times, but poor results were obtained using a [ 3 H]-labelled cRNA probe transcribed from the plasmid with the 5S gene insert. (author)

LNA probe-based assay for the detection of Tomato black ring virus isolates.

Science.gov (United States)

Hasiów-Jaroszewska, Beata; Rymelska, Natalia; Borodynko, Natasza

2015-02-01

Tomato black ring virus (TBRV) infects a wide range of economically important plant species worldwide. In the present study we developed a locked nucleic acid (LNA) real-time RT-PCR assay for accurate detection of genetically diverse TBRV isolates collected from different hosts. The assay based on the LNA probe has a wide detection range, high sensitivity, stability and amplification efficiency. The assay amplified all tested TBRV isolates, but no signal was observed for the RNA from other nepoviruses and healthy plant species. Under optimum reaction conditions, the detection limit was estimated around 17 copies of the TBRV target region in total RNA. Real-time RT-PCR with the LNA probe described in this paper will serve as a valuable tool for robust, sensitive and reliable detection of TBRV isolates. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparison of two commercial carbapenemase gene confirmatory assays in multiresistant Enterobacteriaceae and Acinetobacter baumannii-complex.

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Rösner, Stephan; Gehlweiler, Kevin; Küsters, Uta; Kolbert, Mathias; Hübner, Kirsten; Pfennigwerth, Niels; Mack, Dietrich

2018-01-01

Multidrug-resistant Gram-negative bacilli (MDR-GNB) producing carbapenemases are increasing at an alarming speed. Rapid confirmation of carbapenemase type will be an important diagnostic step in clinical microbiology laboratories not only to reduce the risk of transmissions but also for optimising antibiotic therapy in the future. We compared diagnostic reliability of two commercially available molecular assays (Check-Direct CPE vs. AID line probe assay) for detection and typing of carbapenemase genes in 80 well-characterized isolates of MDR-GNB. Respective strains were isolated in various clinical specimens at our clinical microbiology laboratory. The reference standard included confirmation of carbapenemase-production at the molecular level at the German National Reference Laboratory for Multidrug-resistant Gram-negative bacteria (Ruhr-University Bochum, Germany). 53 Enterobacteriaceae and 27 members of the A. baumannii-complex were used in this study. The tested assays appeared highly reliable to confirm carbapenemase-producing Enterobacteriaceae (CPE) with respective sensitivities of 97.7%, but are currently unsuitable for analysis of members of the A. baumannii-complex. Both assays are easy to perform and rapid tools for confirmation and typing of the most common carbapenemase genes in Enterobacteriaceae. Implementation should be possible for any clinical microbiology laboratory with Check-Direct CPE being easier to handle and having less technological requirements.

Comparison of two commercial carbapenemase gene confirmatory assays in multiresistant Enterobacteriaceae and Acinetobacter baumannii-complex.

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Stephan Rösner

Full Text Available Multidrug-resistant Gram-negative bacilli (MDR-GNB producing carbapenemases are increasing at an alarming speed. Rapid confirmation of carbapenemase type will be an important diagnostic step in clinical microbiology laboratories not only to reduce the risk of transmissions but also for optimising antibiotic therapy in the future. We compared diagnostic reliability of two commercially available molecular assays (Check-Direct CPE vs. AID line probe assay for detection and typing of carbapenemase genes in 80 well-characterized isolates of MDR-GNB. Respective strains were isolated in various clinical specimens at our clinical microbiology laboratory. The reference standard included confirmation of carbapenemase-production at the molecular level at the German National Reference Laboratory for Multidrug-resistant Gram-negative bacteria (Ruhr-University Bochum, Germany. 53 Enterobacteriaceae and 27 members of the A. baumannii-complex were used in this study. The tested assays appeared highly reliable to confirm carbapenemase-producing Enterobacteriaceae (CPE with respective sensitivities of 97.7%, but are currently unsuitable for analysis of members of the A. baumannii-complex. Both assays are easy to perform and rapid tools for confirmation and typing of the most common carbapenemase genes in Enterobacteriaceae. Implementation should be possible for any clinical microbiology laboratory with Check-Direct CPE being easier to handle and having less technological requirements.

Multi-targeted priming for genome-wide gene expression assays

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Adomas Aleksandra B

2010-08-01

Full Text Available Abstract Background Complementary approaches to assaying global gene expression are needed to assess gene expression in regions that are poorly assayed by current methodologies. A key component of nearly all gene expression assays is the reverse transcription of transcribed sequences that has traditionally been performed by priming the poly-A tails on many of the transcribed genes in eukaryotes with oligo-dT, or by priming RNA indiscriminately with random hexamers. We designed an algorithm to find common sequence motifs that were present within most protein-coding genes of Saccharomyces cerevisiae and of Neurospora crassa, but that were not present within their ribosomal RNA or transfer RNA genes. We then experimentally tested whether degenerately priming these motifs with multi-targeted primers improved the accuracy and completeness of transcriptomic assays. Results We discovered two multi-targeted primers that would prime a preponderance of genes in the genomes of Saccharomyces cerevisiae and Neurospora crassa while avoiding priming ribosomal RNA or transfer RNA. Examining the response of Saccharomyces cerevisiae to nitrogen deficiency and profiling Neurospora crassa early sexual development, we demonstrated that using multi-targeted primers in reverse transcription led to superior performance of microarray profiling and next-generation RNA tag sequencing. Priming with multi-targeted primers in addition to oligo-dT resulted in higher sensitivity, a larger number of well-measured genes and greater power to detect differences in gene expression. Conclusions Our results provide the most complete and detailed expression profiles of the yeast nitrogen starvation response and N. crassa early sexual development to date. Furthermore, our multi-targeting priming methodology for genome-wide gene expression assays provides selective targeting of multiple sequences and counter-selection against undesirable sequences, facilitating a more complete and

Bremsstrahlung-induced highly penetrating probes for nondestructive assay and defect analysis

CERN Document Server

Selim, F A; Harmon, J F; Kwofie, J; Spaulding, R; Erickson, G; Roney, T

2002-01-01

Nondestructive assay and defect analysis probes based on bremsstrahlung-induced processes have been developed to identify elements and probe defects in large volume samples. Bremsstrahlung beams from (electron accelerators) with end-point energies both above and below neutron emission threshold have been used. Below neutron emission threshold these beams (from 6 MeV small pulsed linacs), which exhibit high penetration, create positrons via pair production inside the material and produce X-ray fluorescence (XRF) radiation. Chemical assays of heavy elements in thick samples up to 10 g/cm sup 2 thick are provided by energy dispersive XRF measurements. The pair-produced positrons annihilate within the material, thereby emitting 511 keV gamma radiation. Doppler broadening spectroscopy of the 511 keV radiation can be performed to characterize the material and measure defects in samples of any desired thickness. This technique has successfully measured induced strain due to tensile stress in steel samples of 0.64 cm...

Uropathogenic Escherichia coli virulence genes: invaluable approaches for designing DNA microarray probes.

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Jahandeh, Nadia; Ranjbar, Reza; Behzadi, Payam; Behzadi, Elham

2015-01-01

The pathotypes of uropathogenic Escherichia coli (UPEC) cause different types of urinary tract infections (UTIs). The presence of a wide range of virulence genes in UPEC enables us to design appropriate DNA microarray probes. These probes, which are used in DNA microarray technology, provide us with an accurate and rapid diagnosis and definitive treatment in association with UTIs caused by UPEC pathotypes. The main goal of this article is to introduce the UPEC virulence genes as invaluable approaches for designing DNA microarray probes. Main search engines such as Google Scholar and databases like NCBI were searched to find and study several original pieces of literature, review articles, and DNA gene sequences. In parallel with in silico studies, the experiences of the authors were helpful for selecting appropriate sources and writing this review article. There is a significant variety of virulence genes among UPEC strains. The DNA sequences of virulence genes are fabulous patterns for designing microarray probes. The location of virulence genes and their sequence lengths influence the quality of probes. The use of selected virulence genes for designing microarray probes gives us a wide range of choices from which the best probe candidates can be chosen. DNA microarray technology provides us with an accurate, rapid, cost-effective, sensitive, and specific molecular diagnostic method which is facilitated by designing microarray probes. Via these tools, we are able to have an accurate diagnosis and a definitive treatment regarding UTIs caused by UPEC pathotypes.

Evaluation of Myc Gene Amplification in Prostate Cancer Using a Dual Color Chromogenic in-Situ Hybridization (Dual CISH Assay

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Daniel Lerda

2013-04-01

Full Text Available Objetive: The overall purpose of the study was to demonstrate applicability of the Dako dual-color chromogenic in situ hybridization (CISH assay (DAKO Denmark, Glostrup with respect to fluorescence in situ hybridization (FISH probes MYC-C. Methods: MYC gene amplification by FISH and Dako dual-color CISH Results: The study showed that the dual-color CISH assay can convert Texas red and fluorescein isothiocyanate (FITC signals into chromogenic signals. The dual –color CISH assay was performed on 40 cases of prostate cancer. Amplification was identified in 12 of 40 (30% tumors. No amplification was seen in 28 of 40 (70% tumors. FISH data were available in total of 40 tumors. All tumors showed concordant results between dual-color CISH and FISH for classifying a tumor as MYC amplified or not amplified. Conclusions: We conclude that dual-color Dako CISH assay is an accurate method for determining MYC gene amplification with added advantages that make it a more practically useful method. [J Interdiscipl Histopathol 2013; 1(2.000: 81-84

Gene probes : principles and protocols [Methods in molecular biology, v. 179

National Research Council Canada - National Science Library

Rapley, Ralph; Aquino de Muro, Marilena

2002-01-01

... of labeled DNA has allowed genes to be mapped to single chromosomes and in many cases to a single chromosome band, promoting significant advance in human genome mapping. Gene Probes: Principles and Protocols presents the principles for gene probe design, labeling, detection, target format, and hybridization conditions together with detailed protocols, accom...

A multiplex branched DNA assay for parallel quantitative gene expression profiling.

Science.gov (United States)

Flagella, Michael; Bui, Son; Zheng, Zhi; Nguyen, Cung Tuong; Zhang, Aiguo; Pastor, Larry; Ma, Yunqing; Yang, Wen; Crawford, Kimberly L; McMaster, Gary K; Witney, Frank; Luo, Yuling

2006-05-01

We describe a novel method to quantitatively measure messenger RNA (mRNA) expression of multiple genes directly from crude cell lysates and tissue homogenates without the need for RNA purification or target amplification. The multiplex branched DNA (bDNA) assay adapts the bDNA technology to the Luminex fluorescent bead-based platform through the use of cooperative hybridization, which ensures an exceptionally high degree of assay specificity. Using in vitro transcribed RNA as reference standards, we demonstrated that the assay is highly specific, with cross-reactivity less than 0.2%. We also determined that the assay detection sensitivity is 25,000 RNA transcripts with intra- and interplate coefficients of variance of less than 10% and less than 15%, respectively. Using three 10-gene panels designed to measure proinflammatory and apoptosis responses, we demonstrated sensitive and specific multiplex gene expression profiling directly from cell lysates. The gene expression change data demonstrate a high correlation coefficient (R(2)=0.94) compared with measurements obtained using the single-plex bDNA assay. Thus, the multiplex bDNA assay provides a powerful means to quantify the gene expression profile of a defined set of target genes in large sample populations.

Spectrophotometric, colorimetric and visually detection of Pseudomonas aeruginosa ETA gene based gold nanoparticles DNA probe and endonuclease enzyme

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Amini, Bahram; Kamali, Mehdi; Salouti, Mojtaba; Yaghmaei, Parichehreh

2018-06-01

Colorimetric DNA detection is preferred over other methods for clinical molecular diagnosis because it does not require expensive equipment. In the present study, the colorimetric method based on gold nanoparticles (GNPs) and endonuclease enzyme was used for the detection of P. aeruginosa ETA gene. Firstly, the primers and probe for P. aeruginosa exotoxin A (ETA) gene were designed and checked for specificity by the PCR method. Then, GNPs were synthesized using the citrate reduction method and conjugated with the prepared probe to develop the new nano-biosensor. Next, the extracted target DNA of the bacteria was added to GNP-probe complex to check its efficacy for P. aeruginosa ETA gene diagnosis. A decrease in absorbance was seen when GNP-probe-target DNA cleaved into the small fragments of BamHI endonuclease due to the weakened electrostatic interaction between GNPs and the shortened DNA. The right shift of the absorbance peak from 530 to 562 nm occurred after adding the endonuclease. It was measured using a UV-VIS absorption spectroscopy that indicates the existence of the P. aeruginosa ETA gene. Sensitivity was determined in the presence of different concentrations of target DNA of P. aeruginosa. The results obtained from the optimized conditions showed that the absorbance value has linear correlation with concentration of target DNA (R: 0.9850) in the range of 10-50 ng mL-1 with the limit detection of 9.899 ng mL-1. Thus, the specificity of the new method for detection of P. aeruginosa was established in comparison with other bacteria. Additionally, the designed assay was quantitatively applied to detect the P. aeruginosa ETA gene from 103 to 108 CFU mL-1 in real samples with a detection limit of 320 CFU mL-1.

Development of a multiplex assay for genus- and species-specific detection of Phytophthora based on differences in mitochondrial gene order.

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Bilodeau, Guillaume J; Martin, Frank N; Coffey, Michael D; Blomquist, Cheryl L

2014-07-01

A molecular diagnostic assay for Phytophthora spp. that is specific, sensitive, has both genus- and species-specific detection capabilities multiplexed, and can be used to systematically develop markers for detection of a wide range of species would facilitate research and regulatory efforts. To address this need, a marker system was developed based on the high copy sequences of the mitochondrial DNA utilizing gene orders that were highly conserved in the genus Phytophthora but different in the related genus Pythium and plants to reduce the importance of highly controlled annealing temperatures for specificity. An amplification primer pair designed from conserved regions of the atp9 and nad9 genes produced an amplicon of ≈340 bp specific for the Phytophthora spp. tested. The TaqMan probe for the genus-specific Phytophthora test was designed from a conserved portion of the atp9 gene whereas variable intergenic spacer sequences were used for designing the species-specific TaqMan probes. Specific probes were developed for 13 species and the P. citricola species complex. In silico analysis suggests that species-specific probes could be developed for at least 70 additional described and provisional species; the use of locked nucleic acids in TaqMan probes should expand this list. A second locus spanning three tRNAs (trnM-trnP-trnM) was also evaluated for genus-specific detection capabilities. At 206 bp, it was not as useful for systematic development of a broad range of species-specific probes as the larger 340-bp amplicon. All markers were validated against a test panel that included 87 Phytophthora spp., 14 provisional Phytophthora spp., 29 Pythium spp., 1 Phytopythium sp., and 39 plant species. Species-specific probes were validated further against a range of geographically diverse isolates to ensure uniformity of detection at an intraspecific level, as well as with other species having high levels of sequence similarity to ensure specificity. Both diagnostic

Variations in CCL3L gene cluster sequence and non-specific gene copy numbers

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Edberg Jeffrey C

2010-03-01

Full Text Available Abstract Background Copy number variations (CNVs of the gene CC chemokine ligand 3-like1 (CCL3L1 have been implicated in HIV-1 susceptibility, but the association has been inconsistent. CCL3L1 shares homology with a cluster of genes localized to chromosome 17q12, namely CCL3, CCL3L2, and, CCL3L3. These genes are involved in host defense and inflammatory processes. Several CNV assays have been developed for the CCL3L1 gene. Findings Through pairwise and multiple alignments of these genes, we have shown that the homology between these genes ranges from 50% to 99% in complete gene sequences and from 70-100% in the exonic regions, with CCL3L1 and CCL3L3 being identical. By use of MEGA 4 and BioEdit, we aligned sense primers, anti-sense primers, and probes used in several previously described assays against pre-multiple alignments of all four chemokine genes. Each set of probes and primers aligned and matched with overlapping sequences in at least two of the four genes, indicating that previously utilized RT-PCR based CNV assays are not specific for only CCL3L1. The four available assays measured median copies of 2 and 3-4 in European and African American, respectively. The concordance between the assays ranged from 0.44-0.83 suggesting individual discordant calls and inconsistencies with the assays from the expected gene coverage from the known sequence. Conclusions This indicates that some of the inconsistencies in the association studies could be due to assays that provide heterogenous results. Sequence information to determine CNV of the three genes separately would allow to test whether their association with the pathogenesis of a human disease or phenotype is affected by an individual gene or by a combination of these genes.

Evaluation of 5 ' nuclease assay for detection of Actinobacillus pleuropneumoniae

DEFF Research Database (Denmark)

Angen, Øystein; Jensen, J.; Lavritsen, D. T.

2001-01-01

Sequence detection by the 5' nuclease TaqMan assay uses online detection of internal fluorogenic probes in closed PCR tubes. Primers and probe were chosen from a part of the omlA gene common to all serotypes of Actinobacillus pleuropneumoniae, which gave an amplicon of 92 bp, The test was evaluat...

Mapping of gene transcripts by nuclease protection assays and cDNA primer extension

International Nuclear Information System (INIS)

Calzone, F.J.; Britten, R.J.; Davidson, E.J.

1987-01-01

An important problem often faced in the molecular characterization of genes is the precise mapping of those genomic sequences transcribed into RNA. This requires identification of the genomic site initiating gene transcription, the location of genomic sequences removed from the primary gene transcript during RNA processing, and knowledge of sequences terminating the processed gene transcript. The objective of the protocols described here is the generation of transcription maps utilizing relatively uncharacterized gene fragments. The basic approach is hybridization of a single-stranded DNA probe with cellular RNA, followed by treatment with a single-strand-specific nuclease that does not attack DNA-RNA hybrids, in order to destroy any unreacted probe sequences. Thus the probe sequences included in the hybrid duplexes are protected from nuclease digestion. The sizes of the protected probe fragments determined by gel electrophoresis correspond to the lengths of the hybridized sequence elements

Alternative mapping of probes to genes for Affymetrix chips

DEFF Research Database (Denmark)

Gautier, Laurent; Møller, M.; Friis-Hansen, L.

2004-01-01

transcripts: the NCBI RefSeq database. We also built mappings and used them in place of the original probe to genes associations provided by the manufacturer of the arrays. Results: In a large number of cases, 36%, the probes matching a reference sequence were consistent with the grouping of probes...... by the manufacturer of the chips. For the remaining cases there were discrepancies and we show how that can affect the analysis of data. Conclusions: While the probes on Affymetrix arrays remain the same for several years, the biological knowledge concerning the genomic sequences evolves rapidly. Using up...

Development of a PCR/LDR/flow-through hybridization assay using a capillary tube, probe DNA-immobilized magnetic beads and chemiluminescence detection.

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Hommatsu, Manami; Okahashi, Hisamitsu; Ohta, Keisuke; Tamai, Yusuke; Tsukagoshi, Kazuhiko; Hashimoto, Masahiko

2013-01-01

A polymerase chain reaction (PCR)/ligase detection reaction (LDR)/flow-through hybridization assay using chemiluminescence (CL) detection was developed for analyzing point mutations in gene fragments with high diagnostic value for colorectal cancers. A flow-through hybridization format using a capillary tube, in which probe DNA-immobilized magnetic beads were packed, provided accelerated hybridization kinetics of target DNA (i.e. LDR product) to the probe DNA. Simple fluid manipulations enabled both allele-specific hybridization and the removal of non-specifically bound DNA in the wash step. Furthermore, the use of CL detection greatly simplified the detection scheme, since CL does not require a light source for excitation of the fluorescent dye tags on the LDR products. Preliminary results demonstrated that this analytical system could detect both homozygous and heterozygous mutations, without the expensive instrumentation and cumbersome procedures required by conventional DNA microarray-based methods.

Real-time polymerase chain reaction assay for endogenous reference gene for specific detection and quantification of common wheat-derived DNA (Triticum aestivum L.).

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Vautrin, Sonia; Zhang, David

2007-01-01

A species-specific endogenous reference gene system was developed for polymerase chain reaction (PCR)-based analysis in common wheat (Triticum aestivum L.) by targeting the ALMT1 gene, an aluminium-activated malate transporter. The primers and probe were elaborated for real-time PCR-based qualitative and quantitative assay. The size of amplified product is 95 base pairs. The specificity was assessed on 17 monocot and dicot plant species. The established real-time PCR assay amplified only T. aestivum-derived DNA; no amplification occurred on other phylogenetically related species, including durum wheat (T. durum). The robustness of the system was tested on the DNA of 15 common wheat cultivars using 20 000 genomic copies per PCR the mean cycle threshold (Ct) values of 24.02 +/- 0.251 were obtained. The absolute limits of detection and quantification of the real-time PCR assay were estimated to 2 and 20 haploid genome copies of common wheat, respectively. The linearity was experimentally validated on 2-fold serial dilutions of DNA from 650 to 20 000 haploid genome copies. All these results show that the real-time PCR assay developed on the ALMT1 gene is suitable to be used as an endogenous reference gene for PCR-based specific detection and quantification of T. aestivum-derived DNA in various applications, in particular for the detection and quantification of genetically modified materials in common wheat.

Probing intracellular motor protein activity using an inducible cargo trafficking assay.

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Kapitein, Lukas C; Schlager, Max A; van der Zwan, Wouter A; Wulf, Phebe S; Keijzer, Nanda; Hoogenraad, Casper C

2010-10-06

Although purified cytoskeletal motor proteins have been studied extensively with the use of in vitro approaches, a generic approach to selectively probe actin and microtubule-based motor protein activity inside living cells is lacking. To examine specific motor activity inside living cells, we utilized the FKBP-rapalog-FRB heterodimerization system to develop an in vivo peroxisomal trafficking assay that allows inducible recruitment of exogenous and endogenous kinesin, dynein, and myosin motors to drive specific cargo transport. We demonstrate that cargo rapidly redistributes with distinct dynamics for each respective motor, and that combined (antagonistic) actions of more complex motor combinations can also be probed. Of importance, robust cargo redistribution is readily achieved by one type of motor protein and does not require the presence of opposite-polarity motors. Simultaneous live-cell imaging of microtubules and kinesin or dynein-propelled peroxisomes, combined with high-resolution particle tracking, revealed that peroxisomes frequently pause at microtubule intersections. Titration and washout experiments furthermore revealed that motor recruitment by rapalog-induced heterodimerization is dose-dependent but irreversible. Our assay directly demonstrates that robust cargo motility does not require the presence of opposite-polarity motors, and can therefore be used to characterize the motile properties of specific types of motor proteins. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A minor groove binder probe real-time PCR assay for discrimination between type 2-based vaccines and field strains of canine parvovirus.

Science.gov (United States)

Decaro, Nicola; Elia, Gabriella; Desario, Costantina; Roperto, Sante; Martella, Vito; Campolo, Marco; Lorusso, Alessio; Cavalli, Alessandra; Buonavoglia, Canio

2006-09-01

A minor groove binder (MGB) probe assay was developed to discriminate between type 2-based vaccines and field strains of canine parvovirus (CPV). Considering that most of the CPV vaccines contain the old type 2, no longer circulating in canine population, two MGB probes specific for CPV-2 and the antigenic variants (types 2a, 2b and 2c), respectively, were labeled with different fluorophores. The MGB probe assay was able to discriminate correctly between the old type and the variants, with a detection limit of 10(1) DNA copies and a good reproducibility. Quantitation of the viral DNA loads was accurate, as demonstrated by comparing the CPV DNA titres to those calculated by means of the TaqMan assay recognising all CPV types. This assay will ensure resolution of most diagnostic problems in dogs showing CPV disease shortly after CPV vaccination, although it does not discriminate between field strains and type 2b-based vaccines, recently licensed to market in some countries.

Micro-fluidic module for blood cell separation for gene expression radiobiological assays

International Nuclear Information System (INIS)

Brengues, Muriel; Gu, Jian; Zenhausern, Frederic

2015-01-01

Advances in molecular techniques have improved discovery of biomarkers associated with radiation exposure. Gene expression techniques have been demonstrated as effective tools for biodosimetry, and different assay platforms with different chemistries are now available. One of the main challenges is to integrate the sample preparation processing of these assays into micro-fluidic platforms to be fully automated for point-of-care medical countermeasures in the case of a radiological event. Most of these assays follow the same workflow processing that comprises first the collection of blood samples followed by cellular and molecular sample preparation. The sample preparation is based on the specific reagents of the assay system and depends also on the different subsets of cells population and the type of biomarkers of interest. In this article, the authors present a module for isolation of white blood cells from peripheral blood as a prerequisite for automation of gene expression assays on a micro-fluidic cartridge. For each sample condition, the gene expression platform can be adapted to suit the requirements of the selected assay chemistry (authors)

Cloned polynucleotide and synthetic oligonucleotide probes used in colony hybridization are equally efficient in the identification of enterotoxigenic Escherichia coli

International Nuclear Information System (INIS)

Sommerfelt, H.; Kalland, K.H.; Raj, P.; Moseley, S.L.; Bhan, M.K.; Bjorvatn, B.

1988-01-01

Restriction endonuclease-generated polynucleotide and synthetically produced oligonucleotide gene probes used in colony hybridization assays proved to be efficient for the detection and differentiation of enterotoxigenic Escherichia coli. To compare their relative efficiencies, these two sets of probes were radiolabeled with 32 P and were applied to 74 strains of E. coli with known enterotoxin profiles and to 156 previously unexamined E. coli isolates. The enterotoxigenic bacteria Vibrio cholerae O1, Vibrio cholerae non-O1 (NAG), Yersinia enterocolitica, and E. coli harboring the plasmid vectors of the polynucleotide gene probes were examined for further evaluation of probe specificity. The two classes of probes showed a perfect concordance in their specific detection and differentiation of enterotoxigenic E. coli. In the analysis of six strains, the signal strength on autoradiography after hybridization with oligonucleotides was weaker than that obtained after hybridization with polynucleotide probes. The probes did not hybridize with DNA from V. cholerae O1, V. cholerae non-O1 (NAG), or Y. enterocolitica. The strains of E. coli harboring the plasmid vectors of the polynucleotide gene probes were, likewise, negative in the hybridization assays

Cloned polynucleotide and synthetic oligonucleotide probes used in colony hybridization are equally efficient in the identification of enterotoxigenic Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Sommerfelt, H.; Kalland, K.H.; Raj, P.; Moseley, S.L.; Bhan, M.K.; Bjorvatn, B.

1988-11-01

Restriction endonuclease-generated polynucleotide and synthetically produced oligonucleotide gene probes used in colony hybridization assays proved to be efficient for the detection and differentiation of enterotoxigenic Escherichia coli. To compare their relative efficiencies, these two sets of probes were radiolabeled with /sup 32/P and were applied to 74 strains of E. coli with known enterotoxin profiles and to 156 previously unexamined E. coli isolates. The enterotoxigenic bacteria Vibrio cholerae O1, Vibrio cholerae non-O1 (NAG), Yersinia enterocolitica, and E. coli harboring the plasmid vectors of the polynucleotide gene probes were examined for further evaluation of probe specificity. The two classes of probes showed a perfect concordance in their specific detection and differentiation of enterotoxigenic E. coli. In the analysis of six strains, the signal strength on autoradiography after hybridization with oligonucleotides was weaker than that obtained after hybridization with polynucleotide probes. The probes did not hybridize with DNA from V. cholerae O1, V. cholerae non-O1 (NAG), or Y. enterocolitica. The strains of E. coli harboring the plasmid vectors of the polynucleotide gene probes were, likewise, negative in the hybridization assays.

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

Directory of Open Access Journals (Sweden)

Lena Poulsen

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

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

Science.gov (United States)

Ogrean, Christy; Jackson, Ben; Covino, James

2010-01-01

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to >98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts. Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process. Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method. PMID:20567213

Specific and straightforward molecular investigation of β-thalassemia mutations in the Malaysian Malays and Chinese using direct TaqMan genotyping assays.

Science.gov (United States)

Kho, S L; Chua, K H; George, E; Tan, J A M A

2013-07-15

Beta-thalassemia is a life-threatening inherited blood disorder. Rapid characterization of β-globin gene mutations is necessary because of the high frequency of Malaysian β-thalassemia carriers. A combination real-time polymerase chain reaction genotyping assay using TaqMan probes was developed to confirm β-globin gene mutations. In this study, primers and probes were designed to specifically identify 8 common β-thalassemia mutations in the Malaysian Malay and Chinese ethnic groups using the Primer Express software. "Blind tests" using DNA samples from healthy individuals and β-thalassemia patients with different genotypes were performed to determine the specificity and sensitivity of this newly designed assay. Our results showed 100% sensitivity and specificity for this novel assay. In conclusion, the TaqMan genotyping assay is a straightforward assay that allows detection of β-globin gene mutations in less than 40 min. The simplicity and reproducibility of the TaqMan genotyping assay permit its use in laboratories as a rapid and cost-effective diagnostic tool for confirmation of common β-thalassemia mutations in Malaysia.

Development of a duplex real-time RT-qPCR assay to monitor genome replication, gene expression and gene insert stability during in vivo replication of a prototype live attenuated canine distemper virus vector encoding SIV gag.

Science.gov (United States)

Coleman, John W; Wright, Kevin J; Wallace, Olivia L; Sharma, Palka; Arendt, Heather; Martinez, Jennifer; DeStefano, Joanne; Zamb, Timothy P; Zhang, Xinsheng; Parks, Christopher L

2015-03-01

Advancement of new vaccines based on live viral vectors requires sensitive assays to analyze in vivo replication, gene expression and genetic stability. In this study, attenuated canine distemper virus (CDV) was used as a vaccine delivery vector and duplex 2-step quantitative real-time RT-PCR (RT-qPCR) assays specific for genomic RNA (gRNA) or mRNA have been developed that concurrently quantify coding sequences for the CDV nucleocapsid protein (N) and a foreign vaccine antigen (SIV Gag). These amplicons, which had detection limits of about 10 copies per PCR reaction, were used to show that abdominal cavity lymphoid tissues were a primary site of CDV vector replication in infected ferrets, and importantly, CDV gRNA or mRNA was undetectable in brain tissue. In addition, the gRNA duplex assay was adapted for monitoring foreign gene insert genetic stability during in vivo replication by analyzing the ratio of CDV N and SIV gag genomic RNA copies over the course of vector infection. This measurement was found to be a sensitive probe for assessing the in vivo genetic stability of the foreign gene insert. Copyright © 2014 Elsevier B.V. All rights reserved.

GeneChip microarrays-signal intensities, RNA concentrations and probe sequences

International Nuclear Information System (INIS)

Binder, Hans; Preibisch, Stephan

2006-01-01

GeneChip microarrays consist of hundreds of thousands of oligonucleotide probes. The transformation of their signal intensities into RNA transcript concentrations requires the knowledge of the response function of the measuring device. We analysed the 'apparatus' function of perfect match (PM) and mismatched (MM) oligonucleotide probes of GeneChip microarrays after changes of the target concentration using the results of a spiked-in experiment. In agreement with previous studies we found that a competitive two-species Langmuir-adsorption model describes the probe intensities well. Each PM and MM probe is characterized by two hybridization constants which specify the propensity of the probe to bind specific and non-specific transcripts. The affinity for non-specific hybridization is on average equal for PM and MM. The purine-pyrimidine asymmetry of base pair interaction strengths, however, causes a characteristic PM-MM intensity difference, the sign of which depends on the middle base of the probe. The affinity for specific hybridization of the PM exceeds that of the MM on average by nearly one order of magnitude because the central mismatched base only weakly contributes to the stability of the probe/target duplexes. For the first time we differentiate between the free energy parameters related to the 64 possible middle-triples of DNA/RNA oligomer duplexes with a central Watson-Crick pairing and a central mismatched pairing. Both the PM and MM probes respond to the concentration of specific transcripts, which can be estimated from the PM and MM probe intensities using the Langmuir-model. The analysis of the PM-MM intensity difference provides at least no loss of accuracy and precision of the estimated concentration compared with the PM-only estimates which in turn outperform the MM-only estimates. The results show that the processing of the PM-MM intensity difference requires the consideration of a background term due to non-specific hybridization, which is

Probe colorimeter for quantitating enzyme-linked immunosorbent assays and other colorimetric assays performed with microplates.

Science.gov (United States)

Ackerman, S B; Kelley, E A

1983-03-01

The performance of a fiberoptic probe colorimeter (model PC800; Brinkmann Instruments, Inc., Westbury, N.Y.) for quantitating enzymatic or colorimetric assays in 96-well microtiter plates was compared with the performances of a spectrophotometer (model 240; Gilford Instrument Laboratories, Inc., Oberlin, Ohio) and a commercially available enzyme immunoassay reader (model MR590; Dynatech Laboratories, Inc., Alexandria, Va.). Alkaline phosphatase-p-nitrophenyl phosphate in 3 M NaOH was used as the chromophore source. Six types of plates were evaluated for use with the probe colorimeter; they generated reproducibility values (100% coefficient of variation) ranging from 91 to 98% when one individual made 24 independent measurements on the same dilution of chromophore on each plate. Eleven individuals each performed 24 measurements with the colorimeter on either a visually light (absorbance of 0.10 at 420 nm) or a dark (absorbance of 0.80 at 420 nm) dilution of chromophore; reproducibilities averaged 87% for the light dilution and 97% for the dark dilution. When one individual measured the same chromophore sample at least 20 times in the colorimeter, in the spectrophotometer or in the enzyme immunoassay reader, reproducibility for each instrument was greater than 99%. Measurements of a dilution series of chromophore in a fixed volume indicated that the optical responses of each instrument were linear in a range of 0.05 to 1.10 absorbance units.

Time-gated luminescence assay using nonmetal probes for determination of protein kinase activity-based disease markers.

Science.gov (United States)

Kasari, Marje; Padrik, Peeter; Vaasa, Angela; Saar, Kristi; Leppik, Krista; Soplepmann, Jaan; Uri, Asko

2012-03-15

A novel nonmetal optical probe ARC-1063 whose long-lifetime luminescence is induced by association with the target protein kinase is used for the measurement of the concentration of catalytic subunit of protein kinase A (PKAc) in complicated biological solutions. High affinity (K(D) = 10 pM toward PKAc) and unique optical properties of the probe enable its application for the measurement of picomolar concentrations of PKAc in the presence of high concentrations of other proteins. The described assay is applicable in the high-throughput format with the instrument setups designed for lanthanide-based time-gated (time-resolved) luminescence methods. The assay is used for demonstration that extracellular PKAc (ECPKA) is present in plasma samples of all healthy persons and cancer patients but great care must be taken for procedures of treatment of blood samples to avoid disruption, damage, or activation of platelets in the course of plasma (or serum) preparation and conservation. Copyright © 2012 Elsevier Inc. All rights reserved.

Rapid identification of carbapenemase genes in gram-negative bacteria with an oligonucleotide microarray-based assay.

Directory of Open Access Journals (Sweden)

Sascha D Braun

Full Text Available Rapid molecular identification of carbapenemase genes in Gram-negative bacteria is crucial for infection control and prevention, surveillance and for epidemiological purposes. Furthermore, it may have a significant impact upon determining the appropriate initial treatment and greatly benefit for critically ill patients. A novel oligonucleotide microarray-based assay was developed to simultaneously detect genes encoding clinically important carbapenemases as well as selected extended (ESBL and narrow spectrum (NSBL beta-lactamases directly from clonal culture material within few hours. Additionally, a panel of species specific markers was included to identify Escherichia coli, Pseudomonas aeruginosa, Citrobacter freundii/braakii, Klebsiella pneumoniae and Acinetobacter baumannii. The assay was tested using a panel of 117 isolates collected from urinary, blood and stool samples. For these isolates, phenotypic identifications and susceptibility tests were available. An independent detection of carbapenemase, ESBL and NSBL genes was carried out by various external reference laboratories using PCR methods. In direct comparison, the microarray correctly identified 98.2% of the covered carbapenemase genes. This included blaVIM (13 out of 13, blaGIM (2/2, blaKPC (27/27, blaNDM (5/5, blaIMP-2/4/7/8/13/14/15/16/31 (10/10, blaOXA-23 (12/13, blaOXA-40-group (7/7, blaOXA-48-group (32/33, blaOXA-51 (1/1 and blaOXA-58 (1/1. Furthermore, the test correctly identified additional beta-lactamases [blaOXA-1 (16/16, blaOXA-2 (4/4, blaOXA-9 (33/33, OXA-10 (3/3, blaOXA-51 (25/25, blaOXA-58 (2/2, CTX-M1/M15 (17/17 and blaVIM (1/1]. In direct comparison to phenotypical identification obtained by VITEK or MALDI-TOF systems, 114 of 117 (97.4% isolates, including Acinetobacter baumannii (28/28, Enterobacter spec. (5/5, Escherichia coli (4/4, Klebsiella pneumoniae (62/63, Klebsiella oxytoca (0/2, Pseudomonas aeruginosa (12/12, Citrobacter freundii (1/1 and Citrobacter

A probe-based qRT-PCR method to profile immunological gene expression in blood of captive beluga whales (Delphinapterus leucas

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Ming-An Tsai

2017-09-01

Full Text Available Cytokines are fundamental for a functioning immune system, and thus potentially serve as important indicators of animal health. Quantitation of mRNA using quantitative reverse transcription polymerase chain reaction (qRT-PCR is an established immunological technique. It is particularly suitable for detecting the expression of proteins against which monoclonal antibodies are not available. In this study, we developed a probe-based quantitative gene expression assay for immunological assessment of captive beluga whales (Delphinapterus leucas that is one of the most common cetacean species on display in aquariums worldwide. Six immunologically relevant genes (IL-2Rα, -4, -10, -12, TNFα, and IFNγ were selected for analysis, and two validated housekeeping genes (PGK1 and RPL4 with stable expression were used as reference genes. Sixteen blood samples were obtained from four animals with different health conditions and stored in RNAlater™ solution. These samples were used for RNA extraction followed by qRT-PCR analysis. Analysis of gene transcripts was performed by relative quantitation using the comparative Cq method with the integration of amplification efficiency and two reference genes. The expression levels of each gene in the samples from clinically healthy animals were normally distributed. Transcript outliers for IL-2Rα, IL-4, IL-12, TNFα, and IFNγ were noticed in four samples collected from two clinically unhealthy animals. This assay has the potential to identify immune system deviation from normal state, which is caused by health problems. Furthermore, knowing the immune status of captive cetaceans could help both trainers and veterinarians in implementing preventive approaches prior to disease onset.

Serotype determination of Salmonella by xTAG assay.

Science.gov (United States)

Zheng, Zhibei; Zheng, Wei; Wang, Haoqiu; Pan, Jincao; Pu, Xiaoying

2017-10-01

Currently, no protocols or commercial kits are available to determine the serotypes of Salmonella by using Luminex MAGPIX®. In this study, an xTAG assay for serotype determination of Salmonella suitable for Luminex MAGPIX® is described and 228 Salmonella isolates were serotype determined by this xTAG assay. The xTAG assay consists of two steps: 1) Multiplex PCR to amplify simultaneously O, H and Vi antigen genes of Salmonella, and 2) Magplex-TAG™ microsphere hybridization to identify accurately the specific PCR products of different antigens. Compared with the serotyping results of traditional serum agglutination test, the sensitivity and specificity of the xTAG assay were 95.1% and 100%, respectively. The agreement rate of these two assays was 95.2%. Compared with Luminex xMAP® Salmonella Serotyping Assay (SSA) kit, the advantages of this xTAG assay are: First, the magnetic beads make it applicable to both the Luminex®100/200™ and MAGPIX® systems. Second, only primers rather than both primers and probes are needed in the xTAG assay, and the process of coupling antigen-specific oligonucleotide probes to beads is circumvented, which make the xTAG assay convenient to be utilized by other laboratories. The xTAG assay may serve as a rapid alternative or complementary method for traditional Salmonella serotyping tests, especially for laboratories that utilize the MAGPIX® systems. Copyright © 2017 Elsevier B.V. All rights reserved.

A general framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina.

Science.gov (United States)

Bidard, Frédérique; Imbeaud, Sandrine; Reymond, Nancie; Lespinet, Olivier; Silar, Philippe; Clavé, Corinne; Delacroix, Hervé; Berteaux-Lecellier, Véronique; Debuchy, Robert

2010-06-18

The development of new microarray technologies makes custom long oligonucleotide arrays affordable for many experimental applications, notably gene expression analyses. Reliable results depend on probe design quality and selection. Probe design strategy should cope with the limited accuracy of de novo gene prediction programs, and annotation up-dating. We present a novel in silico procedure which addresses these issues and includes experimental screening, as an empirical approach is the best strategy to identify optimal probes in the in silico outcome. We used four criteria for in silico probe selection: cross-hybridization, hairpin stability, probe location relative to coding sequence end and intron position. This latter criterion is critical when exon-intron gene structure predictions for intron-rich genes are inaccurate. For each coding sequence (CDS), we selected a sub-set of four probes. These probes were included in a test microarray, which was used to evaluate the hybridization behavior of each probe. The best probe for each CDS was selected according to three experimental criteria: signal-to-noise ratio, signal reproducibility, and representative signal intensities. This procedure was applied for the development of a gene expression Agilent platform for the filamentous fungus Podospora anserina and the selection of a single 60-mer probe for each of the 10,556 P. anserina CDS. A reliable gene expression microarray version based on the Agilent 44K platform was developed with four spot replicates of each probe to increase statistical significance of analysis.

A general framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina

Directory of Open Access Journals (Sweden)

Bidard Frédérique

2010-06-01

Full Text Available Abstract Background The development of new microarray technologies makes custom long oligonucleotide arrays affordable for many experimental applications, notably gene expression analyses. Reliable results depend on probe design quality and selection. Probe design strategy should cope with the limited accuracy of de novo gene prediction programs, and annotation up-dating. We present a novel in silico procedure which addresses these issues and includes experimental screening, as an empirical approach is the best strategy to identify optimal probes in the in silico outcome. Findings We used four criteria for in silico probe selection: cross-hybridization, hairpin stability, probe location relative to coding sequence end and intron position. This latter criterion is critical when exon-intron gene structure predictions for intron-rich genes are inaccurate. For each coding sequence (CDS, we selected a sub-set of four probes. These probes were included in a test microarray, which was used to evaluate the hybridization behavior of each probe. The best probe for each CDS was selected according to three experimental criteria: signal-to-noise ratio, signal reproducibility, and representative signal intensities. This procedure was applied for the development of a gene expression Agilent platform for the filamentous fungus Podospora anserina and the selection of a single 60-mer probe for each of the 10,556 P. anserina CDS. Conclusions A reliable gene expression microarray version based on the Agilent 44K platform was developed with four spot replicates of each probe to increase statistical significance of analysis.

Interphase FISH detection of BCL2 rearrangement in follicular lymphoma using breakpoint-flanking probes

NARCIS (Netherlands)

Vaandrager, J W; Schuuring, E; Raap, T; Philippo, K; Kleiverda, K; Kluin, P

Rearrangement of the BCL2 gene is an important parameter for the differential diagnosis of non-Hodgkin lymphomas. Although a relatively large proportion of breakpoints is clustered, many are missed by standard PCR. A FISH assay is therefore desired. Up to now, a lack of probes flanking the BCL2 gene

Application of a 5 ' nuclease assay for detection of Lawsonia intracellularis in fecal samples from pigs

DEFF Research Database (Denmark)

Lindecrona, R. H.; Jensen, Tim Kåre; Andersen, P. H.

2002-01-01

A 5' nuclease assay was developed to detect Lawsonia intracellularis in porcine fecal samples. The specific probe and primers were chosen by using the 16S ribosomal DNA gene as a target. The 5' nuclease assay was used with a total of 204 clinical samples, and the results were compared to those of...

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.

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Lingxiang Zhu

Full Text Available 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.

Mechanism Profiling of Hepatotoxicity Caused by Oxidative Stress Using Antioxidant Response Element Reporter Gene Assay Models and Big Data.

Science.gov (United States)

Kim, Marlene Thai; Huang, Ruili; Sedykh, Alexander; Wang, Wenyi; Xia, Menghang; Zhu, Hao

2016-05-01

Hepatotoxicity accounts for a substantial number of drugs being withdrawn from the market. Using traditional animal models to detect hepatotoxicity is expensive and time-consuming. Alternative in vitro methods, in particular cell-based high-throughput screening (HTS) studies, have provided the research community with a large amount of data from toxicity assays. Among the various assays used to screen potential toxicants is the antioxidant response element beta lactamase reporter gene assay (ARE-bla), which identifies chemicals that have the potential to induce oxidative stress and was used to test > 10,000 compounds from the Tox21 program. The ARE-bla computational model and HTS data from a big data source (PubChem) were used to profile environmental and pharmaceutical compounds with hepatotoxicity data. Quantitative structure-activity relationship (QSAR) models were developed based on ARE-bla data. The models predicted the potential oxidative stress response for known liver toxicants when no ARE-bla data were available. Liver toxicants were used as probe compounds to search PubChem Bioassay and generate a response profile, which contained thousands of bioassays (> 10 million data points). By ranking the in vitro-in vivo correlations (IVIVCs), the most relevant bioassay(s) related to hepatotoxicity were identified. The liver toxicants profile contained the ARE-bla and relevant PubChem assays. Potential toxicophores for well-known toxicants were created by identifying chemical features that existed only in compounds with high IVIVCs. Profiling chemical IVIVCs created an opportunity to fully explore the source-to-outcome continuum of modern experimental toxicology using cheminformatics approaches and big data sources. Kim MT, Huang R, Sedykh A, Wang W, Xia M, Zhu H. 2016. Mechanism profiling of hepatotoxicity caused by oxidative stress using antioxidant response element reporter gene assay models and big data. Environ Health Perspect 124:634-641;�

Selective activation of SHP2 activity by cisplatin revealed by a novel chemical probe-based assay

International Nuclear Information System (INIS)

Kuo, Chun-Chen; Chu, Chi-Yuan; Lin, Jing-Jer; Lo, Lee-Chiang

2010-01-01

Src homology-2 (SH2) domain-containing phosphatase 2 (SHP2) is known to participate in several different signaling pathways to mediate cell growth, survival, migration, and differentiation. However, due to the lack of proper analytical tools, it is unclear whether the phosphatase activity of SHP2 is activated in most studies. We have previously developed an activity-based probe LCL2 that formed covalent linkage with catalytically active protein tyrosine phosphatases (PTPs). Here, by combining LCL2 with a SHP2 specific antibody, we established an assay system that enables the direct monitoring of SHP2 activity upon cisplatin treatment of cancer cells. The protocol is advantageous over conventional colorimetric or in-gel PTP assays as it is specific and does not require the use of radioisotope reagents. Using this assay, we found SHP2 activity was selectively activated by cisplatin. Moreover, the activation of SHP2 appeared to be specific for cisplatin as other DNA damage agents failed to activate the activity. Although the role of SHP2 activation by cisplatin treatments is still unclear to us, our results provide the first direct evidence for the activation of SHP2 during cisplatin treatments. More importantly, the concept of using activity-based probe in conjunction with target-specific antibodies could be extended to other enzyme classes.

Identification of listeria species isolated in Tunisia by Microarray based assay : results of a preliminary study

International Nuclear Information System (INIS)

Hmaied, Fatma; Helel, Salma; Barkallah, Insaf; Leberre, V.; Francois, J.M.; Kechrid, A.

2008-01-01

Microarray-based assay is a new molecular approach for genetic screening and identification of microorganisms. We have developed a rapid microarray-based assay for the reliable detection and discrimination of Listeria spp. in food and clinical isolates from Tunisia. The method used in the present study is based on the PCR amplification of a virulence factor gene (iap gene). the PCR mixture contained cyanine Cy5labeled dCTP. Therefore, The PCR products were fluorescently labeled. The presence of multiple species-specific sequences within the iap gene enabled us to design different oligoprobes per species. The species-specific sequences of the iap gene used in this study were obtained from genBank and then aligned for phylogenetic analysis in order to identify and retrieve the sequences of homologues of the amplified iap gene analysed. 20 probes were used for detection and identification of 22 food isolates and clinical isolates of Listeria spp (L. monocytogenes, L. ivanovi), L. welshimeri, L. seeligeri, and L. grayi). Each bacterial gene was identified by hybridization to oligoprobes specific for each Listeria species and immobilized on a glass surface. The microarray analysis showed that 5 clinical isolates and 2 food isolates were identified listeria monocytogenes. Concerning the remaining 15 food isolates; 13 were identified listeria innocua and 2 isolates could not be identified by microarray based assay. Further phylogenetic and molecular analysis are required to design more species-specific probes for the identification of Listeria spp. Microarray-based assay is a simple and rapid method used for Listeria species discrimination

Preclinical detection of porcine circovirus type 2 infection using an ultrasensitive nanoparticle DNA probe-based PCR assay.

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Yong Huang

Full Text Available Porcine circovirus type 2 (PCV2 has emerged as one of the most important pathogens affecting swine production globally. Preclinical identification of PCV2 is very important for effective prophylaxis of PCV2-associated diseases. In this study, we developed an ultrasensitive nanoparticle DNA probe-based PCR assay (UNDP-PCR for PCV2 detection. Magnetic microparticles coated with PCV2 specific DNA probes were used to enrich PCV2 DNA from samples, then gold nanoparticles coated with PCV2 specific oligonucleotides were added to form a sandwich nucleic acid-complex. After the complex was formed, the oligonucleotides were released and characterized by PCR. This assay exhibited about 500-fold more sensitive than conventional PCR, with a detection limit of 2 copies of purified PCV2 genomic DNA and 10 viral copies of PCV2 in serum. The assay has a wide detection range for all of PCV2 genotypes with reliable reproducibility. No cross-reactivity was observed from the samples of other related viruses including porcine circovirus type 1, porcine parvovirus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus and classical swine fever virus. The positive detection rate of PCV2 specific UNDP-PCR in 40 preclinical field samples was 27.5%, which appeared greater than that by conventional and real-time PCR and appeared application potency in evaluation of the viral loads levels of preclinical infection samples. The UNDP-PCR assay reported here can reliably rule out false negative results from antibody-based assays, provide a nucleic acid extraction free, specific, ultrasensitive, economic and rapid diagnosis method for preclinical PCV2 infection in field, which may help prevent large-scale outbreaks.

A novel, multiplexed, probe-based quantitative PCR assay for the soybean root- and stem-rot pathogen, Phytophthora sojae, utilizes its transposable element.

Science.gov (United States)

Haudenshield, James S; Song, Jeong Y; Hartman, Glen L

2017-01-01

Phytophthora root rot of soybean [Glycine max (L.) Merr.] is caused by the oomycete Phytophthora sojae (Kaufm. & Gerd.). P. sojae has a narrow host range, consisting primarily of soybean, and it is a serious pathogen worldwide. It exists in root and stem tissues as mycelium, wherein it can form oospores which subsequently germinate to release motile, infectious zoospores. Molecular assays detecting DNA of P. sojae are useful in disease diagnostics, and for determining the presence of the organism in host tissues, soils, and runoff or ponded water from potentially infested fields. Such assays as published have utilized ITS sequences from the nuclear ribosomal RNA genes in conventional PCR or dye-binding quantitative PCR (Q-PCR) but are not amenable to multiplexing, and some of these assays did not utilize control strategies for type I or type II errors. In this study, we describe primers and a bifunctional probe with specificity to a gypsy-like retroelement in the P. sojae genome to create a fluorogenic 5'-exonuclease linear hydrolysis assay, with a multiplexed internal control reaction detecting an exogenous target to validate negative calls, and with uracil-deglycosylase-mediated protection against carryover contamination. The assay specifically detected 13 different P. sojae isolates, and excluded 17 other Phytophthora species along with 20 non-Phytophthora fungal and oomycete species pathogenic on soybean. A diagnostic limit of detection of 34 fg total P. sojae DNA was observed in serial dilutions, equivalent to 0.3 genome, and a practical detection sensitivity of four zoospores per sample was achieved, despite losses during DNA extraction.

Gene probes : principles and protocols [Methods in molecular biology, v. 179

National Research Council Canada - National Science Library

Rapley, Ralph; Aquino de Muro, Marilena

2002-01-01

"Senior scientists Marilena Aquino de Muro and Ralph Rapley have brought together an outstanding collection of time-tested protocols for designing and using genes probes in a wide variety of applications...

Ultraspecific probes for high throughput HLA typing

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Eggers Rick

2009-02-01

Full Text Available Abstract Background The variations within an individual's HLA (Human Leukocyte Antigen genes have been linked to many immunological events, e.g. susceptibility to disease, response to vaccines, and the success of blood, tissue, and organ transplants. Although the microarray format has the potential to achieve high-resolution typing, this has yet to be attained due to inefficiencies of current probe design strategies. Results We present a novel three-step approach for the design of high-throughput microarray assays for HLA typing. This approach first selects sequences containing the SNPs present in all alleles of the locus of interest and next calculates the number of base changes necessary to convert a candidate probe sequences to the closest subsequence within the set of sequences that are likely to be present in the sample including the remainder of the human genome in order to identify those candidate probes which are "ultraspecific" for the allele of interest. Due to the high specificity of these sequences, it is possible that preliminary steps such as PCR amplification are no longer necessary. Lastly, the minimum number of these ultraspecific probes is selected such that the highest resolution typing can be achieved for the minimal cost of production. As an example, an array was designed and in silico results were obtained for typing of the HLA-B locus. Conclusion The assay presented here provides a higher resolution than has previously been developed and includes more alleles than previously considered. Based upon the in silico and preliminary experimental results, we believe that the proposed approach can be readily applied to any highly polymorphic gene system.

A gene-protein assay for human epidermal growth factor receptor 2 (HER2: brightfield tricolor visualization of HER2 protein, the HER2 gene, and chromosome 17 centromere (CEN17 in formalin-fixed, paraffin-embedded breast cancer tissue sections

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Nitta Hiroaki

2012-05-01

Full Text Available Abstract Background The eligibility of breast cancer patients for human epidermal growth factor receptor 2 (HER2-directed therapies is determined by the HER2 gene amplification and/or HER2 protein overexpression status of the breast tumor as determined by in situ hybridization (ISH or immunohistochemistry (IHC, respectively. Our objective was to combine the US Food and Drug Administration (FDA-approved HER2 & chromosome 17 centromere (CEN17 brightfield ISH (BISH and HER2 IHC assays into a single automated HER2 gene-protein assay allowing simultaneous detection of all three targets in a single tissue section. Methods The HER2 gene-protein assay was optimized using formalin-fixed, paraffin-embedded (FFPE samples of the xenograft tumors MCF7 [HER2 negative (non-amplified gene, protein negative] and Calu-3 [HER2 positive (amplified gene, protein positive]. HER2 IHC was performed using a rabbit monoclonal anti-HER2 antibody (clone 4B5 and a conventional 3,3'-diaminobenzidine IHC detection. The HER2 & CEN17 BISH signals were visualized using horseradish peroxidase-based silver and alkaline phosphatase-based red detection systems, respectively with a cocktail of 2,4-dinitrophenyl-labeled HER2 and digoxigenin-labeled CEN17 probes. The performance of the gene-protein assay on tissue microarray slides containing 189 randomly selected FFPE clinical breast cancer tissue cores was compared to that of the separate HER2 IHC and HER2 & CEN17 BISH assays. Results HER2 protein detection was optimal when the HER2 IHC protocol was used before (rather than after the BISH protocol. The sequential use of HER2 IHC and HER2 & CEN17 BISH detection steps on FFPE xenograft tumor sections appropriately co-localized the HER2 protein, HER2 gene, and CEN17 signals after mitigating the silver background staining by using a naphthol phosphate-containing hybridization buffer for the hybridization step. The HER2 protein and HER2 gene status obtained using the multiplex HER2 gene

Line probe assay for differentiation within Mycobacterium tuberculosis complex. Evaluation on clinical specimens and isolates including Mycobacterium pinnipedii

DEFF Research Database (Denmark)

Kjeldsen, Marianne Kirstine; Bek, Dorte; Rasmussen, Erik Michael

2009-01-01

A line probe assay (GenoType MTBC) was evaluated for species differentiation within the Mycobacterium tuberculosis complex (MTBC). We included 387 MTBC isolates, 43 IS6110 low-copy MTBC isolates, 28 clinical specimens with varying microscopy grade, and 30 isolates of non-tuberculous mycobacteria...

High-throughput platform assay technology for the discovery of pre-microrna-selective small molecule probes.

Science.gov (United States)

Lorenz, Daniel A; Song, James M; Garner, Amanda L

2015-01-21

MicroRNAs (miRNA) play critical roles in human development and disease. As such, the targeting of miRNAs is considered attractive as a novel therapeutic strategy. A major bottleneck toward this goal, however, has been the identification of small molecule probes that are specific for select RNAs and methods that will facilitate such discovery efforts. Using pre-microRNAs as proof-of-concept, herein we report a conceptually new and innovative approach for assaying RNA-small molecule interactions. Through this platform assay technology, which we term catalytic enzyme-linked click chemistry assay or cat-ELCCA, we have designed a method that can be implemented in high throughput, is virtually free of false readouts, and is general for all nucleic acids. Through cat-ELCCA, we envision the discovery of selective small molecule ligands for disease-relevant miRNAs to promote the field of RNA-targeted drug discovery and further our understanding of the role of miRNAs in cellular biology.

Use of a real time PCR assay for detection of the ctxA gene of Vibrio cholerae in an environmental survey of Mobile Bay.

Science.gov (United States)

Blackstone, George M; Nordstrom, Jessica L; Bowen, Michael D; Meyer, Richard F; Imbro, Paula; DePaola, Angelo

2007-02-01

Toxigenic Vibrio cholerae, the etiological agent of cholera, is a natural inhabitant of the marine environment and causes severe diarrheal disease affecting thousands of people each year in developing countries. It is the subject of extensive testing of shrimp produced and exported from these countries. We report the development of a real time PCR (qPCR) assay to detect the gene encoding cholera toxin, ctxA, found in toxigenic V. cholerae strains. This assay was tested against DNA isolated from soil samples collected from diverse locations in the US, a panel of eukaryotic DNA from various sources, and prokaryotic DNA from closely related and unrelated bacterial sources. Only Vibrio strains known to contain ctxA generated a fluorescent signal with the 5' nuclease probe targeting the ctxA gene, thus confirming the specificity of the assay. In addition, the assay was quantitative in pure culture across a six-log dynamic range down to <10 CFU per reaction. To test the robustness of this assay, oysters, aquatic sediments, and seawaters from Mobile Bay, AL, were analyzed by qPCR and traditional culture methods. The assay was applied to overnight alkaline peptone water enrichments of these matrices after boiling the enrichments for 10 min. Toxigenic V. cholerae strains were not detected by either qPCR or conventional methods in the 16 environmental samples examined. A novel exogenous internal amplification control developed by us to prevent false negatives identified the samples that were inhibitory to the PCR. This assay, with the incorporated internal control, provides a highly specific, sensitive, and rapid detection method for the detection of toxigenic strains of V. cholerae.

Methods for transient assay of gene function in floral tissues

Directory of Open Access Journals (Sweden)

Pathirana Nilangani N

2007-01-01

Full Text Available Abstract Background There is considerable interest in rapid assays or screening systems for assigning gene function. However, analysis of gene function in the flowers of some species is restricted due to the difficulty of producing stably transformed transgenic plants. As a result, experimental approaches based on transient gene expression assays are frequently used. Biolistics has long been used for transient over-expression of genes of interest, but has not been exploited for gene silencing studies. Agrobacterium-infiltration has also been used, but the focus primarily has been on the transient transformation of leaf tissue. Results Two constructs, one expressing an inverted repeat of the Antirrhinum majus (Antirrhinum chalcone synthase gene (CHS and the other an inverted repeat of the Antirrhinum transcription factor gene Rosea1, were shown to effectively induce CHS and Rosea1 gene silencing, respectively, when introduced biolistically into petal tissue of Antirrhinum flowers developing in vitro. A high-throughput vector expressing the Antirrhinum CHS gene attached to an inverted repeat of the nos terminator was also shown to be effective. Silencing spread systemically to create large zones of petal tissue lacking pigmentation, with transmission of the silenced state spreading both laterally within the affected epidermal cell layer and into lower cell layers, including the epidermis of the other petal surface. Transient Agrobacterium-mediated transformation of petal tissue of tobacco and petunia flowers in situ or detached was also achieved, using expression of the reporter genes GUS and GFP to visualise transgene expression. Conclusion We demonstrate the feasibility of using biolistics-based transient RNAi, and transient transformation of petal tissue via Agrobacterium infiltration to study gene function in petals. We have also produced a vector for high throughput gene silencing studies, incorporating the option of using T-A cloning to

Evaluation of four endogenous reference genes and their real-time PCR assays for common wheat quantification in GMOs detection.

Science.gov (United States)

Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao

2013-01-01

Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat.

Evaluation of four endogenous reference genes and their real-time PCR assays for common wheat quantification in GMOs detection.

Directory of Open Access Journals (Sweden)

Huali Huang

Full Text Available Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L. DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat.

Evaluation of Four Endogenous Reference Genes and Their Real-Time PCR Assays for Common Wheat Quantification in GMOs Detection

Science.gov (United States)

Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao

2013-01-01

Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat. PMID:24098735

A probe-based quantitative PCR assay for detecting Tetracapsuloides bryosalmonae in fish tissue and environmental DNA water samples

Science.gov (United States)

Hutchins, Patrick; Sepulveda, Adam; Martin, Renee; Hopper, Lacey

2017-01-01

A probe-based quantitative real-time PCR assay was developed to detect Tetracapsuloides bryosalmonae, which causes proliferative kidney disease in salmonid fish, in kidney tissue and environmental DNA (eDNA) water samples. The limits of detection and quantification were 7 and 100 DNA copies for calibration standards and T. bryosalmonae was reliably detected down to 100 copies in tissue and eDNA samples. The assay presented here is a highly sensitive and quantitative tool for detecting T. bryosalmonae with potential applications for tissue diagnostics and environmental detection.

Development and evaluation of Taqman assays for the differentiation of Dickeya (sub)species

NARCIS (Netherlands)

Wolf, van der J.M.; Haas, de B.H.; Hoof, van R.A.; Haan, de E.G.; Bovenkamp, van den G.W.

2014-01-01

TaqMan assays were developed for the detection of seven Dickeya species, namely D. dianthicola, D. dadantii, D. paradisiaca, D. chrysanthemi, D. zeae, D. dieffenbachiae and D. solani. Sequences of the gene coding for dnaX were used for the design of primers and probes. In studies with axenic

A homogeneous and “off–on” fluorescence aptamer-based assay for chloramphenicol using vesicle quantum dot-gold colloid composite probes

Energy Technology Data Exchange (ETDEWEB)

Miao, Yang-Bao [State Key Laboratory Base of Novel Functional Materials and Preparation Science, Faculty of Materials Science and Chemical Engineering, Ningbo University, Ningbo 315211 (China); Ren, Hong-Xia [Key Laboratory of Asymmetric Synthesis and Chirotechnology of Sichuan Province, Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041 (China); University of Chinese Academy of Sciences, Beijing 10049 (China); Gan, Ning, E-mail: ganning@nbu.edu.cn [State Key Laboratory Base of Novel Functional Materials and Preparation Science, Faculty of Materials Science and Chemical Engineering, Ningbo University, Ningbo 315211 (China); Zhou, You [State Key Laboratory Base of Novel Functional Materials and Preparation Science, Faculty of Materials Science and Chemical Engineering, Ningbo University, Ningbo 315211 (China); Cao, Yuting, E-mail: caoyuting@nbu.edu.cn [State Key Laboratory Base of Novel Functional Materials and Preparation Science, Faculty of Materials Science and Chemical Engineering, Ningbo University, Ningbo 315211 (China); Li, Tianhua [State Key Laboratory Base of Novel Functional Materials and Preparation Science, Faculty of Materials Science and Chemical Engineering, Ningbo University, Ningbo 315211 (China); Chen, Yinji [Faculty of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing 210000 (China)

2016-07-27

In this work, a novel homogeneous and signal “off–on” aptamer based fluorescence assay was successfully developed to detect chloramphenicol (CAP) residues in food based on the fluorescence resonance energy transfer (FRET). The vesicle nanotracer was prepared through labeling single stranded DNA binding protein (SSB) on limposome-CdSe/ZnS quantum dot (SSB/L-QD) complexes. It was worth mentioning that the signal tracer (SSB/L-QD) with vesicle shape, which was fabricated being encapsulated with a number of quantum dots and SSB. The nanotracer has excellent signal amplification effects. The vesicle composite probe was formed by combining aptamer labeled nano-gold (Au-Apt) and SSB/L-QD. Which based on SSB's specific affinity towards aptamer. This probe can't emit fluoresce which is in “off” state because the signal from SSB/L-QD as donor can be quenched by the Au-aptas acceptor. When CAP was added in the composite probe solution, the aptamer on the Au-Apt can be preferentially bounded with CAP then release from the composite probe, which can turn the “off” signal of SSB/L-QD tracer into “on” state. The assay indicates excellent linear response to CAP from 0.001 nM to 10 nM and detection limit down to 0.3 pM. The vesicle probes with size of 88 nm have strong signal amplification. Because a larger number of QDs can be labeled inside the double phosphorus lipid membrane. Besides, it was employed to detect CAP residues in the milk samples with results being agreed well with those from ELISA, verifying its accuracy and reliability. - Highlights: • Homogeneous and “off–on” fluorescence aptamer-based assay was developed to detect chloramphenicol (CAP) residues in food. • This probe was fabricated based on a vesicle QDs signal tracer (SSB/L-QD) combining with Au-Aptamer. • The detection mechanism was based on FRET with high specificity. • The results for CAP detection in the milk samples agreed well with those from ELISA, while

Comparative evaluation of GenoType MTBDRplus line probe assay with solid culture method in early diagnosis of multidrug resistant tuberculosis (MDR-TB at a tertiary care centre in India.

Directory of Open Access Journals (Sweden)

Raj N Yadav

Full Text Available The objectives of the study were to compare the performance of line probe assay (GenoType MTBDRplus with solid culture method for an early diagnosis of multidrug resistant tuberculosis (MDR-TB, and to study the mutation patterns associated with rpoB, katG and inhA genes at a tertiary care centre in north India.In this cross-sectional study, 269 previously treated sputum-smear acid-fast bacilli (AFB positive MDR-TB suspects were enrolled from January to September 2012 at the All India Institute of Medical Sciences hospital, New Delhi. Line probe assay (LPA was performed directly on the sputum specimens and the results were compared with that of conventional drug susceptibility testing (DST on solid media [Lowenstein Jensen (LJ method].DST results by LPA and LJ methods were compared in 242 MDR-TB suspects. The LPA detected rifampicin (RIF resistance in 70 of 71 cases, isoniazid (INH resistance in 86 of 93 cases, and MDR-TB in 66 of 68 cases as compared to the conventional method. Overall (rifampicin, isoniazid and MDR-TB concordance of the LPA with the conventional DST was 96%. Sensitivity and specificity were 98% and 99% respectively for detection of RIF resistance; 92% and 99% respectively for detection of INH resistance; 97% and 100% respectively for detection of MDR-TB. Frequencies of katG gene, inhA gene and combined katG and inhA gene mutations conferring all INH resistance were 72/87 (83%, 10/87 (11% and 5/87 (6% respectively. The turnaround time of the LPA test was 48 hours.The LPA test provides an early diagnosis of monoresistance to isoniazid and rifampicin and is highly sensitive and specific for an early diagnosis of MDR-TB. Based on these findings, it is concluded that the LPA test can be useful in early diagnosis of drug resistant TB in high TB burden countries.

Detection of enteric Adenoviruses in South-African waters using gene probes

CSIR Research Space (South Africa)

Genthe, Bettina

1995-01-01

Full Text Available Gene probes developed locally for both enteric Adenoviruses 40 and 41 were used to determine whether these viruses were present in both raw and treated waters. Approximately sixty water samples were concentrated by ultra filtration and analysed...

Multiplex ligation-dependent probe amplification (MLPA) assay for blood group genotyping, copy number quantification, and analysis of RH variants

NARCIS (Netherlands)

Veldhuisen, Barbera; van der Schoot, C. E.; de Haas, Masja

2015-01-01

The blood group multiplex ligation-dependent probe amplification (MLPA) is a comprehensive assay, developed for genotyping the majority of clinically relevant blood group antigens in both patients and donors. The MLPA is an easy method to apply and only requires a thermal cycler and capillary

Development of a real-time RT-PCR assay based on primer-probe energy transfer for the detection of all serotypes of bluetongue virus

DEFF Research Database (Denmark)

Leblanc, N; Rasmussen, Thomas Bruun; Fernandez, J

2010-01-01

A real-time RT-PCR assay based on the primer–probe energy transfer (PriProET) was developed to detect all 24 serotypes of bluetongue virus (BTV). BTV causes serious disease, primarily in sheep, but in other ruminants as well. A distinguishing characteristic of the assay is its tolerance toward...

An Aptamer Bio-barCode (ABC) assay using SPR, RNase H, and probes with RNA and gold-nanorods for anti-cancer drug screening.

Science.gov (United States)

Loo, Jacky Fong-Chuen; Yang, Chengbin; Tsang, Hing Lun; Lau, Pui Man; Yong, Ken-Tye; Ho, Ho Pui; Kong, Siu Kai

2017-10-07

With modifications to an ultra-sensitive bio-barcode (BBC) assay, we have developed a next generation aptamer-based bio-barcode (ABC) assay to detect cytochrome-c (Cyto-c), a cell death marker released from cancer cells, for anti-cancer drug screening. An aptamer is a short single-stranded DNA selected from a synthetic DNA library that is capable of binding to its target with high affinity and specificity based on its unique DNA sequence and 3D structure after folding. Similar to the BBC assay, Cyto-c is captured by a micro-magnetic particle (MMP) coated with capturing antibodies (Ab) and an aptamer specifically against Cyto-c to form sandwich structures ([MMP-Ab]-[Cyto-c]-[Aptamer]). After washing and melting, our aptamers, acting as a DNA bio-barcode, are released from the sandwiches and hybridized with the probes specially designed for RNase H for surface plasmon resonance (SPR) sensing. In an aptamer-probe duplex, RNase H digests the RNA in the probe and releases the intact aptamer for another round of hybridization and digestion. With signal enhancement effects from gold-nanorods (Au-NRs) on probes for SPR sensing, the detection limit was found to be 1 nM for the aptamer and 80 pM for Cyto-c. Without the time-consuming DNA amplification steps by PCR, the detection process of this new ABC assay can be completed within three hours. As a proof-of-concept, phenylarsine oxide was found to be a potent agent to kill liver cancer cells with multi-drug resistance at the nano-molar level. This approach thus provides a fast, sensitive and robust tool for anti-cancer drug screening.

Development and characterization of a magnetic bead-quantum dot nanoparticles based assay capable of Escherichia coli O157:H7 quantification

Energy Technology Data Exchange (ETDEWEB)

Kim, Gha-Young [Department of Civil Engineering, Auburn University, Auburn, AL 36849 (United States); Son, Ahjeong, E-mail: ason@auburn.edu [Department of Civil Engineering, Auburn University, Auburn, AL 36849 (United States)

2010-09-10

The development and characterization of a magnetic bead (MB)-quantum dot (QD) nanoparticles based assay capable of quantifying pathogenic bacteria is presented here. The MB-QD assay operates by having a capturing probe DNA selectively linked to the signaling probe DNA via the target genomic DNA (gDNA) during DNA hybridization. The signaling probe DNA is labeled with fluorescent QD{sub 565} which serves as a reporter. The capturing probe DNA is conjugated simultaneously to a MB and another QD{sub 655}, which serve as a carrier and an internal standard, respectively. Successfully captured target gDNA is separated using a magnetic field and is quantified via a spectrofluorometer. The use of QDs (i.e., QD{sub 565}/QD{sub 655}) as both a fluorescence label and an internal standard increased the sensitivity of the assay. The passivation effect and the molar ratio between QD and DNA were optimized. The MB-QD assay demonstrated a detection limit of 890 zeptomolar (i.e., 10{sup -21} mol L{sup -1}) concentration for the linear single stranded DNA (ssDNA). It also demonstrated a detection limit of 87 gene copies for double stranded DNA (dsDNA) eaeA gene extracted from pure Escherichia coli (E. coli) O157:H7 culture. Its corresponding dynamic range, sensitivity, and selectivity were also presented. Finally, the bacterial gDNA of E. coli O157:H7 was used to highlight the MB-QD assay's ability to detect below the minimum infective dose (i.e., 100 organisms) of E. coli O157:H7 in water environment.

Biomarker discovery for colon cancer using a 761 gene RT-PCR assay

Directory of Open Access Journals (Sweden)

Hackett James R

2007-08-01

Full Text Available Abstract Background Reverse transcription PCR (RT-PCR is widely recognized to be the gold standard method for quantifying gene expression. Studies using RT-PCR technology as a discovery tool have historically been limited to relatively small gene sets compared to other gene expression platforms such as microarrays. We have recently shown that TaqMan® RT-PCR can be scaled up to profile expression for 192 genes in fixed paraffin-embedded (FPE clinical study tumor specimens. This technology has also been used to develop and commercialize a widely used clinical test for breast cancer prognosis and prediction, the Onco typeDX™ assay. A similar need exists in colon cancer for a test that provides information on the likelihood of disease recurrence in colon cancer (prognosis and the likelihood of tumor response to standard chemotherapy regimens (prediction. We have now scaled our RT-PCR assay to efficiently screen 761 biomarkers across hundreds of patient samples and applied this process to biomarker discovery in colon cancer. This screening strategy remains attractive due to the inherent advantages of maintaining platform consistency from discovery through clinical application. Results RNA was extracted from formalin fixed paraffin embedded (FPE tissue, as old as 28 years, from 354 patients enrolled in NSABP C-01 and C-02 colon cancer studies. Multiplexed reverse transcription reactions were performed using a gene specific primer pool containing 761 unique primers. PCR was performed as independent TaqMan® reactions for each candidate gene. Hierarchal clustering demonstrates that genes expected to co-express form obvious, distinct and in certain cases very tightly correlated clusters, validating the reliability of this technical approach to biomarker discovery. Conclusion We have developed a high throughput, quantitatively precise multi-analyte gene expression platform for biomarker discovery that approaches low density DNA arrays in numbers of

Identification of pathogenic Nocardia species by reverse line blot hybridization targeting the 16S rRNA and 16S-23S rRNA gene spacer regions.

Science.gov (United States)

Xiao, Meng; Kong, Fanrong; Sorrell, Tania C; Cao, Yongyan; Lee, Ok Cha; Liu, Ying; Sintchenko, Vitali; Chen, Sharon C A

2010-02-01

Although 16S rRNA gene sequence analysis is employed most often for the definitive identification of Nocardia species, alternate molecular methods and polymorphisms in other gene targets have also enabled species determinations. We evaluated a combined Nocardia PCR-based reverse line blot (RLB) hybridization assay based on 16S and 16S-23S rRNA gene spacer region polymorphisms to identify 12 American Type Culture Collection and 123 clinical Nocardia isolates representing 14 species; results were compared with results from 16S rRNA gene sequencing. Thirteen 16S rRNA gene-based (two group-specific and 11 species-specific) and five 16S-23S spacer-targeted (two taxon-specific and three species-specific) probes were utilized. 16S rRNA gene-based probes correctly identified 124 of 135 isolates (sensitivity, 92%) but were unable to identify Nocardia paucivorans strains (n = 10 strains) and a Nocardia asteroides isolate with a novel 16S rRNA gene sequence. Nocardia farcinica and Nocardia cyriacigeorgica strains were identified by the sequential use of an N. farcinica-"negative" probe and a combined N. farcinica/N. cyriacigeorgica probe. The assay specificity was high (99%) except for weak cross-reactivity between the Nocardia brasiliensis probe with the Nocardia thailandica DNA product; however, cross-hybridization with closely related nontarget species may occur. The incorporation of 16S-23S rRNA gene spacer-based probes enabled the identification of all N. paucivorans strains. The overall sensitivity using both probe sets was >99%. Both N. farcinica-specific 16S-23S rRNA gene spacer-directed probes were required to identify all N. farcinica stains by using this probe set. The study demonstrates the utility of a combined PCR/RLB assay for the identification of clinically relevant Nocardia species and its potential for studying subtypes of N. farcinica. Where species assignment is ambiguous or not possible, 16S rRNA gene sequencing is recommended.

Sensitive detection of novel Indian isolate of BTV 21 using ns1 gene based real-time PCR assay

Directory of Open Access Journals (Sweden)

Gaya Prasad

2013-06-01

Full Text Available Aim: The study was conducted to develop ns1 gene based sensitive real-time RT-PCR assay for diagnosis of India isolates of bluetongue virus (BTV. Materials and Methods: The BTV serotype 21 isolate (KMNO7 was isolated from Andhra Pradesh and propagated in BHK-21 cell line in our laboratory. The Nucleic acid (dsRNA of virus was extracted using Trizol method and cDNA was prepared using a standard protocol. The cDNA was allowed to ns1 gene based group specific PCR to confirm the isolate as BTV. The viral RNA was diluted 10 folds and the detection limit of ns1 gene based RT-PCR was determined. Finally the tenfold diluted viral RNA was subjected to real-time RT-PCR using ns1 gene primer and Taq man probe to standardized the reaction and determine the detection limit. Results: The ns1 gene based group specific PCR showed a single 366bp amplicon in agarose gel electrophoresis confirmed the sample as BTV. The ns1 gene RT-PCR using tenfold diluted viral RNA showed the detection limit of 70.0 fg in 1%agarose gel electrophoresis. The ns1 gene based real time RT-PCR was successfully standardized and the detection limit was found to be 7.0 fg. Conclusion: The ns1 gene based real-time RT-PCR was successfully standardized and it was found to be 10 times more sensitive than conventional RT-PCR. Key words: bluetongue, BTV21, RT-PCR, Real time RT-PCR, ns1 gene [Vet World 2013; 6(8.000: 554-557

Evaluation of a polymerase chain reaction reverse hybridization line probe assay for the detection and identification of medically important fungi in bronchoalveolar lavage fluids.

NARCIS (Netherlands)

Meletiadis, J.; Melchers, W.J.G.; Meis, J.F.G.M.; Hurk, P.J.J.C. van den; Jannes, G.; Verweij, P.E.

2003-01-01

An assay system in which polymerase chain reaction (PCR) amplification of the ITS-1 region of ribosomal DNA (rDNA) is combined with a reverse-hybridization line probe assay (LiPA) was used for the identification of six Candida species and four Aspergillus species in pure cultures of clinical

Identification of Five Novel Salmonella Typhi-Specific Genes as Markers for Diagnosis of Typhoid Fever Using Single-Gene Target PCR Assays

Directory of Open Access Journals (Sweden)

Yuan Xin Goay

2016-01-01

Full Text Available Salmonella Typhi (S. Typhi causes typhoid fever which is a disease characterised by high mortality and morbidity worldwide. In order to curtail the transmission of this highly infectious disease, identification of new markers that can detect the pathogen is needed for development of sensitive and specific diagnostic tests. In this study, genomic comparison of S. Typhi with other enteric pathogens was performed, and 6 S. Typhi genes, that is, STY0201, STY0307, STY0322, STY0326, STY2020, and STY2021, were found to be specific in silico. Six PCR assays each targeting a unique gene were developed to test the specificity of these genes in vitro. The diagnostic sensitivities and specificities of each assay were determined using 39 S. Typhi, 62 non-Typhi Salmonella, and 10 non-Salmonella clinical isolates. The results showed that 5 of these genes, that is, STY0307, STY0322, STY0326, STY2020, and STY2021, demonstrated 100% sensitivity (39/39 and 100% specificity (0/72. The detection limit of the 5 PCR assays was 32 pg for STY0322, 6.4 pg for STY0326, STY2020, and STY2021, and 1.28 pg for STY0307. In conclusion, 5 PCR assays using STY0307, STY0322, STY0326, STY2020, and STY2021 were developed and found to be highly specific at single-gene target resolution for diagnosis of typhoid fever.

Identification of Five Novel Salmonella Typhi-Specific Genes as Markers for Diagnosis of Typhoid Fever Using Single-Gene Target PCR Assays.

Science.gov (United States)

Goay, Yuan Xin; Chin, Kai Ling; Tan, Clarissa Ling Ling; Yeoh, Chiann Ying; Ja'afar, Ja'afar Nuhu; Zaidah, Abdul Rahman; Chinni, Suresh Venkata; Phua, Kia Kien

2016-01-01

Salmonella Typhi ( S . Typhi) causes typhoid fever which is a disease characterised by high mortality and morbidity worldwide. In order to curtail the transmission of this highly infectious disease, identification of new markers that can detect the pathogen is needed for development of sensitive and specific diagnostic tests. In this study, genomic comparison of S . Typhi with other enteric pathogens was performed, and 6 S . Typhi genes, that is, STY0201, STY0307, STY0322, STY0326, STY2020, and STY2021, were found to be specific in silico . Six PCR assays each targeting a unique gene were developed to test the specificity of these genes in vitro . The diagnostic sensitivities and specificities of each assay were determined using 39 S . Typhi, 62 non-Typhi Salmonella , and 10 non- Salmonella clinical isolates. The results showed that 5 of these genes, that is, STY0307, STY0322, STY0326, STY2020, and STY2021, demonstrated 100% sensitivity (39/39) and 100% specificity (0/72). The detection limit of the 5 PCR assays was 32 pg for STY0322, 6.4 pg for STY0326, STY2020, and STY2021, and 1.28 pg for STY0307. In conclusion, 5 PCR assays using STY0307, STY0322, STY0326, STY2020, and STY2021 were developed and found to be highly specific at single-gene target resolution for diagnosis of typhoid fever.

Impact of inter-genotypic recombination and probe cross-reactivity on the performance of the Abbott RealTime HCV Genotype II assay for hepatitis C genotyping.

Science.gov (United States)

Sridhar, Siddharth; Yip, Cyril C Y; Chan, Jasper F W; To, Kelvin K W; Cheng, Vincent C C; Yuen, Kwok-Yung

2018-05-01

The Abbott RealTime HCV Genotype II assay (Abbott-RT-HCV assay) is a real-time PCR based genotyping method for hepatitis C virus (HCV). This study measured the impact of inter-genotypic recombination and probe cross-reactivity on the performance of the Abbott-RT-HCV assay. 517 samples were genotyped using the Abbott-RT-HCV assay over a one-year period, 34 (6.6%) were identified as HCV genotype 1 without further subtype designation raising the possibility of inaccurate genotyping. These samples were subjected to confirmatory sequencing. 27 of these 34 (79%) samples were genotype 1b while five (15%) were genotype 6. One HCV isolate was an inter-genotypic 1a/4o recombinant. This is a novel natural HCV recombinant that has never been reported. Inter-genotypic recombination and probe cross-reactivity can affect the accuracy of the Abbott-RT-HCV assay, both of which have significant implications on antiviral regimen choice. Confirmatory sequencing of ambiguous results is crucial for accurate genotyping. Copyright © 2018 Elsevier Inc. All rights reserved.

Detection of exogenous gene doping of IGF-I by a real-time quantitative PCR assay.

Science.gov (United States)

Zhang, Jin-Ju; Xu, Jing-Feng; Shen, Yong-Wei; Ma, Shi-Jiao; Zhang, Ting-Ting; Meng, Qing-Lin; Lan, Wen-Jun; Zhang, Chun; Liu, Xiao-Mei

2017-07-01

Gene doping can be easily concealed since its product is similar to endogenous protein, making its effective detection very challenging. In this study, we selected insulin-like growth factor I (IGF-I) exogenous gene for gene doping detection. First, the synthetic IGF-I gene was subcloned to recombinant adeno-associated virus (rAAV) plasmid to produce recombinant rAAV2/IGF-I-GFP vectors. Second, in an animal model, rAAV2/IGF-I-GFP vectors were injected into the thigh muscle tissue of mice, and then muscle and blood specimens were sampled at different time points for total DNA isolation. Finally, real-time quantitative PCR was employed to detect the exogenous gene doping of IGF-I. In view of the characteristics of endogenous IGF-I gene sequences, a TaqMan probe was designed at the junction of exons 2 and 3 of IGF-I gene to distinguish it from the exogenous IGF-I gene. In addition, an internal reference control plasmid and its probe were used in PCR to rule out false-positive results through comparison of their threshold cycle (Ct) values. Thus, an accurate exogenous IGF-I gene detection approach was developed in this study. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

A comparison of 12-gene colon cancer assay gene expression in African American and Caucasian patients with stage II colon cancer.

Science.gov (United States)

Govindarajan, Rangaswamy; Posey, James; Chao, Calvin Y; Lu, Ruixiao; Jadhav, Trafina; Javed, Ahmed Y; Javed, Awais; Mahmoud, Fade A; Osarogiagbon, Raymond U; Manne, Upender

2016-06-18

African American (AA) colon cancer patients have a worse prognosis than Caucasian (CA) colon cancer patients, however, reasons for this disparity are not well understood. To determine if tumor biology might contribute to differential prognosis, we measured recurrence risk and gene expression using the Oncotype DX® Colon Cancer Assay (12-gene assay) and compared the Recurrence Score results and gene expression profiles between AA patients and CA patients with stage II colon cancer. We retrieved demographic, clinical, and archived tumor tissues from stage II colon cancer patients at four institutions. The 12-gene assay and mismatch repair (MMR) status were performed by Genomic Health (Redwood City, California). Student's t-test and the Wilcoxon rank sum test were used to compare Recurrence Score data and gene expression data from AA and CA patients (SAS Enterprise Guide 5.1). Samples from 122 AA and 122 CA patients were analyzed. There were 118 women (63 AA, 55 CA) and 126 men (59 AA, 67 CA). Median age was 66 years for AA patients and 68 for CA patients. Age, gender, year of surgery, pathologic T-stage, tumor location, the number of lymph nodes examined, lymphovascular invasion, and MMR status were not significantly different between groups (p = 0.93). The mean Recurrence Score result for AA patients (27.9 ± 12.8) and CA patients (28.1 ± 11.8) was not significantly different and the proportions of patients with high Recurrence Score values (≥41) were similar between the groups (17/122 AA; 15/122 CA). None of the gene expression variables, either single genes or gene groups (cell cycle group, stromal group, BGN1, FAP, INHBA1, Ki67, MYBL2, cMYC and GADD45B), was significantly different between the racial groups. After controlling for clinical and pathologic covariates, the means and distributions of Recurrence Score results and gene expression profiles showed no statistically significant difference between patient groups. The distribution of

A comparison of 12-gene colon cancer assay gene expression in African American and Caucasian patients with stage II colon cancer

International Nuclear Information System (INIS)

Govindarajan, Rangaswamy; Posey, James; Chao, Calvin Y.; Lu, Ruixiao; Jadhav, Trafina; Javed, Ahmed Y.; Javed, Awais; Mahmoud, Fade A.; Osarogiagbon, Raymond University; Manne, Upender

2016-01-01

African American (AA) colon cancer patients have a worse prognosis than Caucasian (CA) colon cancer patients, however, reasons for this disparity are not well understood. To determine if tumor biology might contribute to differential prognosis, we measured recurrence risk and gene expression using the Oncotype DX® Colon Cancer Assay (12-gene assay) and compared the Recurrence Score results and gene expression profiles between AA patients and CA patients with stage II colon cancer. We retrieved demographic, clinical, and archived tumor tissues from stage II colon cancer patients at four institutions. The 12-gene assay and mismatch repair (MMR) status were performed by Genomic Health (Redwood City, California). Student’s t-test and the Wilcoxon rank sum test were used to compare Recurrence Score data and gene expression data from AA and CA patients (SAS Enterprise Guide 5.1). Samples from 122 AA and 122 CA patients were analyzed. There were 118 women (63 AA, 55 CA) and 126 men (59 AA, 67 CA). Median age was 66 years for AA patients and 68 for CA patients. Age, gender, year of surgery, pathologic T-stage, tumor location, the number of lymph nodes examined, lymphovascular invasion, and MMR status were not significantly different between groups (p = 0.93). The mean Recurrence Score result for AA patients (27.9 ± 12.8) and CA patients (28.1 ± 11.8) was not significantly different and the proportions of patients with high Recurrence Score values (≥41) were similar between the groups (17/122 AA; 15/122 CA). None of the gene expression variables, either single genes or gene groups (cell cycle group, stromal group, BGN1, FAP, INHBA1, Ki67, MYBL2, cMYC and GADD45B), was significantly different between the racial groups. After controlling for clinical and pathologic covariates, the means and distributions of Recurrence Score results and gene expression profiles showed no statistically significant difference between patient groups. The distribution of Recurrence Score

A Novel Complementation Assay for Quick and Specific Screen of Genes Encoding Glycerol-3-Phosphate Acyltransferases

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Jie Lei

2018-03-01

Full Text Available The initial step in glycerolipid biosynthesis, especially in diverse allopolyploid crop species, is poorly understood, mainly due to the lack of an effective and convenient method for functional characterization of genes encoding glycerol-3-phosphate acyltransferases (GPATs catalyzing this reaction. Here we present a novel complementation assay for quick and specific characterization of GPAT-encoding genes. Its key design involves rational construction of yeast conditional lethal gat1Δgat2Δ double mutant bearing the heterologous Arabidopsis AtGPAT1 gene whose leaky expression under repressed conditions does not support any non-specific growth, thereby circumventing the false positive problem encountered with the system based on the gat1Δgat2Δ mutant harboring the native episomal GAT1 gene whose leaky expression appears to be sufficient for generating enough GPAT activities for the non-specific restoration of the mutant growth. A complementation assay developed based on this novel mutant enables quick phenotypic screen of GPAT sequences. A high degree of specificity of our assay was exemplified by its ability to differentiate effectively GPAT-encoding genes from those of other fatty acyltransferases and lipid-related sequences. Using this assay, we show that Arabidopsis AtGPAT1, AtGPAT5, and AtGPAT7 can complement the phosphatidate biosynthetic defect in the double mutants. Collectively, our assay provides a powerful tool for rapid screening, validation and optimization of GPAT sequences, aiding future engineering of the initial step of the triacylglycerol biosynthesis in oilseeds.

Characterization of vitellogenin gene expression in round goby (Neogobius melanostomus) using a quantitative polymerase chain reaction assay.

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Bowley, Lucas A; Alam, Farhana; Marentette, Julie R; Balshine, Sigal; Wilson, Joanna Y

2010-12-01

A growing concern over endocrine disruption in aquatic species has prompted the development of molecular assays to monitor environmental impacts. This study describes the development of quantitative polymerase chain reaction (qPCR) assays to characterize the expression of two vitellogenin (Vtg) genes in the invasive round goby (Neogobius melanostomus). Fragments from the 18SrRNA (housekeeping gene), Vtg II, and Vtg III genes were cloned and sequenced. The qPCR assays were developed to detect hepatic Vtg expression in goby. The assays detected induction of both Vtg genes in nonreproductive males following a two-week laboratory exposure to 17β-estradiol (≥1 mg/kg i.p. injection). The assays were applied to goby from Hamilton Harbour, Lake Ontario (Canada), including those from sites where feminization and intersex of goby has been documented. Both Vtg genes had significantly higher expression in females compared to males. Male reproductive goby adopt either parental or sneaker tactics; Vtg II expression was higher in sneaker than in parental males but parental and nonreproductive males did not differ from each other. The Vtg III expression was significantly higher in sneaker males followed by parental males and nonreproductive males, respectively. The Vtg II and III expression in nonreproductive males was elevated in the contaminated site with documented intersex. This assay provides an important tool for the use of an invasive species in monitoring endocrine disruption in the Great Lakes region. Copyright © 2010 SETAC.

Comprehensive genotyping for 18 blood group systems using a multiplex ligation-dependent probe amplification assay shows a high degree of accuracy

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Haer-Wigman, Lonneke; Ji, Yanli; Lodén, Martin; de Haas, Masja; van der Schoot, C. Ellen; Veldhuisen, Barbera

2013-01-01

In recent years genotyping methods have been implemented in blood banks as alternative to comprehensive serologic typing. We evaluated a newly developed assay for convenient and comprehensive genotyping of blood group alleles based on multiplex ligation-dependent probe amplification (MLPA)

Primary Multidrug Resistant Tuberculosis and Utility of Line Probe Assay for Its Detection in Smear-Positive Sputum Samples in a Tertiary Care Hospital in South India

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Fahmiya Leena Yacoob

2016-01-01

Full Text Available In a high tuberculosis burdened country like India, rapid, cost-effective, and reliable diagnostic tools for tuberculosis are an urgent need of the hour to prevent inappropriate treatment strategies and further spread of resistance. This study aimed to estimate the proportion of new smear-positive tuberculosis cases with primary resistance to rifampicin and/or isoniazid as well as identify the common mutations associated with it. Sputum of 200 newly diagnosed smear-positive cases of 1+ score and above was directly subjected to Line Probe Assay using the GenoType MTBDRplus assay kit. All samples were inoculated onto solid media and 61 samples were inoculated in automated liquid culture also. The Line Probe Assay gave hundred percent interpretable results with 2.5% of the study population showing resistant pattern. Only 1% of the cases were primary multidrug resistant tuberculosis and 1.5% showed isoniazid monoresistance. S531L and C15T were the most common genetic mutations seen for rifampicin and isoniazid resistance, respectively. 40% had absent rpoB wild type 8 band indicating probable silent mutation after clinical correlation. The average turnaround time for Line Probe Assay was far less (3.8 days as compared to solid and liquid cultures (35.6 days and 13.5 days, resp..

Primary Multidrug Resistant Tuberculosis and Utility of Line Probe Assay for Its Detection in Smear-Positive Sputum Samples in a Tertiary Care Hospital in South India.

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Yacoob, Fahmiya Leena; Philomina Jose, Beena; Karunakaran Lelitha, Sarada Devi; Sreenivasan, Sreelatha

2016-01-01

In a high tuberculosis burdened country like India, rapid, cost-effective, and reliable diagnostic tools for tuberculosis are an urgent need of the hour to prevent inappropriate treatment strategies and further spread of resistance. This study aimed to estimate the proportion of new smear-positive tuberculosis cases with primary resistance to rifampicin and/or isoniazid as well as identify the common mutations associated with it. Sputum of 200 newly diagnosed smear-positive cases of 1+ score and above was directly subjected to Line Probe Assay using the GenoType MTBDRplus assay kit. All samples were inoculated onto solid media and 61 samples were inoculated in automated liquid culture also. The Line Probe Assay gave hundred percent interpretable results with 2.5% of the study population showing resistant pattern. Only 1% of the cases were primary multidrug resistant tuberculosis and 1.5% showed isoniazid monoresistance. S531L and C15T were the most common genetic mutations seen for rifampicin and isoniazid resistance, respectively. 40% had absent rpoB wild type 8 band indicating probable silent mutation after clinical correlation. The average turnaround time for Line Probe Assay was far less (3.8 days) as compared to solid and liquid cultures (35.6 days and 13.5 days, resp.).

Endogenous Locus Reporter Assays.

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Liu, Yaping; Hermes, Jeffrey; Li, Jing; Tudor, Matthew

2018-01-01

Reporter gene assays are widely used in high-throughput screening (HTS) to identify compounds that modulate gene expression. Traditionally a reporter gene assay is built by cloning an endogenous promoter sequence or synthetic response elements in the regulatory region of a reporter gene to monitor transcriptional activity of a specific biological process (exogenous reporter assay). In contrast, an endogenous locus reporter has a reporter gene inserted in the endogenous gene locus that allows the reporter gene to be expressed under the control of the same regulatory elements as the endogenous gene, thus more accurately reflecting the changes seen in the regulation of the actual gene. In this chapter, we introduce some of the considerations behind building a reporter gene assay for high-throughput compound screening and describe the methods we have utilized to establish 1536-well format endogenous locus reporter and exogenous reporter assays for the screening of compounds that modulate Myc pathway activity.

Detection of selected antibiotic resistance genes using multiplex PCR assay in mastitis pathogens in the Czech Republic

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Vladimir Pyatov

2017-01-01

Full Text Available The aim of this research was to develop multiplex polymerase chain reaction assays for the detection of aminoglycoside (strA, strB, sulphonamide (sulI, sulII, tetracycline (tetA, tetB, tetK, tetM, tetO, macrolide and lincosamide (msrA, ermA, ermB, ermC, mefA/E genes of resistance in mastitis pathogens (Escherichia coli, Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae and Streptococcus dysgalactiae. Applying the established assays, we investigated the distribution of antibiotic resistance genes in the above mentioned species isolated from milk samples in the Czech Republic. Each assay consisted of seven pairs of primers. Six of them amplified fragments of antibiotic resistance genes and one pair a fragment of a species specific gene. Polymerase chain reaction conditions were optimized to amplify seven gene fragments simultaneously in one reaction. In total, 249 isolates were used, among which 111 were positive for E. coli, 52 for S. aureus and 86 for Streptococcus spp. The majority (60.2% of bacteria carried at least one antibiotic resistance gene and 44.6% were multidrug-resistant. The designed multiplex polymerase chain reaction assays may be applied as diagnostic method to replace or complement standard techniques of antibiotic susceptibility testing in the mentioned pathogens.

Detection of single-copy functional genes in prokaryotic cells by two-pass TSA-FISH with polynucleotide probes.

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Kawakami, Shuji; Hasegawa, Takuya; Imachi, Hiroyuki; Yamaguchi, Takashi; Harada, Hideki; Ohashi, Akiyoshi; Kubota, Kengo

2012-02-01

In situ detection of functional genes with single-cell resolution is currently of interest to microbiologists. Here, we developed a two-pass tyramide signal amplification (TSA)-fluorescence in situ hybridization (FISH) protocol with PCR-derived polynucleotide probes for the detection of single-copy genes in prokaryotic cells. The mcrA gene and the apsA gene in methanogens and sulfate-reducing bacteria, respectively, were targeted. The protocol showed bright fluorescence with a good signal-to-noise ratio and achieved a high efficiency of detection (>98%). The discrimination threshold was approximately 82-89% sequence identity. Microorganisms possessing the mcrA or apsA gene in anaerobic sludge samples were successfully detected by two-pass TSA-FISH with polynucleotide probes. The developed protocol is useful for identifying single microbial cells based on functional gene sequences. Copyright © 2011 Elsevier B.V. All rights reserved.

Multiple-endpoints gene alteration-based (MEGA) assay: A toxicogenomics approach for water quality assessment of wastewater effluents.

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Fukushima, Toshikazu; Hara-Yamamura, Hiroe; Nakashima, Koji; Tan, Lea Chua; Okabe, Satoshi

2017-12-01

Wastewater effluents contain a significant number of toxic contaminants, which, even at low concentrations, display a wide variety of toxic actions. In this study, we developed a multiple-endpoints gene alteration-based (MEGA) assay, a real-time PCR-based transcriptomic analysis, to assess the water quality of wastewater effluents for human health risk assessment and management. Twenty-one genes from the human hepatoblastoma cell line (HepG2), covering the basic health-relevant stress responses such as response to xenobiotics, genotoxicity, and cytotoxicity, were selected and incorporated into the MEGA assay. The genes related to the p53-mediated DNA damage response and cytochrome P450 were selected as markers for genotoxicity and response to xenobiotics, respectively. Additionally, the genes that were dose-dependently regulated by exposure to the wastewater effluents were chosen as markers for cytotoxicity. The alterations in the expression of an individual gene, induced by exposure to the wastewater effluents, were evaluated by real-time PCR and the results were validated by genotoxicity (e.g., comet assay) and cell-based cytotoxicity tests. In summary, the MEGA assay is a real-time PCR-based assay that targets cellular responses to contaminants present in wastewater effluents at the transcriptional level; it is rapid, cost-effective, and high-throughput and can thus complement any chemical analysis for water quality assessment and management. Copyright © 2017 Elsevier Ltd. All rights reserved.

Establishment of recombinase polymerase amplification assay for five hemorrhagic fever-related viruses

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Xue-feng CAO

2017-08-01

Full Text Available Objective To establish a one-step recombinase polymerase amplification (RPA method for pathogen screening and rapid detection in the field targeting for five hemorrhagic fever related viruses (Zaire ebola virus, Sudan ebola virus, Marburg virus, Lassa virus and Yellow fever virus. Methods The specific nucleic acid (NA fragments of each virus were selected as target genes by genome sequence analysis, and the primers and probes for RPA assays were designed according to the sequence. A series of diluted template genes were used for RPA detection to determine the sensitivity. The hemorrhagic fever-related viral nucleic acids were used for RPA detection to determine the specificity. The amplification experiments were carried out at different temperature ranging from 37℃ to 42℃ to validate the reaction temperature range. Results The RPA reaction systems of the five hemorrhagic fever viruses could effectively amplify the target genes, the sensitivities were between 1.5×102 and 1.5×103 copies. No cross reactions existed with the other hemorrhagic fever-related viral genes. Meanwhile, RPA assay could effectively amplify the target genes at 37-42℃. Conclusion The isothermal RPA assays of five hemorrhagic fever viruses are established, which may amply target genes fast and react at a wide temperature range, and be potentially useful for in field pathogens detection. DOI: 10.11855/j.issn.0577-7402.2017.06.09

Detection of the Light Organ Symbiont, Vibrio fischeri, in Hawaiian Seawater by Using lux Gene Probes.

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Lee, K H; Ruby, E G

1992-03-01

Symbiotic bacteria that inhabit the light-emitting organ of the Hawaiian squid Euprymna scolopes are distinctive from typical Vibrio fischeri organisms in that they are not visibly luminous when grown in laboratory culture. Therefore, the abundance of these bacteria in seawater samples cannot be estimated simply by identifying them among luminous colonies that arise on nutrient agar plates. Instead, we have used luxR and polymerase chain reaction generated luxA gene probes to identify both luminous and non-visibly luminous V. fischeri colonies by DNA-DNA hybridization. The probes were specific, hybridizing at least 50 to 100 times more strongly to immobilized DNAs from V. fischeri strains than to those of pure cultures of other related species. Thus, even non-visibly luminous V. fischeri colonies could be identified among colonies obtained from natural seawater samples by their probe-positive reaction. Bacteria in seawater samples, obtained either within or distant from squid habitats, were collected on membrane filters and incubated until colonies appeared. The filters were then observed for visibly luminous V. fischeri colonies and hybridized with the lux gene probes to determine the number of total V. fischeri colonies (both luminous and non-visibly luminous). We detected no significant differences in the abundance of luminous V. fischeri CFU in any of the water samples observed (probe-positive colonies of V. fischeri (up to 900 CFU/100 ml) were found only in seawater collected from within the natural habitats of the squids. A number of criteria were used to confirm that these probe-positive strains were indistinguishable from symbiotic V. fischeri. Therefore, the luxA and luxR gene probes were species specific and gave a reliable estimate of the number of culturable V. fischeri colonies in natural water samples.

Improving colorimetric assays through protein enzyme-assisted gold nanoparticle amplification.

Science.gov (United States)

Xie, Xiaoji; Xu, Wei; Liu, Xiaogang

2012-09-18

and function with high specificity, offering substantial advantages in both sensitivity and specificity over conventional detection methods. The screening of nuclease, methyltransferase, protease, and kinase activities can be colorimetrically performed in a straightforward manner. Finally, we discuss examples of colorimetric assays for metal ions and small molecules that constitute important advances toward visual monitoring of enzyme catalytic functions and gene expression. Although these enzyme-assisted assay methods hold great promise for myriad applications in biomedicine and bioimaging, the application of the described techniques in vivo faces formidable challenges. In addition, researchers do not fully understand the interactions of gold nanoparticles with enzyme molecules. This understanding will require the development of new techniques to probe enzyme substrate dynamics at the particle interface with higher spatial resolution and chemical specificity.

Whole-genome gene expression profiling of formalin-fixed, paraffin-embedded tissue samples.

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Craig April

2009-12-01

Full Text Available We have developed a gene expression assay (Whole-Genome DASL, capable of generating whole-genome gene expression profiles from degraded samples such as formalin-fixed, paraffin-embedded (FFPE specimens.We demonstrated a similar level of sensitivity in gene detection between matched fresh-frozen (FF and FFPE samples, with the number and overlap of probes detected in the FFPE samples being approximately 88% and 95% of that in the corresponding FF samples, respectively; 74% of the differentially expressed probes overlapped between the FF and FFPE pairs. The WG-DASL assay is also able to detect 1.3-1.5 and 1.5-2 -fold changes in intact and FFPE samples, respectively. The dynamic range for the assay is approximately 3 logs. Comparing the WG-DASL assay with an in vitro transcription-based labeling method yielded fold-change correlations of R(2 approximately 0.83, while fold-change comparisons with quantitative RT-PCR assays yielded R(2 approximately 0.86 and R(2 approximately 0.55 for intact and FFPE samples, respectively. Additionally, the WG-DASL assay yielded high self-correlations (R(2>0.98 with low intact RNA inputs ranging from 1 ng to 100 ng; reproducible expression profiles were also obtained with 250 pg total RNA (R(2 approximately 0.92, with approximately 71% of the probes detected in 100 ng total RNA also detected at the 250 pg level. When FFPE samples were assayed, 1 ng total RNA yielded self-correlations of R(2 approximately 0.80, while still maintaining a correlation of R(2 approximately 0.75 with standard FFPE inputs (200 ng.Taken together, these results show that WG-DASL assay provides a reliable platform for genome-wide expression profiling in archived materials. It also possesses utility within clinical settings where only limited quantities of samples may be available (e.g. microdissected material or when minimally invasive procedures are performed (e.g. biopsied specimens.

Development and Characterization of Probe-Based Real Time Quantitative RT-PCR Assays for Detection and Serotyping of Foot-And-Mouth Disease Viruses Circulating in West Eurasia.

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Jamal, Syed M; Belsham, Graham J

2015-01-01

Rapid and accurate diagnosis of foot-and-mouth disease (FMD) and virus serotyping are of paramount importance for control of this disease in endemic areas where vaccination is practiced. Ideally this virus characterization should be achieved without the need for virus amplification in cell culture. Due to the heterogeneity of FMD viruses (FMDVs) in different parts of the world, region specific diagnostic tests are required. In this study, hydrolysable probe-based real time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays were developed for specific detection and serotyping of the FMDVs currently circulating in West Eurasia. These assays were evaluated, in parallel with pan-FMDV diagnostic assays and earlier serotype-specific assays, using field samples originating from Pakistan and Afghanistan containing FMD viruses belonging to different sublineages of O-PanAsia, A-Iran05 and Asia-1 (Group-II and Group-VII (Sindh-08)). In addition, field samples from Iran and Bulgaria, containing FMDVs belonging to the O-PanAsiaANT-10 sublineage were also tested. Each of the three primer/probe sets was designed to be specific for just one of the serotypes O, A and Asia-1 of FMDV and detected the RNA from the target viruses with cycle threshold (CT) values comparable with those obtained with the serotype-independent pan-FMDV diagnostic assays. No cross-reactivity was observed in these assays between the heterotypic viruses circulating in the region. The assays reported here have higher diagnostic sensitivity (100% each for serotypes O and Asia-1, and 92% [95% CI = 81.4-100%] for serotype A positive samples) and specificity (100% each for serotypes O, A and Asia-1 positive samples) for the viruses currently circulating in West Eurasia compared to the serotyping assays reported earlier. Comparisons of the sequences of the primers and probes used in these assays and the corresponding regions of the circulating viruses provided explanations for the poor

Development and Characterization of Probe-Based Real Time Quantitative RT-PCR Assays for Detection and Serotyping of Foot-And-Mouth Disease Viruses Circulating in West Eurasia.

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Syed M Jamal

Full Text Available Rapid and accurate diagnosis of foot-and-mouth disease (FMD and virus serotyping are of paramount importance for control of this disease in endemic areas where vaccination is practiced. Ideally this virus characterization should be achieved without the need for virus amplification in cell culture. Due to the heterogeneity of FMD viruses (FMDVs in different parts of the world, region specific diagnostic tests are required. In this study, hydrolysable probe-based real time reverse transcription quantitative polymerase chain reaction (RT-qPCR assays were developed for specific detection and serotyping of the FMDVs currently circulating in West Eurasia. These assays were evaluated, in parallel with pan-FMDV diagnostic assays and earlier serotype-specific assays, using field samples originating from Pakistan and Afghanistan containing FMD viruses belonging to different sublineages of O-PanAsia, A-Iran05 and Asia-1 (Group-II and Group-VII (Sindh-08. In addition, field samples from Iran and Bulgaria, containing FMDVs belonging to the O-PanAsiaANT-10 sublineage were also tested. Each of the three primer/probe sets was designed to be specific for just one of the serotypes O, A and Asia-1 of FMDV and detected the RNA from the target viruses with cycle threshold (CT values comparable with those obtained with the serotype-independent pan-FMDV diagnostic assays. No cross-reactivity was observed in these assays between the heterotypic viruses circulating in the region. The assays reported here have higher diagnostic sensitivity (100% each for serotypes O and Asia-1, and 92% [95% CI = 81.4-100%] for serotype A positive samples and specificity (100% each for serotypes O, A and Asia-1 positive samples for the viruses currently circulating in West Eurasia compared to the serotyping assays reported earlier. Comparisons of the sequences of the primers and probes used in these assays and the corresponding regions of the circulating viruses provided explanations for

Comparison of CpG island methylator phenotype (CIMP) frequency in colon cancer using different probe- and gene-specific scoring alternatives on recommended multi-gene panels.

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Berg, Marianne; Hagland, Hanne R; Søreide, Kjetil

2014-01-01

In colorectal cancer a distinct subgroup of tumours demonstrate the CpG island methylator phenotype (CIMP). However, a consensus of how to score CIMP is not reached, and variation in definition may influence the reported CIMP prevalence in tumours. Thus, we sought to compare currently suggested definitions and cut-offs for methylation markers and how they influence CIMP classification in colon cancer. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), with subsequent fragment analysis, was used to investigate methylation of tumour samples. In total, 31 CpG sites, located in 8 different genes (RUNX3, MLH1, NEUROG1, CDKN2A, IGF2, CRABP1, SOCS1 and CACNA1G) were investigated in 64 distinct colon cancers and 2 colon cancer cell lines. The Ogino gene panel includes all 8 genes, in addition to the Weisenberger panel of which only 5 of the 8 genes included were investigated. In total, 18 alternative combinations of scoring of CIMP positivity on probe-, gene-, and panel-level were analysed and compared. For 47 samples (71%), the CIMP status was constant and independent of criteria used for scoring; 34 samples were constantly scored as CIMP negative, and 13 (20%) consistently scored as CIMP positive. Only four of 31 probes (13%) investigated showed no difference in the numbers of positive samples using the different cut-offs. Within the panels a trend was observed that increasing the gene-level stringency resulted in a larger difference in CIMP positive samples than increasing the probe-level stringency. A significant difference between positive samples using 'the most stringent' as compared to 'the least stringent' criteria (20% vs 46%, respectively; pCIMP depending on the cut-offs and genes included in a panel was found, with twice as many positives samples by least compared to most stringent definition used.

A specific endogenous reference for genetically modified common bean (Phaseolus vulgaris L.) DNA quantification by real-time PCR targeting lectin gene.

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Venturelli, Gustavo L; Brod, Fábio C A; Rossi, Gabriela B; Zimmermann, Naíra F; Oliveira, Jaison P; Faria, Josias C; Arisi, Ana C M

2014-11-01

The Embrapa 5.1 genetically modified (GM) common bean was approved for commercialization in Brazil. Methods for the quantification of this new genetically modified organism (GMO) are necessary. The development of a suitable endogenous reference is essential for GMO quantification by real-time PCR. Based on this, a new taxon-specific endogenous reference quantification assay was developed for Phaseolus vulgaris L. Three genes encoding common bean proteins (phaseolin, arcelin, and lectin) were selected as candidates for endogenous reference. Primers targeting these candidate genes were designed and the detection was evaluated using the SYBR Green chemistry. The assay targeting lectin gene showed higher specificity than the remaining assays, and a hydrolysis probe was then designed. This assay showed high specificity for 50 common bean samples from two gene pools, Andean and Mesoamerican. For GM common bean varieties, the results were similar to those obtained for non-GM isogenic varieties with PCR efficiency values ranging from 92 to 101 %. Moreover, this assay presented a limit of detection of ten haploid genome copies. The primers and probe developed in this work are suitable to detect and quantify either GM or non-GM common bean.

Nucleic acid hybridization assays employing dA-tailed capture probes. II. Advanced multiple capture methods

International Nuclear Information System (INIS)

Hunsaker, W.R.; Badri, H.; Lombardo, M.; Collins, M.L.

1989-01-01

A fourth capture is added to the reversible target capture procedure. This results in an improved radioisotopic detection limit of 7.3 x 10(-21) mol of target. In addition, the standard triple capture method is converted into a nonradioactive format with a detection limit of under 1 amol of target. The principal advantage of nonradioactive detection is that the entire assay can be performed in about 1 h. Nucleic acids are released from cells in the presence of the (capture probe) which contains a 3'-poly(dA) sequence and the (labeled probe) which contains a detectable nonradioactive moiety such as biotin. After a brief hybridization in solution, the target is captured on oligo(dT) magnetic particles. The target is further purified from sample impurities and excess labeled probe by recapture either once or twice more on fresh magnetic particles. The highly purified target is then concentrated to 200 nl by recapture onto a poly(dT) nitrocellulose filter and rapidly detected with streptavidin-alkaline phosphatase using bromochloroindolyl phosphate and nitroblue tetrazolium. Using this procedure, as little as 0.25 amol of a target plasmid has been detected nonradioactively in crude samples in just 1 h without prior purification of the DNA and RNA. Finally, a new procedure called background capture is introduced to complement the background-reducing power of RTC

Development of an event-specific hydrolysis probe quantitative real-time polymerase chain reaction assay for Embrapa 5.1 genetically modified common bean (Phaseolus vulgaris).

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Treml, Diana; Venturelli, Gustavo L; Brod, Fábio C A; Faria, Josias C; Arisi, Ana C M

2014-12-10

A genetically modified (GM) common bean event, namely Embrapa 5.1, resistant to the bean golden mosaic virus (BGMV), was approved for commercialization in Brazil. Brazilian regulation for genetically modified organism (GMO) labeling requires that any food containing more than 1% GMO be labeled. The event-specific polymerase chain reaction (PCR) method has been the primary trend for GMO identification and quantitation because of its high specificity based on the flanking sequence. This work reports the development of an event-specific assay, named FGM, for Embrapa 5.1 detection and quantitation by use of SYBR Green or hydrolysis probe. The FGM assay specificity was tested for Embrapa 2.3 event (a noncommercial GM common bean also resistant to BGMV), 46 non-GM common bean varieties, and other crop species including maize, GM maize, soybean, and GM soybean. The FGM assay showed high specificity to detect the Embrapa 5.1 event. Standard curves for the FGM assay presented a mean efficiency of 95% and a limit of detection (LOD) of 100 genome copies in the presence of background DNA. The primers and probe developed are suitable for the detection and quantitation of Embrapa 5.1.

Avoiding inconsistencies over time and tracking difficulties in Applied Biosystems AB1700™/Panther™ probe-to-gene annotations

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Benecke Arndt

2005-12-01

Full Text Available Abstract Background Significant inconsistencies between probe-to-gene annotations between different releases of probe set identifiers by commercial microarray platform solutions have been reported. Such inconsistencies lead to misleading or ambiguous interpretation of published gene expression results. Results We report here similar inconsistencies in the probe-to-gene annotation of Applied Biosystems AB1700 data, demonstrating that this is not an isolated concern. Moreover, the online information source PANTHER does not provide information required to track such inconsistencies, hence, even correctly annotated datasets, when resubmitted after PANTHER was updated to a new probe-to-gene annotation release, will generate differing results without any feedback on the origin of the change. Conclusion The importance of unequivocal annotation of microarray experiments can not be underestimated. Inconsistencies greatly diminish the usefulness of the technology. Novel methods in the analysis of transcriptome profiles often rely on large disparate datasets stemming from multiple sources. The predictive and analytic power of such approaches rapidly diminishes if only least-common subsets can be used for analysis. We present here the information that needs to be provided together with the raw AB1700 data, and the information required together with the biologic interpretation of such data to avoid inconsistencies and tracking difficulties.

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

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Wilson Zoe A

2008-06-01

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

Oligonucleotide PIK3CA/Chromosome 3 Dual in Situ Hybridization Automated Assay with Improved Signals, One-Hour Hybridization, and No Use of Blocking DNA.

Science.gov (United States)

Zhang, Wenjun; Hubbard, Antony; Baca-Parkinson, Leslie; Stanislaw, Stacey; Vladich, Frank; Robida, Mark D; Grille, James G; Maxwell, Daniel; Tsao, Tsu-Shuen; Carroll, William; Gardner, Tracie; Clements, June; Singh, Shalini; Tang, Lei

2015-09-01

The PIK3CA gene at chromosome 3q26.32 was found to be amplified in up to 45% of patients with squamous cell carcinoma of the lung. The strong correlation between PIK3CA amplification and increased phosphatidylinositol 3-kinase (PI3K) pathway activities suggested that PIK3CA gene copy number is a potential predictive biomarker for PI3K inhibitors. Currently, all microscopic assessments of PIK3CA and chromosome 3 (CHR3) copy numbers use fluorescence in situ hybridization. PIK3CA probes are derived from bacterial artificial chromosomes whereas CHR3 probes are derived mainly from the plasmid pHS05. These manual fluorescence in situ hybridization assays mandate 12- to 18-hour hybridization and use of blocking DNA from human sources. Moreover, fluorescence in situ hybridization studies provide limited morphologic assessment and suffer from signal decay. We developed an oligonucleotide-based bright-field in situ hybridization assay that overcomes these shortcomings. This assay requires only a 1-hour hybridization with no need for blocking DNA followed by indirect chromogenic detection. Oligonucleotide probes produced discrete and uniform CHR3 stains superior to those from the pHS05 plasmid. This assay achieved successful staining in 100% of the 195 lung squamous cell carcinoma resections and in 94% of the 33 fine-needle aspirates. This robust automated bright-field dual in situ hybridization assay for the simultaneous detection of PIK3CA and CHR3 centromere provides a potential clinical diagnostic method to assess PIK3CA gene abnormality in lung tumors. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Real-time RPA assay for rapid detection and differentiation of wild-type pseudorabies and gE-deleted vaccine viruses.

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Wang, Jianchang; Liu, Libing; Wang, Jinfeng; Pang, Xiaoyu; Yuan, Wanzhe

2018-02-15

The objective of this study was to develop a dual real-time recombinase polymerase amplification (RPA) assay using exo probes for the detection and differentiation of pseudorabies virus (PRV). Specific RPA primers and probes were designed for gB and gE genes of PRV within the conserved region of viral genome. The reaction process can be completed in 20 min at 39 °C. The dual real-time RPA assay performed in the single tube was capable of specific detecting and differentiating of the wild-type PRV and gE-deleted vaccine strains, without cross-reactions with other non-targeted pig viruses. The analytical sensitivity of the assay was 10 2 copies for gB and gE genes. The dual real-time RPA demonstrated a 100% diagnostic agreement with the real-time PCR on 4 PRV strains and 37 clinical samples. Through the linear regression analysis, the R 2 value of the real-time RPA and the real-time PCR for gB and gE was 0.983 and 0.992, respectively. The dual real-time RPA assay provides an alternative useful tool for rapid, simple, and reliable detection and differentiation of PRV, especially in remote and rural areas. Copyright © 2017 Elsevier Inc. All rights reserved.

Multiplex real-time PCR assay for the detection of extended-spectrum β-lactamase and carbapenemase genes using melting curve analysis.

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Singh, Prashant; Pfeifer, Yvonne; Mustapha, Azlin

2016-05-01

Real-time PCR melt curve assays for the detection of β-lactamase, extended-spectrum β-lactamase and carbapenemase genes in Gram-negative bacteria were developed. Two multiplex real-time PCR melt curve assays were developed for the detection of ten common β-lactamase genes: blaKPC-like, blaOXA-48-like, blaNDM-like, blaVIM-like, blaIMP-like, blaCTX-M-1+2-group, blaCMY-like, blaACC-like, blaSHV-like and blaTEM-like. The assays were evaluated using 25 bacterial strains and 31 DNA samples (total n=56) comprising different Enterobacteriaceae genera and Pseudomonas spp. These strains were previously characterized at five research institutes. Each resistance gene targeted in this study generated a non-overlapping and distinct melt curve peak. The assay worked effectively and detected the presence of additional resistance genes in 23 samples. The assays developed in this study offer a simple, low cost method for the detection of prevalent β-lactamase, ESBL and carbapenemase genes among Gram-negative pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.

TaqMan MGB probe fluorescence real-time quantitative PCR for rapid detection of Chinese Sacbrood virus.

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Ma Mingxiao

Full Text Available Sacbrood virus (SBV is a picorna-like virus that affects honey bees (Apis mellifera and results in the death of the larvae. Several procedures are available to detect Chinese SBV (CSBV in clinical samples, but not to estimate the level of CSBV infection. The aim of this study was develop an assay for rapid detection and quantification of this virus. Primers and probes were designed that were specific for CSBV structural protein genes. A TaqMan minor groove binder (MGB probe-based, fluorescence real-time quantitative PCR was established. The specificity, sensitivity and stability of the assay were assessed; specificity was high and there were no cross-reactivity with healthy larvae or other bee viruses. The assay was applied to detect CSBV in 37 clinical samples and its efficiency was compared with clinical diagnosis, electron microscopy observation, and conventional RT-PCR. The TaqMan MGB-based probe fluorescence real-time quantitative PCR for CSBV was more sensitive than other methods tested. This assay was a reliable, fast, and sensitive method that was used successfully to detect CSBV in clinical samples. The technology can provide a useful tool for rapid detection of CSBV. This study has established a useful protocol for CSBV testing, epidemiological investigation, and development of animal models.

Detection of the Light Organ Symbiont, Vibrio fischeri, in Hawaiian Seawater by Using lux Gene Probes †

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Lee, Kyu-Ho; Ruby, Edward G.

1992-01-01

Symbiotic bacteria that inhabit the light-emitting organ of the Hawaiian squid Euprymna scolopes are distinctive from typical Vibrio fischeri organisms in that they are not visibly luminous when grown in laboratory culture. Therefore, the abundance of these bacteria in seawater samples cannot be estimated simply by identifying them among luminous colonies that arise on nutrient agar plates. Instead, we have used luxR and polymerase chain reaction generated luxA gene probes to identify both luminous and non-visibly luminous V. fischeri colonies by DNA-DNA hybridization. The probes were specific, hybridizing at least 50 to 100 times more strongly to immobilized DNAs from V. fischeri strains than to those of pure cultures of other related species. Thus, even non-visibly luminous V. fischeri colonies could be identified among colonies obtained from natural seawater samples by their probe-positive reaction. Bacteria in seawater samples, obtained either within or distant from squid habitats, were collected on membrane filters and incubated until colonies appeared. The filters were then observed for visibly luminous V. fischeri colonies and hybridized with the lux gene probes to determine the number of total V. fischeri colonies (both luminous and non-visibly luminous). We detected no significant differences in the abundance of luminous V. fischeri CFU in any of the water samples observed (≤1 to 3 CFU/100 ml). However, probe-positive colonies of V. fischeri (up to 900 CFU/100 ml) were found only in seawater collected from within the natural habitats of the squids. A number of criteria were used to confirm that these probe-positive strains were indistinguishable from symbiotic V. fischeri. Therefore, the luxA and luxR gene probes were species specific and gave a reliable estimate of the number of culturable V. fischeri colonies in natural water samples. Images PMID:16348678

The detection and differentiation of methicillin-resistant and methicillin-susceptible Staphylococcus aureus endocarditis by using the BD GeneOhm StaphSR Assay.

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Frey, Amy B; Wilson, Deborah A; LaSalvia, Margaret M; Tan, Carmela D; Rodriguez, E Rene; Shrestha, Nabin K; Hall, Gerri S; Procop, Gary W

2011-11-01

We use the BD GeneOhm StaphSR Assay (BD Diagnostics, Oakville, Canada) to screen for Staphylococcus aureus nasal colonization and sought to evaluate this assay for the assessment of valve specimens from patients with endocarditis. We examined 23 paired fresh and formalin-fixed, paraffin-embedded cardiac valve tissue samples, 12 of which had S aureus endocarditis, using the BD GeneOhm StaphSR Assay for the detection and differentiation of methicillin-susceptible and methicillin-resistant S aureus. This assay appropriately characterized all specimens with respect to the presence or absence of S aureus. There was an 87.5% correlation between the presence or absence of the mecA gene and the oxacillin susceptibility results for the S aureus isolates studied. The GeneOhm StaphSR assay accurately detected S aureus in cardiac valve tissue samples. Rare discordances were observed between oxacillin susceptibility status and mecA gene detection by this assay.

A novel approach to simultaneously scan genes at fragile sites

International Nuclear Information System (INIS)

Willem, Pascale; Brown, Jacqueline; Schouten, Jan

2006-01-01

Fragile sites are regions of the genome sensitive to replication stress and to exposure to environmental carcinogens. The two most commonly expressed fragile sites FRA3B and FRA16D host the histidine triad (FHIT) and WW domain containing oxidoreductase (WWOX) genes respectively. There is growing evidence that both genes contribute to cancer development and they are frequently altered by allelic and homozygous deletions in a variety of tumors. Their status is linked to prognosis in several malignancies and they are thought to be involved in early tumorigenesis. The loci for FHIT and WWOX both span over a megabase but the genes encode for small transcripts. Thus the screening of intragenic deletion can be difficult and has relied on loss of heterozygosity LOH assays, or genomic arrays. Multiplex ligation dependent probe amplification MLPA, allows for the detection of deletions/duplications and relative quantification of up to 40 specific probes in a single assay. A FHIT/WWOX MLPA assay was designed, applied and validated in five esophageal squamous cell carcinoma ESCC, cell lines established in South Africa where this cancer is of high prevalence. Sixteen probes covered all FHIT exons and 7 probes covered WWOX. Both homozygous and hemizygous deletions were detected in FHIT, in four of the cell lines with a preferential deletion of exons 5 and 4. Chromosome 3 short arm was present in normal copy number indicating that deletions were site specific. In contrast WWOX was not altered in any cell lines. RT-PCR expression pattern paralleled the pattern of deletions. Ten primary ESCC tumor specimens were subsequently screened with this assay. FHIT exon deletions were found in four of them. This method offers an alternative to loss of heterozygosity studies. Simultaneous scanning of FHIT and WWOX exons in the context of early tumorigenesis and tumor progression, may help clarify the mechanistic events related to cancer development which are not revealed by imuno

Advance in plasma SEPT9 gene methylation assay for colorectal cancer early detection.

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Wang, Yu; Chen, Pei-Min; Liu, Rong-Bin

2018-01-15

This review article summarizes the research advances of the plasma-based SEPT9 gene methylation assay for the clinical detection of colorectal cancer and its limitations. Colorectal cancer is a common malignancy with a poor prognosis and a high mortality, for which early detection and diagnosis are particularly crucial for the high-risk groups. Increasing evidence supported that SEPT9 gene methylation is associated with the pathogenesis of colorectal cancer and that detecting the level of methylation of SEPT9 in the peripheral blood can be used for screening of colorectal cancer in susceptible populations. In recent years, the data obtained in clinical studies demonstrated that the SEPT9 gene methylation assay has a good diagnostic performance with regard to both sensitivity and specificity with the advantage of better acceptability, convenience and compliance with serological testing compared with fecal occult blood tests and carcinoembryonic antigen for colorectal cancer (CRC). Furthermore, the combination of multiple methods or markers has become a growing trend for CRC detection and screening. Nevertheless, the clinical availability of the methylated SEPT9 assay is still limited because of the large degree of sample heterogeneity caused by demographic characteristics, pathological features, comorbidities and/or technique selection. Another factor is the cost-effectiveness of colorectal cancer screening strategies that hinders its large-scale application. In addition, improvements in its accuracy in detecting adenomas and premalignant polyps are required.

Continuously tunable nucleic acid hybridization probes.

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Wu, Lucia R; Wang, Juexiao Sherry; Fang, John Z; Evans, Emily R; Pinto, Alessandro; Pekker, Irena; Boykin, Richard; Ngouenet, Celine; Webster, Philippa J; Beechem, Joseph; Zhang, David Yu

2015-12-01

In silico-designed nucleic acid probes and primers often do not achieve favorable specificity and sensitivity tradeoffs on the first try, and iterative empirical sequence-based optimization is needed, particularly in multiplexed assays. We present a novel, on-the-fly method of tuning probe affinity and selectivity by adjusting the stoichiometry of auxiliary species, which allows for independent and decoupled adjustment of the hybridization yield for different probes in multiplexed assays. Using this method, we achieved near-continuous tuning of probe effective free energy. To demonstrate our approach, we enforced uniform capture efficiency of 31 DNA molecules (GC content, 0-100%), maximized the signal difference for 11 pairs of single-nucleotide variants and performed tunable hybrid capture of mRNA from total RNA. Using the Nanostring nCounter platform, we applied stoichiometric tuning to simultaneously adjust yields for a 24-plex assay, and we show multiplexed quantitation of RNA sequences and variants from formalin-fixed, paraffin-embedded samples.

Comparison of CpG island methylator phenotype (CIMP frequency in colon cancer using different probe- and gene-specific scoring alternatives on recommended multi-gene panels.

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Marianne Berg

Full Text Available BACKGROUND: In colorectal cancer a distinct subgroup of tumours demonstrate the CpG island methylator phenotype (CIMP. However, a consensus of how to score CIMP is not reached, and variation in definition may influence the reported CIMP prevalence in tumours. Thus, we sought to compare currently suggested definitions and cut-offs for methylation markers and how they influence CIMP classification in colon cancer. METHODS: Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA, with subsequent fragment analysis, was used to investigate methylation of tumour samples. In total, 31 CpG sites, located in 8 different genes (RUNX3, MLH1, NEUROG1, CDKN2A, IGF2, CRABP1, SOCS1 and CACNA1G were investigated in 64 distinct colon cancers and 2 colon cancer cell lines. The Ogino gene panel includes all 8 genes, in addition to the Weisenberger panel of which only 5 of the 8 genes included were investigated. In total, 18 alternative combinations of scoring of CIMP positivity on probe-, gene-, and panel-level were analysed and compared. RESULTS: For 47 samples (71%, the CIMP status was constant and independent of criteria used for scoring; 34 samples were constantly scored as CIMP negative, and 13 (20% consistently scored as CIMP positive. Only four of 31 probes (13% investigated showed no difference in the numbers of positive samples using the different cut-offs. Within the panels a trend was observed that increasing the gene-level stringency resulted in a larger difference in CIMP positive samples than increasing the probe-level stringency. A significant difference between positive samples using 'the most stringent' as compared to 'the least stringent' criteria (20% vs 46%, respectively; p<0.005 was demonstrated. CONCLUSIONS: A statistical significant variation in the frequency of CIMP depending on the cut-offs and genes included in a panel was found, with twice as many positives samples by least compared to most stringent definition

Probe-level linear model fitting and mixture modeling results in high accuracy detection of differential gene expression

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Lemieux Sébastien

2006-08-01

Full Text Available Abstract Background The identification of differentially expressed genes (DEGs from Affymetrix GeneChips arrays is currently done by first computing expression levels from the low-level probe intensities, then deriving significance by comparing these expression levels between conditions. The proposed PL-LM (Probe-Level Linear Model method implements a linear model applied on the probe-level data to directly estimate the treatment effect. A finite mixture of Gaussian components is then used to identify DEGs using the coefficients estimated by the linear model. This approach can readily be applied to experimental design with or without replication. Results On a wholly defined dataset, the PL-LM method was able to identify 75% of the differentially expressed genes within 10% of false positives. This accuracy was achieved both using the three replicates per conditions available in the dataset and using only one replicate per condition. Conclusion The method achieves, on this dataset, a higher accuracy than the best set of tools identified by the authors of the dataset, and does so using only one replicate per condition.

Identification of differentially expressed genes between developing seeds of different soybean cultivars

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Rongshuang Lin

2015-12-01

Full Text Available Soybean is a major source of protein and oil and a primary feedstock for biodiesel production. Research on soybean seed composition and yield has revealed that protein, oil and yield are controlled quantitatively and quantitative trait loci (QTL have been identified for each of these traits. However, very limited information is available regarding the genetic mechanisms controlling seed composition and yield. To help address this deficiency, we used Affymetrix Soybean GeneChips® to identify genes that are differentially expressed between developing seeds of the Minsoy and Archer soybean cultivars, which differ in seed weight, yield, protein content and oil content. A total of 700 probe sets were found to be expressed at significantly different (defined as having an adjusted p-value below or equal to 0.05 and an at least 2-fold difference levels between the two cultivars at one or more of the three developmental stages and in at least one of the two years assayed. Comparison of data from soybeans collected in two different years revealed that 97 probe sets were expressed at significantly different levels in both years. Functional annotations were assigned to 78% of these 97 probe sets based on the SoyBase Affymetrix™ GeneChip® Soybean Genome Array Annotation. Genes involved in receptor binding/activity and protein binding are overrepresented among the group of 97 probe sets that were differentially expressed in both years assayed. Probe sets involved in growth/development, signal transduction, transcription, defense/stress response and protein and lipid metabolism were also identified among the 97 probe sets and their possible implications in the regulation of agronomic traits are discussed. As the Minsoy and Archer soybean cultivars differ with respect to seed size, yield, protein content and lipid content, some of the differentially expressed probe sets identified in this study may thus play important roles in controlling these traits

Reverse line blot probe design and polymerase chain reaction optimization for bloodmeal analysis of ticks from the eastern United States.

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Scott, M C; Harmon, J R; Tsao, J I; Jones, C J; Hickling, G J

2012-05-01

Determining the host preference of vector ticks is vital to elucidating the eco-epidemiology of the diseases they spread. Detachment of ticks from captured hosts can provide evidence of feeding on those host species, but only for those species that are feasible to capture. Recently developed, highly sensitive molecular assays show great promise in allowing host selection to be determined from minute traces of host DNA that persist in recently molted ticks. Using methods developed in Europe as a starting-point, we designed 12S rDNA mitochondrial gene probes suitable for use in a reverse line blot (RLB) assay of ticks feeding on common host species in the eastern United States. This is the first study to use the 12S mitochondrial gene in a RLB bloodmeal assay in North America. The assay combines conventional PCR with a biotin-labeled primer and reverse line blots that can be stripped and rehybridized up to 20 times, making the method less expensive and more straightforward to interpret than previous methods of tick bloodmeal identification. Probes were designed that target the species, genus, genus group, family, order, or class of eight reptile, 13 birds, and 32 mammal hosts. After optimization, the RLB assay correctly identified the current hostspecies for 99% of ticks [Amblyomma americanum (L.) and eight other ixodid tick species] collected directly from known hosts. The method identified previous-host DNA for approximately half of all questing ticks assayed. Multiple bloodmeal determinations were obtained in some instances from feeding and questing ticks; this pattern is consistent with previous RLB studies but requires further investigation. Development of this probe library, suitable for eastern U.S. ecosystems, opens new avenues for eco-epidemiological investigations of this region's tick-host systems.

Rapid detection of pathological mutations and deletions of the haemoglobin beta gene (HBB) by High Resolution Melting (HRM) analysis and Gene Ratio Analysis Copy Enumeration PCR (GRACE-PCR).

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Turner, Andrew; Sasse, Jurgen; Varadi, Aniko

2016-10-19

Inherited disorders of haemoglobin are the world's most common genetic diseases, resulting in significant morbidity and mortality. The large number of mutations associated with the haemoglobin beta gene (HBB) makes gene scanning by High Resolution Melting (HRM) PCR an attractive diagnostic approach. However, existing HRM-PCR assays are not able to detect all common point mutations and have only a very limited ability to detect larger gene rearrangements. The aim of the current study was to develop a HBB assay, which can be used as a screening test in highly heterogeneous populations, for detection of both point mutations and larger gene rearrangements. The assay is based on a combination of conventional HRM-PCR and a novel Gene Ratio Analysis Copy Enumeration (GRACE) PCR method. HRM-PCR was extensively optimised, which included the use of an unlabelled probe and incorporation of universal bases into primers to prevent interference from common non-pathological polymorphisms. GRACE-PCR was employed to determine HBB gene copy numbers relative to a reference gene using melt curve analysis to detect rearrangements in the HBB gene. The performance of the assay was evaluated by analysing 410 samples. A total of 44 distinct pathological genotypes were detected. In comparison with reference methods, the assay has a sensitivity of 100 % and a specificity of 98 %. We have developed an assay that detects both point mutations and larger rearrangements of the HBB gene. This assay is quick, sensitive, specific and cost effective making it suitable as an initial screening test that can be used for highly heterogeneous cohorts.

Putative and unique gene sequence utilization for the design of species specific probes as modeled by Lactobacillus plantarum

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The concept of utilizing putative and unique gene sequences for the design of species specific probes was tested. The abundance profile of assigned functions within the Lactobacillus plantarum genome was used for the identification of the putative and unique gene sequence, csh. The targeted gene (cs...

Comparison of gull-specific assays targeting 16S rRNA gene of Catellicoccus marimammalium and Streptococcus spp.

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Gulls have been implicated as a source of fecal contamination in inland and coastal waters. Only one gull-specific assay is currently available (i.e., gull2 qPCR assay). This assay is based on the 16S rRNA gene of Catellicocclls marimammalium and has showed a high level of host-s...

Assay for identification of heterozygous single-nucleotide polymorphism (Ala67Thr in human poliovirus receptor gene

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Shyam Sundar Nandi

2016-01-01

Results: A new SNP assay for detection of heterozygous Ala67Thr genotype was developed and validated by testing 150 DNA samples. Heterozygous CD155 was detected in 27.33 per cent (41/150 of DNA samples tested by both SNP detection assay and sequencing. Interpretation & conclusions: The SNP detection assay was successfully developed for identification of Ala67Thr polymorphism in human PVR/CD155 gene. The SNP assay will be useful for large scale screening of DNA samples.

Rapid assessment of repair of ultraviolet DNA damage with a modified host-cell reactivation assay using a luciferase reporter gene and correlation with polymorphisms of DNA repair genes in normal human lymphocytes

Energy Technology Data Exchange (ETDEWEB)

Qiao Yawei; Spitz, Margaret R.; Guo Zhaozheng; Hadeyati, Mohammad; Grossman, Lawrence; Kraemer, Kenneth H.; Wei Qingyi

2002-11-30

As DNA repair plays an important role in genetic susceptibility to cancer, assessment of the DNA repair phenotype is critical for molecular epidemiological studies of cancer. In this report, we compared use of the luciferase (luc) reporter gene in a host-cell reactivation (HCR) (LUC) assay of repair of ultraviolet (UV) damage to DNA to use of the chloramphenicol (cat) gene-based HCR (CAT) assay we used previously for case-control studies. We performed both the assays on cryopreserved lymphocytes from 102 healthy non-Hispanic white subjects. There was a close correlation between DNA repair capacity (DRC) as measured by the LUC and CAT assays. Although these two assays had similar variation, the LUC assay was faster and more sensitive. We also analyzed the relationship between DRC and the subjects' previously determined genotypes for four polymorphisms of two nucleotide-excision repair (NER) genes (in intron 9 of xeroderma pigmentosum (XP) C and exons 6, 10 and 23 of XPD) and one polymorphism of a base-excision repair gene in exon 10 of X-ray complementing group 1 (XRCC1). The DRC was significantly lower in subjects homozygous for one or more polymorphisms of the two NER genes than in subjects with other genotypes (P=0.010). In contrast, the polymorphic XRCC1 allele had no significant effect on DRC. These results suggest that the post-UV LUC assay measures NER phenotype and that polymorphisms of XPC and XPD genes modulate DRC. For population studies of the DNA repair phenotype, many samples need to be evaluated, and so the LUC assay has several advantages over the CAT assay: the LUC assay was more sensitive, had less variation, was not radioactive, was easier to perform, and required fewer cryopreserved cells. These features make the LUC-based HCR assay suitable for molecular epidemiological studies.

Use of gene probes to assess the impact and effectiveness of aerobic in situ bioremediation of TCE

Energy Technology Data Exchange (ETDEWEB)

Hazen, Terry C.; Chakraborty, Romy; Fleming, James M.; Gregory, Ingrid R.; Bowman, John P.; Jimenez, Luis; Zhang, Dai; Pfiffner, Susan M.; Brockman, Fred J.; Sayler, Gary S.

2009-03-15

Gene probe hybridization was used to determine distribution and expression of co-metabolic genes at a contaminated site as it underwent in situ methanotrophic bioremediation of trichloroethylene (TCE). The bioremediation strategies tested included a series of air, air:methane, and air:methane:nutrient pulses of the test plot using horizontal injection wells. During the test period, the levels of TCE reduced drastically in almost all test samples. Sediment core samples (n = 367) taken from 0 m (surface)-43 m depth were probed for gene coding for methanotrophic soluble methane monooxygenase (sMMO) and heterotrophic toluene dioxygenase (TOD), which are known to co-metabolize TCE. The same sediment samples were also probed for genes coding for methanol dehydrogenase (MDH) (catalyzing the oxidation of methanol to formaldehyde) to assess specifically changes in methylotrophic bacterial populations in the site. Gene hybridization results showed that the frequency of detection of sMMO genes were stimulated approximately 250% following 1% methane:air (v/v) injection. Subsequent injection of 4% methane:air (v/v) resulted in an 85% decline probably due to nutrient limitations, since addition of nutrients (gaseous nitrogen and phosphorus) thereafter caused an increase in the frequency of detection of sMMO genes. Detection of TOD genes declined during the process, and eventually they were non-detectable by the final treatment, suggesting that methanotrophs displaced the TOD gene containing heterotrophs. Active transcription of sMMO and TOD was evidenced by hybridization to mRNA. These analyses combined with results showing the concomitant decline in TCE concentrations, increases in chloride concentration and increases in methanotroph viable counts, provide multiple lines of evidence that TCE remediation was caused specifically by methanotrophs. Our results suggest that sMMO genes are responsible for most, if not all, of the observed biodegradation of TCE. This study

Homogeneous versus heterogeneous probes for microbial ecological microarrays.

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Bae, Jin-Woo; Park, Yong-Ha

2006-07-01

Microbial ecological microarrays have been developed for investigating the composition and functions of microorganism communities in environmental niches. These arrays include microbial identification microarrays, which use oligonucleotides, gene fragments or microbial genomes as probes. In this article, the advantages and disadvantages of each type of probe are reviewed. Oligonucleotide probes are currently useful for probing uncultivated bacteria that are not amenable to gene fragment probing, whereas the functional gene fragments amplified randomly from microbial genomes require phylogenetic and hierarchical categorization before use as microbial identification probes, despite their high resolution for both specificity and sensitivity. Until more bacteria are sequenced and gene fragment probes are thoroughly validated, heterogeneous bacterial genome probes will provide a simple, sensitive and quantitative tool for exploring the ecosystem structure.

Gene-mutation assays in lambda-lacZ transgenic mice : comparison of lacZ with endogenous genes in splenocytes and small intestinal epithelium

NARCIS (Netherlands)

Delft, J.H.M. van; Bergmans, A.; Dam, F.J. van; Tates, A.D.; Howard, L.; Winton, D.J.; Baan, R.A.

1998-01-01

Comparison of results derived from transgenic animal gene-mutation assays with those from mutation analyses in endogenous genes is an important step in the validation of the former. We have used λlacZ transgenic mice to study alkylation-induced mutagenesis in vivo in (a) lacZ and hprt in spleen

Diagnostic performance of automated liquid culture and molecular line probe assay in smear-negative pulmonary tuberculosis.

Science.gov (United States)

Kotwal, Aarti; Biswas, Debasis; Raghuvanshi, Shailendra; Sindhwani, Girish; Kakati, Barnali; Sharma, Shweta

2017-04-01

The diagnosis of smear-negative pulmonary tuberculosis (PTB) is particularly challenging, and automated liquid culture and molecular line probe assays (LPA) may prove particularly useful. The objective of our study was to evaluate the diagnostic potential of automated liquid culture (ALC) technology and commercial LPA in sputum smear-negative PTB suspects. Spot sputum samples were collected from 145 chest-symptomatic smear-negative patients and subjected to ALC, direct drug susceptibility test (DST) testing and LPA, as per manufacturers' instructions. A diagnostic yield of 26.2% was observed among sputum smear-negative TB suspects with 47.4% of the culture isolates being either INH- and/or rifampicin-resistant. Complete agreement was observed between the results of ALC assay and LPA except for two isolates which demonstrated sensitivity to INH and rifampicin at direct DST but were rifampicin-resistant in LPA. Two novel mutations were also detected among the multidrug isolates by LPA. In view of the diagnostic challenges associated with the diagnosis of TB in sputum smear-negative patients, our study demonstrates the applicability of ALC and LPA in establishing diagnostic evidence of TB.

Detecting variants with Metabolic Design, a new software tool to design probes for explorative functional DNA microarray development

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Gravelat Fabrice

2010-09-01

Full Text Available Abstract Background Microorganisms display vast diversity, and each one has its own set of genes, cell components and metabolic reactions. To assess their huge unexploited metabolic potential in different ecosystems, we need high throughput tools, such as functional microarrays, that allow the simultaneous analysis of thousands of genes. However, most classical functional microarrays use specific probes that monitor only known sequences, and so fail to cover the full microbial gene diversity present in complex environments. We have thus developed an algorithm, implemented in the user-friendly program Metabolic Design, to design efficient explorative probes. Results First we have validated our approach by studying eight enzymes involved in the degradation of polycyclic aromatic hydrocarbons from the model strain Sphingomonas paucimobilis sp. EPA505 using a designed microarray of 8,048 probes. As expected, microarray assays identified the targeted set of genes induced during biodegradation kinetics experiments with various pollutants. We have then confirmed the identity of these new genes by sequencing, and corroborated the quantitative discrimination of our microarray by quantitative real-time PCR. Finally, we have assessed metabolic capacities of microbial communities in soil contaminated with aromatic hydrocarbons. Results show that our probe design (sensitivity and explorative quality can be used to study a complex environment efficiently. Conclusions We successfully use our microarray to detect gene expression encoding enzymes involved in polycyclic aromatic hydrocarbon degradation for the model strain. In addition, DNA microarray experiments performed on soil polluted by organic pollutants without prior sequence assumptions demonstrate high specificity and sensitivity for gene detection. Metabolic Design is thus a powerful, efficient tool that can be used to design explorative probes and monitor metabolic pathways in complex environments

Unlabeled probes for the detection and typing of herpes simplex virus.

Science.gov (United States)

Dames, Shale; Pattison, David C; Bromley, L Kathryn; Wittwer, Carl T; Voelkerding, Karl V

2007-10-01

Unlabeled probe detection with a double-stranded DNA (dsDNA) binding dye is one method to detect and confirm target amplification after PCR. Unlabeled probes and amplicon melting have been used to detect small deletions and single-nucleotide polymorphisms in assays where template is in abundance. Unlabeled probes have not been applied to low-level target detection, however. Herpes simplex virus (HSV) was chosen as a model to compare the unlabeled probe method to an in-house reference assay using dual-labeled, minor groove binding probes. A saturating dsDNA dye (LCGreen Plus) was used for real-time PCR. HSV-1, HSV-2, and an internal control were differentiated by PCR amplicon and unlabeled probe melting analysis after PCR. The unlabeled probe technique displayed 98% concordance with the reference assay for the detection of HSV from a variety of archived clinical samples (n = 182). HSV typing using unlabeled probes was 99% concordant (n = 104) to sequenced clinical samples and allowed for the detection of sequence polymorphisms in the amplicon and under the probe. Unlabeled probes and amplicon melting can be used to detect and genotype as few as 10 copies of target per reaction, restricted only by stochastic limitations. The use of unlabeled probes provides an attractive alternative to conventional fluorescence-labeled, probe-based assays for genotyping and detection of HSV and might be useful for other low-copy targets where typing is informative.

Using Morpholinos to Probe Gene Networks in Sea Urchin.

Science.gov (United States)

Materna, Stefan C

2017-01-01

The control processes that underlie the progression of development can be summarized in maps of gene regulatory networks (GRNs). A critical step in their assembly is the systematic perturbation of network candidates. In sea urchins the most important method for interfering with expression in a gene-specific way is application of morpholino antisense oligonucleotides (MOs). MOs act by binding to their sequence complement in transcripts resulting in a block in translation or a change in splicing and thus result in a loss of function. Despite the tremendous success of this technology, recent comparisons to mutants generated by genome editing have led to renewed criticism and challenged its reliability. As with all methods based on sequence recognition, MOs are prone to off-target binding that may result in phenotypes that are erroneously ascribed to the loss of the intended target. However, the slow progression of development in sea urchins has enabled extremely detailed studies of gene activity in the embryo. This wealth of knowledge paired with the simplicity of the sea urchin embryo enables careful analysis of MO phenotypes through a variety of methods that do not rely on terminal phenotypes. This article summarizes the use of MOs in probing GRNs and the steps that should be taken to assure their specificity.

Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists

DEFF Research Database (Denmark)

Lallemand, Christophe; Kavrochorianou, Nadia; Steenholdt, Casper

2011-01-01

A cell-based assay has been developed for the quantification of the activity of TNFα antagonists based on human erythroleukemic K562 cells transfected with a NFκB regulated firefly luciferase reporter-gene construct. Both drug activity and anti-drug neutralizing antibodies can be quantified...... with a high degree of precision within 2h, and without interference from cytokines and other factors known to activate NFκB. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFα-induced firefly luciferase activity to be normalized...... relative to Renilla luciferase expression. Thus, results are independent of cell number or differences in cell viability, resulting in intra and inter assay coefficients of variation of 10% or less. Normalization of results relative to the expression of an internal standard also provides a means...

Transcript profiling of common bean (Phaseolus vulgaris L. using the GeneChip® Soybean Genome Array: optimizing analysis by masking biased probes

Directory of Open Access Journals (Sweden)

Gronwald John W

2010-05-01

Full Text Available Abstract Background Common bean (Phaseolus vulgaris L. and soybean (Glycine max both belong to the Phaseoleae tribe and share significant coding sequence homology. This suggests that the GeneChip® Soybean Genome Array (soybean GeneChip may be used for gene expression studies using common bean. Results To evaluate the utility of the soybean GeneChip for transcript profiling of common bean, we hybridized cRNAs purified from nodule, leaf, and root of common bean and soybean in triplicate to the soybean GeneChip. Initial data analysis showed a decreased sensitivity and accuracy of measuring differential gene expression in common bean cross-species hybridization (CSH GeneChip data compared to that of soybean. We employed a method that masked putative probes targeting inter-species variable (ISV regions between common bean and soybean. A masking signal intensity threshold was selected that optimized both sensitivity and accuracy of measuring differential gene expression. After masking for ISV regions, the number of differentially-expressed genes identified in common bean was increased by 2.8-fold reflecting increased sensitivity. Quantitative RT-PCR (qRT-PCR analysis of 20 randomly selected genes and purine-ureide pathway genes demonstrated an increased accuracy of measuring differential gene expression after masking for ISV regions. We also evaluated masked probe frequency per probe set to gain insight into the sequence divergence pattern between common bean and soybean. The sequence divergence pattern analysis suggested that the genes for basic cellular functions and metabolism were highly conserved between soybean and common bean. Additionally, our results show that some classes of genes, particularly those associated with environmental adaptation, are highly divergent. Conclusions The soybean GeneChip is a suitable cross-species platform for transcript profiling in common bean when used in combination with the masking protocol described. In

RHD genotype and zygosity analysis in the Chinese Southern Han D plus , D- and D variant donors using the multiplex ligation-dependent probe amplification assay

NARCIS (Netherlands)

Ji, Y. L.; Luo, H.; Wen, J. Z.; Haer-Wigman, L.; Veldhuisen, B.; Wei, L.; Wang, Z.; Ligthart, P.; Lodén-van Straaten, M.; Fu, Y. S.; van der Schoot, C. E.; Luo, G. P.

2017-01-01

Background and ObjectivesSeveral comprehensive genotyping platforms for determining red blood cell (RBC) antigens have been established and validated for use in the Caucasian and Black populations, but not for the Chinese. The multiplex ligation-dependent probe amplification (MLPA) assay was

Collaborative ring trial of the papaya endogenous reference gene and its polymerase chain reaction assays for genetically modified organism analysis.

Science.gov (United States)

Wei, Jiaojun; Li, Feiwu; Guo, Jinchao; Li, Xiang; Xu, Junfeng; Wu, Gang; Zhang, Dabing; Yang, Litao

2013-11-27

The papaya (Carica papaya L.) Chymopapain (CHY) gene has been reported as a suitable endogenous reference gene for genetically modified (GM) papaya detection in previous studies. Herein, we further validated the use of the CHY gene and its qualitative and quantitative polymerase chain reaction (PCR) assays through an interlaboratory collaborative ring trial. A total of 12 laboratories working on detection of genetically modified organisms participated in the ring trial and returned test results. Statistical analysis of the returned results confirmed the species specificity, low heterogeneity, and single-copy number of the CHY gene among different papaya varieties. The limit of detection of the CHY qualitative PCR assay was 0.1%, while the limit of quantification of the quantitative PCR assay was ∼25 copies of haploid papaya genome with acceptable PCR efficiency and linearity. The differences between the tested and true values of papaya content in 10 blind samples ranged from 0.84 to 6.58%. These results indicated that the CHY gene was suitable as an endogenous reference gene for the identification and quantification of GM papaya.

Quenching methods for background reduction in luminescence-based probe-target binding assays

Energy Technology Data Exchange (ETDEWEB)

Cai, Hong [Los Alamos, NM; Goodwin, Peter M [Los Alamos, NM; Keller, Richard A [Los Alamos, NM; Nolan, Rhiannon L [Santa Fe, NM

2007-04-10

Background luminescence is reduced from a solution containing unbound luminescent probes, each having a first molecule that attaches to a target molecule and having an attached luminescent moiety, and luminescent probe/target adducts. Quenching capture reagent molecules are formed that are capable of forming an adduct with the unbound luminescent probes and having an attached quencher material effective to quench luminescence of the luminescent moiety. The quencher material of the capture reagent molecules is added to a solution of the luminescent probe/target adducts and binds in a proximity to the luminescent moiety of the unbound luminescent probes to quench luminescence from the luminescent moiety when the luminescent moiety is exposed to exciting illumination. The quencher capture reagent does not bind to probe molecules that are bound to target molecules and the probe/target adduct emission is not quenched.

PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei

Energy Technology Data Exchange (ETDEWEB)

Müller, Patrick; Rößler, Jens; Schwarz-Finsterle, Jutta [University of Heidelberg, Kirchhoff Institute for Physics, Im Neuenheimer Feld 227, D-69120 Heidelberg (Germany); Schmitt, Eberhard, E-mail: eschmitt@kip.uni-heidelberg.de [University of Heidelberg, Kirchhoff Institute for Physics, Im Neuenheimer Feld 227, D-69120 Heidelberg (Germany); University of Göttingen, Institute for Numerical and Applied Mathematics, Lotzestraße 16-18, D-37083 Göttingen (Germany); Hausmann, Michael, E-mail: hausmann@kip.uni-heidelberg.de [University of Heidelberg, Kirchhoff Institute for Physics, Im Neuenheimer Feld 227, D-69120 Heidelberg (Germany)

2016-07-01

Recently, advantages concerning targeting specificity of PCR constructed oligonucleotide FISH probes in contrast to established FISH probes, e.g. BAC clones, have been demonstrated. These techniques, however, are still using labelling protocols with DNA denaturing steps applying harsh heat treatment with or without further denaturing chemical agents. COMBO-FISH (COMBinatorial Oligonucleotide FISH) allows the design of specific oligonucleotide probe combinations in silico. Thus, being independent from primer libraries or PCR laboratory conditions, the probe sequences extracted by computer sequence data base search can also be synthesized as single stranded PNA-probes (Peptide Nucleic Acid probes). Gene targets can be specifically labelled with at least about 20 PNA-probes obtaining visibly background free specimens. By using appropriately designed triplex forming oligonucleotides, the denaturing procedures can completely be omitted. These results reveal a significant step towards oligonucleotide-FISH maintaining the 3D-nanostructure and even the viability of the cell target. The method is demonstrated with the detection of Her2/neu and GRB7 genes, which are indicators in breast cancer diagnosis and therapy. - Highlights: • Denaturation free protocols preserve 3D architecture of chromosomes and nuclei. • Labelling sets are determined in silico for duplex and triplex binding. • Probes are produced chemically with freely chosen backbones and base variants. • Peptide nucleic acid backbones reduce hindering charge interactions. • Intercalating side chains stabilize binding of short oligonucleotides.

PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei

International Nuclear Information System (INIS)

Müller, Patrick; Rößler, Jens; Schwarz-Finsterle, Jutta; Schmitt, Eberhard; Hausmann, Michael

2016-01-01

Recently, advantages concerning targeting specificity of PCR constructed oligonucleotide FISH probes in contrast to established FISH probes, e.g. BAC clones, have been demonstrated. These techniques, however, are still using labelling protocols with DNA denaturing steps applying harsh heat treatment with or without further denaturing chemical agents. COMBO-FISH (COMBinatorial Oligonucleotide FISH) allows the design of specific oligonucleotide probe combinations in silico. Thus, being independent from primer libraries or PCR laboratory conditions, the probe sequences extracted by computer sequence data base search can also be synthesized as single stranded PNA-probes (Peptide Nucleic Acid probes). Gene targets can be specifically labelled with at least about 20 PNA-probes obtaining visibly background free specimens. By using appropriately designed triplex forming oligonucleotides, the denaturing procedures can completely be omitted. These results reveal a significant step towards oligonucleotide-FISH maintaining the 3D-nanostructure and even the viability of the cell target. The method is demonstrated with the detection of Her2/neu and GRB7 genes, which are indicators in breast cancer diagnosis and therapy. - Highlights: • Denaturation free protocols preserve 3D architecture of chromosomes and nuclei. • Labelling sets are determined in silico for duplex and triplex binding. • Probes are produced chemically with freely chosen backbones and base variants. • Peptide nucleic acid backbones reduce hindering charge interactions. • Intercalating side chains stabilize binding of short oligonucleotides.

A high-throughput fluorescence-based assay system for appetite-regulating gene and drug screening.

Directory of Open Access Journals (Sweden)

Yasuhito Shimada

Full Text Available The increasing number of people suffering from metabolic syndrome and obesity is becoming a serious problem not only in developed countries, but also in developing countries. However, there are few agents currently approved for the treatment of obesity. Those that are available are mainly appetite suppressants and gastrointestinal fat blockers. We have developed a simple and rapid method for the measurement of the feeding volume of Danio rerio (zebrafish. This assay can be used to screen appetite suppressants and enhancers. In this study, zebrafish were fed viable paramecia that were fluorescently-labeled, and feeding volume was measured using a 96-well microplate reader. Gene expression analysis of brain-derived neurotrophic factor (bdnf, knockdown of appetite-regulating genes (neuropeptide Y, preproinsulin, melanocortin 4 receptor, agouti related protein, and cannabinoid receptor 1, and the administration of clinical appetite suppressants (fluoxetine, sibutramine, mazindol, phentermine, and rimonabant revealed the similarity among mechanisms regulating appetite in zebrafish and mammals. In combination with behavioral analysis, we were able to evaluate adverse effects on locomotor activities from gene knockdown and chemical treatments. In conclusion, we have developed an assay that uses zebrafish, which can be applied to high-throughput screening and target gene discovery for appetite suppressants and enhancers.

[Quantitative fluorogenic real-time PCR assay for respiratory syncytial virus detection].

Science.gov (United States)

Zhang, Qi-wei; You, Shang-you; Sun, Ji-min; Wu, Qi; Yu, Chun-hua; Zhang, Chu-yu

2005-07-01

To Establish a rapid and objective quantitative fluorogenic real-time PCR assay for early detection of human respiratory syncytial virus (hRSV). Two pairs of primers and one TaqMan Fluorogenic probe that are specific for the recognition of the most conservative N gene of hRSV for virus detection with LighCycler PCR in 93 nasopharyngeal secretion specimens collected from infants and young children. The assay was compared with virus isolation, routine PCR, nested PCR, and enzyme-linked immunosorbent assay (ELISA). This TaqMan assay had a sensitivity of 1 x 10(2) cDNA copies/microl with a dynamic range between 1 x 10(2) and 1 x 10(7) cDNA copies/microl, which was the same as that of nested PCR, but 10 times more sensitive than routine PCR. The specificity of the assay was evaluated by comparing hRSV with polivirus type 1, coxsackie virus type 2, influenza A, influenza B and adenovirus type 7. A PCR product of the expected size (195 bp) was produced and fluorescence signal detected for hRSV, but not for any of the other viruses. The results in LightCycler and Rotor-Gene instrument were consistent. Forty-four specimens (43.9%) were hRSV-positive with this assay and 4 (4/93,4.3%) were hRSV-positive with ELISA, showing rather low correlation between the two methods. No visible relation was found between the concentration of hRSV RNA and severity of the disease. This assay is rapid, sensitive, specific and quantitative, and has the potential of wide application for early diagnosis of hRSV infection and evaluation of the therapeutic effect.

Measurement of Gene Expression in Archival Paraffin-Embedded Tissues

Science.gov (United States)

Cronin, Maureen; Pho, Mylan; Dutta, Debjani; Stephans, James C.; Shak, Steven; Kiefer, Michael C.; Esteban, Jose M.; Baker, Joffre B.

2004-01-01

Throughout the last decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous in length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10-μm FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RT-PCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests. PMID:14695316

TumorNext-Lynch-MMR: a comprehensive next generation sequencing assay for the detection of germline and somatic mutations in genes associated with mismatch repair deficiency and Lynch syndrome.

Science.gov (United States)

Gray, Phillip N; Tsai, Pei; Chen, Daniel; Wu, Sitao; Hoo, Jayne; Mu, Wenbo; Li, Bing; Vuong, Huy; Lu, Hsiao-Mei; Batth, Navanjot; Willett, Sara; Uyeda, Lisa; Shah, Swati; Gau, Chia-Ling; Umali, Monalyn; Espenschied, Carin; Janicek, Mike; Brown, Sandra; Margileth, David; Dobrea, Lavinia; Wagman, Lawrence; Rana, Huma; Hall, Michael J; Ross, Theodora; Terdiman, Jonathan; Cullinane, Carey; Ries, Savita; Totten, Ellen; Elliott, Aaron M

2018-04-17

The current algorithm for Lynch syndrome diagnosis is highly complex with multiple steps which can result in an extended time to diagnosis while depleting precious tumor specimens. Here we describe the analytical validation of a custom probe-based NGS tumor panel, TumorNext-Lynch-MMR, which generates a comprehensive genetic profile of both germline and somatic mutations that can accelerate and streamline the time to diagnosis and preserve specimen. TumorNext-Lynch-MMR can detect single nucleotide variants, small insertions and deletions in 39 genes that are frequently mutated in Lynch syndrome and colorectal cancer. Moreover, the panel provides microsatellite instability status and detects loss of heterozygosity in the five Lynch genes; MSH2 , MSH6 , MLH1 , PMS2 and EPCAM . Clinical cases are described that highlight the assays ability to differentiate between somatic and germline mutations, precisely classify variants and resolve discordant cases.

Comparison of Gull Feces-specific Assays Targeting the 16S rRNA Gene of Catellicoccus Marimammalium and Streptococcus spp.

Science.gov (United States)

Two novel gull-specific qPCR assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR-green-based assay targeting Streptococcus spp. (i.e., gull3) and a TaqMan qPCR assay targeting Catellicoccus marimammalium (i.e., gull4). The main objectives ...

Development of RNA-FISH Assay for Detection of Oncogenic FGFR3-TACC3 Fusion Genes in FFPE Samples.

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Masahiro Kurobe

Full Text Available Oncogenic FGFR3-TACC3 fusions and FGFR3 mutations are target candidates for small molecule inhibitors in bladder cancer (BC. Because FGFR3 and TACC3 genes are located very closely on chromosome 4p16.3, detection of the fusion by DNA-FISH (fluorescent in situ hybridization is not a feasible option. In this study, we developed a novel RNA-FISH assay using branched DNA probe to detect FGFR3-TACC3 fusions in formaldehyde-fixed paraffin-embedded (FFPE human BC samples.The RNA-FISH assay was developed and validated using a mouse xenograft model with human BC cell lines. Next, we assessed the consistency of the RNA-FISH assay using 104 human BC samples. In this study, primary BC tissues were stored as frozen and FFPE tissues. FGFR3-TACC3 fusions were independently detected in FFPE sections by the RNA-FISH assay and in frozen tissues by RT-PCR. We also analyzed the presence of FGFR3 mutations by targeted sequencing of genomic DNA extracted from deparaffinized FFPE sections.FGFR3-TACC3 fusion transcripts were identified by RNA-FISH and RT-PCR in mouse xenograft FFPE tissues using the human BC cell lines RT112 and RT4. These cell lines have been reported to be fusion-positive. Signals for FGFR3-TACC3 fusions by RNA-FISH were positive in 2/60 (3% of non-muscle-invasive BC (NMIBC and 2/44 (5% muscle-invasive BC (MIBC patients. The results of RT-PCR of all 104 patients were identical to those of RNA-FISH. FGFR3 mutations were detected in 27/60 (45% NMIBC and 8/44 (18% MIBC patients. Except for one NMIBC patient, FGFR3 mutation and FGFR3-TACC3 fusion were mutually exclusive.We developed an RNA-FISH assay for detection of the FGFR3-TACC3 fusion in FFPE samples of human BC tissues. Screening for not only FGFR3 mutations, but also for FGFR3-TACC3 fusion transcripts has the potential to identify additional patients that can be treated with FGFR inhibitors.

Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays

NARCIS (Netherlands)

Legler, J.; Dennekamp, M.; Vethaak, A.D.; Brouwer, A.; Koeman, J.H.; Burg, van der B.; Murk, A.J.

2002-01-01

Sediments may be the ultimate sink for persistent (xeno-) estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The

Detection of Cyanobacteria in Eutrophic Water Using a Portable Electrocoagulator and NanoGene Assay.

Science.gov (United States)

Lee, Eun-Hee; Chua, Beelee; Son, Ahjeong

2018-02-06

We have demonstrated the detection of cyanobacteria in eutrophic water samples using a portable electrocoagulator and NanoGene assay. The electrocoagulator is designed to preconcentrate cyanobacteria from water samples prior to analysis via NanoGene assay. Using Microcystis aeruginosa laboratory culture and environmental samples (cell densities ranging from 1.7 × 10 5 to 4.1 × 10 6 and 6.5 × 10 3 to 6.6 × 10 7 cells·mL -1 , respectively), the electrocoagulator was evaluated and compared with a conventional centrifuge. Varying the operation duration from 0 to 300 s with different cell densities was first investigated. Preconcentration efficiencies (obtained via absorbance measurement) and dry cell weight of preconcentrated cyanobacteria were then obtained and compared. For laboratory samples at cell densities from 3.2 × 10 5 to 4.1 × 10 6 cells·mL -1 , the preconcentration efficiencies of electrocoagulator appeared to be stable at ∼60%. At lower cell densities (1.7 and 2.2 × 10 5 cells·mL -1 ), the preconcentration efficiencies decreased to 33.9 ± 0.2 and 40.4 ± 5.4%, respectively. For environmental samples at cell densities of 2.7 × 10 5 and 6.6 × 10 7 cells·mL -1 , the electrocoagulator maintained its preconcentration efficiency at ∼60%. On the other hand, the centrifuge's preconcentration efficiencies decreased to nondetectable and below 40%, respectively. This shows that the electrocoagulator outperformed the centrifuge when using eutrophic water samples. Finally, the compatibility of the electrocoagulator with the NanoGene assay was verified via the successful detection of the microcystin synthetase D (mcyD) gene in environmental samples. The viability of the electrocoagulator as an in situ compatible alternative to the centrifuge is also discussed.

A Real-Time PCR Assay Based on 5.8S rRNA Gene (5.8S rDNA) for Rapid Detection of Candida from Whole Blood Samples.

Science.gov (United States)

Guo, Yi; Yang, Jing-Xian; Liang, Guo-Wei

2016-06-01

The prevalence of Candida in bloodstream infections (BSIs) has increased. To date, the identification of Candida in BSIs still mainly relies on blood culture and serological tests, but they have various limitations. Therefore, a real-time PCR assay for the detection of Candida from whole blood is presented. The unique primers/probe system was designed on 5.8S rRNA gene (5.8S rDNA) of Candida genus. The analytical sensitivity was determined by numbers of positive PCRs in 12 repetitions. At the concentration of 10(1) CFU/ml blood, positive PCR rates of 100 % were obtained for C. albicans, C. parapsilosis, C. tropicalis, and C. krusei. The detection rate for C. glabrata was 75 % at 10(1) CFU/ml blood. The reaction specificity was 100 % when evaluating the assay using DNA samples from clinical isolates and human blood. The maximum CVs of intra-assay and inter-assay for the detection limit were 1.22 and 2.22 %, respectively. To assess the clinical applicability, 328 blood samples from 82 patients were prospectively tested and real-time PCR results were compared with results from blood culture. Diagnostic sensitivity of the PCR was 100 % using as gold standard blood culture, and specificity was 98.4 %. Our data suggest that the developed assay can be used in clinical laboratories as an accurate and rapid screening test for the Candida from whole blood. Although further evaluation is warranted, our assay holds promise for earlier diagnosis of candidemia.

Shared probe design and existing microarray reanalysis using PICKY

Directory of Open Access Journals (Sweden)

Chou Hui-Hsien

2010-04-01

Full Text Available Abstract Background Large genomes contain families of highly similar genes that cannot be individually identified by microarray probes. This limitation is due to thermodynamic restrictions and cannot be resolved by any computational method. Since gene annotations are updated more frequently than microarrays, another common issue facing microarray users is that existing microarrays must be routinely reanalyzed to determine probes that are still useful with respect to the updated annotations. Results PICKY 2.0 can design shared probes for sets of genes that cannot be individually identified using unique probes. PICKY 2.0 uses novel algorithms to track sharable regions among genes and to strictly distinguish them from other highly similar but nontarget regions during thermodynamic comparisons. Therefore, PICKY does not sacrifice the quality of shared probes when choosing them. The latest PICKY 2.1 includes the new capability to reanalyze existing microarray probes against updated gene sets to determine probes that are still valid to use. In addition, more precise nonlinear salt effect estimates and other improvements are added, making PICKY 2.1 more versatile to microarray users. Conclusions Shared probes allow expressed gene family members to be detected; this capability is generally more desirable than not knowing anything about these genes. Shared probes also enable the design of cross-genome microarrays, which facilitate multiple species identification in environmental samples. The new nonlinear salt effect calculation significantly increases the precision of probes at a lower buffer salt concentration, and the probe reanalysis function improves existing microarray result interpretations.

Detection of Different Genotypes of Clostridium perfringens in Feces of Healthy Dairy Cattle from China using Real-Time Duplex PCR Assay

Directory of Open Access Journals (Sweden)

Guanghua Wang, Jizhang Zhou, Fuying Zheng, Guozhen Lin, Xiaoan Cao, Xiaowei Gong and Changqing Qiu*

2011-04-01

Full Text Available Dual-labeled fluorescence hybridization probe-based multiplex quantitative real-time polymerase chain reaction (qPCR assay was used for the detection of Clostridium perfringens toxin genes alpha (cpa, beta (cpb, iota (ia, epsilon (etx, beta2 (cpb2 and enterotoxin (cpe directly from the feces of cattle. Fecal samples from 261 lactating cattle, belonging to three dairy herds in Ningxia (China, were examined using the developed assays. The duplex qPCR assay revealed that cpa, etx, cpb2 and cpe toxin genes were detected in 176 (100%, 15 (8.5%, 142 (80.7% and 4 (2.3% of 176 PCR positive samples, respectively. The findings of this study revealed that C. perfringens beta2-toxin-producing strains were widely prevalent in lactating cows in Ningxia, possibly playing an important role in C. perfringens-associated diarrheal disease.

Short communication: identification of a novel HIV type 1 subtype H/J recombinant in Canada with discordant HIV viral load (RNA) values in three different commercial assays.

Science.gov (United States)

Kim, John E; Beckthold, Brenda; Chen, Zhaoxia; Mihowich, Jennifer; Malloch, Laurie; Gill, Michael John

2007-11-01

The presence of HIV-1 non-B subtypes is increasing worldwide. This poses challenges to commercial diagnostic and viral load (RNA) monitoring tests that are predominantly based on HIV-1 subtype B strains. Based on phylogenetic analysis of the gag, pol, and env gene regions, we describe the first HIV-1 H/J recombinant in Canada that presented divergent viral load values. DNA sequence analysis of the gag gene region further revealed that genetic diversity between this H/J recombinant and the primers and probes used in the bio-Merieux Nuclisens HIV-1 QT (Nuclisens) and Roche Amplicor Monitor HIV-1, v1.5 (Monitor) viral RNA assays can erroneously lead to undetectable viral load values. This observation appears to be more problematic in the Nuclisens assay. In light of increasing genetic diversity in HIV worldwide we recommend that DNA sequencing of HIV, especially in the gag gene region targeted by primers and probes used in molecular diagnostic and viral load tests, be incorporated into clinical monitoring practices.

Investigating a multigene prognostic assay based on significant pathways for Luminal A breast cancer through gene expression profile analysis.

Science.gov (United States)

Gao, Haiyan; Yang, Mei; Zhang, Xiaolan

2018-04-01

The present study aimed to investigate potential recurrence-risk biomarkers based on significant pathways for Luminal A breast cancer through gene expression profile analysis. Initially, the gene expression profiles of Luminal A breast cancer patients were downloaded from The Cancer Genome Atlas database. The differentially expressed genes (DEGs) were identified using a Limma package and the hierarchical clustering analysis was conducted for the DEGs. In addition, the functional pathways were screened using Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses and rank ratio calculation. The multigene prognostic assay was exploited based on the statistically significant pathways and its prognostic function was tested using train set and verified using the gene expression data and survival data of Luminal A breast cancer patients downloaded from the Gene Expression Omnibus. A total of 300 DEGs were identified between good and poor outcome groups, including 176 upregulated genes and 124 downregulated genes. The DEGs may be used to effectively distinguish Luminal A samples with different prognoses verified by hierarchical clustering analysis. There were 9 pathways screened as significant pathways and a total of 18 DEGs involved in these 9 pathways were identified as prognostic biomarkers. According to the survival analysis and receiver operating characteristic curve, the obtained 18-gene prognostic assay exhibited good prognostic function with high sensitivity and specificity to both the train and test samples. In conclusion the 18-gene prognostic assay including the key genes, transcription factor 7-like 2, anterior parietal cortex and lymphocyte enhancer factor-1 may provide a new method for predicting outcomes and may be conducive to the promotion of precision medicine for Luminal A breast cancer.

ProSeeK: a web server for MLPA probe design.

Science.gov (United States)

Pantano, Lorena; Armengol, Lluís; Villatoro, Sergi; Estivill, Xavier

2008-11-28

The technological evolution of platforms for detecting genome-wide copy number imbalances has allowed the discovery of an unexpected amount of human sequence that is variable in copy number among individuals. This type of human variation can make an important contribution to human diversity and disease susceptibility. Multiplex Ligation-dependent Probe Amplification (MLPA) is a targeted method to assess copy number differences for up to 40 genomic loci in one single experiment. Although specific MLPA assays can be ordered from MRC-Holland (the proprietary company of the MLPA technology), custom designs are also developed in many laboratories worldwide. After our own experience, an important drawback of custom MLPA assays is the time spent during the design of the specific oligonucleotides that are used as probes. Due to the large number of probes included in a single assay, a number of restrictions need to be met in order to maximize specificity and to increase success likelihood. We have developed a web tool for facilitating and optimising custom probe design for MLPA experiments. The algorithm only requires the target sequence in FASTA format and a set of parameters, that are provided by the user according to each specific MLPA assay, to identify the best probes inside the given region. To our knowledge, this is the first available tool for optimizing custom probe design of MLPA assays. The ease-of-use and speed of the algorithm dramatically reduces the turn around time of probe design. ProSeeK will become a useful tool for all laboratories that are currently using MLPA in their research projects for CNV studies.

ProSeeK: A web server for MLPA probe design

Directory of Open Access Journals (Sweden)

Villatoro Sergi

2008-11-01

Full Text Available Abstract Background The technological evolution of platforms for detecting genome-wide copy number imbalances has allowed the discovery of an unexpected amount of human sequence that is variable in copy number among individuals. This type of human variation can make an important contribution to human diversity and disease susceptibility. Multiplex Ligation-dependent Probe Amplification (MLPA is a targeted method to assess copy number differences for up to 40 genomic loci in one single experiment. Although specific MLPA assays can be ordered from MRC-Holland (the proprietary company of the MLPA technology, custom designs are also developed in many laboratories worldwide. After our own experience, an important drawback of custom MLPA assays is the time spent during the design of the specific oligonucleotides that are used as probes. Due to the large number of probes included in a single assay, a number of restrictions need to be met in order to maximize specificity and to increase success likelihood. Results We have developed a web tool for facilitating and optimising custom probe design for MLPA experiments. The algorithm only requires the target sequence in FASTA format and a set of parameters, that are provided by the user according to each specific MLPA assay, to identify the best probes inside the given region. Conclusion To our knowledge, this is the first available tool for optimizing custom probe design of MLPA assays. The ease-of-use and speed of the algorithm dramatically reduces the turn around time of probe design. ProSeeK will become a useful tool for all laboratories that are currently using MLPA in their research projects for CNV studies.

Real-time sequence-validated loop-mediated isothermal amplification assays for detection of Middle East respiratory syndrome coronavirus (MERS-CoV.

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Sanchita Bhadra

Full Text Available The Middle East respiratory syndrome coronavirus (MERS-CoV, an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU (5 to 50 PFU/ml of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens.

Diagnostic PCR: Comparative sensitivity of four probe chemistries

DEFF Research Database (Denmark)

Josefsen, Mathilde Hartmann; Löfström, Charlotta; Sommer, Helle Mølgaard

2009-01-01

Three probe chemistries: locked nucleic acid (LNA), minor groove binder (MGB) and Scorpion were compared with a TaqMan probe in a validated real-time PCR assay for detection of food-borne thermotolerant Campylobacter. The LNA probe produced significantly lower Ct-values and a higher proportion of...

Simultaneous use of multiplex ligation-dependent probe amplification assay and flow cytometric DNA ploidy analysis in patients with acute leukemia.

Science.gov (United States)

Reyes-Núñez, Virginia; Galo-Hooker, Evelyn; Pérez-Romano, Beatriz; Duque, Ricardo E; Ruiz-Arguelles, Alejandro; Garcés-Eisele, Javier

2018-01-01

The aim of this work was to simultaneously use multiplex ligation-dependent probe amplification (MLPA) assay and flow cytometric DNA ploidy analysis (FPA) to detect aneuploidy in patients with newly diagnosed acute leukemia. MLPA assay and propidium iodide FPA were used to test samples from 53 consecutive patients with newly diagnosed acute leukemia referred to our laboratory for immunophenotyping. Results were compared by nonparametric statistics. The combined use of both methods significantly increased the rate of detection of aneuploidy as compared to that obtained by each method alone. The limitations of one method are somehow countervailed by the other and vice versa. MPLA and FPA yield different yet complementary information concerning aneuploidy in acute leukemia. The simultaneous use of both methods might be recommended in the clinical setting. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

A quantitative TaqMan PCR assay for the detection of Ureaplasma diversum.

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Marques, Lucas M; Amorim, Aline T; Martins, Hellen Braga; Rezende, Izadora Souza; Barbosa, Maysa Santos; Lobão, Tassia Neves; Campos, Guilherme B; Timenetsky, Jorge

2013-12-27

Ureaplasma diversum in veterinary studies is an undesirable microbe, which may cause infection in bulls and may result in seminal vesiculitis, balanopostitis, and alterations in spermatozoids, whereas in cows, it may cause placentitis, fetal alveolitis, abortion, and birth of weak calves. U. diversum is released through organic secretions, especially semen, preputial and vaginal mucus, conjunctival secretion, and milk. The aim of the present study was to develop a TaqMan probe, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of U. diversum from genital swabs of bovines. Primers and probes specific to U. diversum 16S rRNA gene were designed. The specificity, detection limit, intra- and inter-assay variability of qPCR to detect this ureaplasma was compared with the results of the conventional PCR assay (cPCR). Swabs of vaginal mucus from 169 cows were tested. The qPCR assay detected as few as 10 copies of U. diversum and was 100-fold more sensitive than the cPCR. No cross-reactivity with other Mollicutes or eubacteria was observed. U. diversum was detected in 79 swabs (46.42%) by qPCR, while using cPCR it was detected in 42 (25%) samples. The difference in cPCR and qPCR ureaplasma detection between healthy and sick animals was not statistically significant. But the U. diversum load in samples from animals with genital disorders was higher than in healthy animals. The qPCR assay developed herein is highly sensitive and specific for the detection and quantification of U. diversum in vaginal bovine samples. Copyright © 2013. Published by Elsevier B.V.

Multiplex real-time PCR (TaqMan) assay for the simultaneous detection and discrimination of potato powdery and common scab diseases and pathogens.

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Qu, X S; Wanner, L A; Christ, B J

2011-03-01

To develop a multiplex real-time PCR assay using TaqMan probes for the simultaneous detection and discrimination of potato powdery scab and common scab, two potato tuber diseases with similar symptoms, and the causal pathogens Spongospora subterranea and plant pathogenic Streptomyces spp. Real-time PCR primers and a probe for S. subterranea were designed based on the DNA sequence of the ribosomal RNA ITS2 region. Primers and a probe for pathogenic Streptomyces were designed based on the DNA sequence of the txtAB genes. The two sets of primer pairs and probes were used in a single real-time PCR assay. The multiplex real-time PCR assay was confirmed to be specific for S. subterranea and pathogenic Streptomyces. The assay detected DNA quantities of 100 fg for each of the two pathogens and linear responses and high correlation coefficients between the amount of DNA and C(t) values for each pathogen were achieved. The presence of two sets of primer pairs and probes and of plant extracts did not alter the sensitivity and efficiency of multiplex PCR amplification. Using the PCR assay, we could discriminate between powdery scab and common scab tubers with similar symptoms. Common scab and powdery scab were detected in some tubers with no visible symptoms. Mixed infections of common scab and powdery scab on single tubers were also revealed. This multiplex real-time PCR assay is a rapid, cost efficient, specific and sensitive tool for the simultaneous detection and discrimination of the two pathogens on infected potato tubers when visual symptoms are inconclusive or not present. Accurate and quick identification and discrimination of the cause of scab diseases on potatoes will provide critical information to potato growers and researchers for disease management. This is important because management strategies for common and powdery scab diseases are very different. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

Development of a Gene Expression Assay for the Diagnosis of Mycobacterium bovis Infection in African Lions (Panthera leo).

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Olivier, T T; Viljoen, I M; Hofmeyr, J; Hausler, G A; Goosen, W J; Tordiffe, A S W; Buss, P; Loxton, A G; Warren, R M; Miller, M A; van Helden, P D; Parsons, S D C

2017-06-01

Mycobacterium bovis infection, the cause of bovine tuberculosis (BTB), is endemic in wildlife in the Kruger National Park (KNP), South Africa. In lions, a high infection prevalence and BTB mortalities have been documented in the KNP; however, the ecological consequences of this disease are currently unknown. Sensitive assays for the detection of this infection in this species are therefore required. Blood from M. bovis-exposed, M. bovis-unexposed, M. tuberculosis-exposed and M. bovis-infected lions was incubated in QuantiFERON ® -TB Gold (QFT) tubes containing either saline or ESAT-6/CFP-10 peptides. Using qPCR, selected reference genes were evaluated for expression stability in these samples and selected target genes were evaluated as markers of antigen-dependent immune activation. The abundance of monokine induced by gamma interferon (MIG/CXCL9) mRNA, measured in relation to that of YWHAZ, was used as a marker of ESAT-6/CFP-10 sensitization. The gene expression assay results were compared between lion groups, and lenient and stringent diagnostic cut-off values were calculated. This CXCL9 gene expression assay combines a highly specific stimulation platform with a sensitive diagnostic marker that allows for discrimination between M. bovis-infected and M. bovis-uninfected lions. © 2015 Blackwell Verlag GmbH.

Development of a novel real-time qPCR assay for the dual detection of canine and phocine distemper virus

DEFF Research Database (Denmark)

Nielsen, Linette Buxbom; Hjulsager, Charlotte Kristiane; Larsen, Helene

conventional PCR assays with real-time PCR assays to obtain a uniform assay palette. The present work describes the development of a novel real-time RT-qPCR assay for the dual detection of canine and phocine distemper virus. The assay is relevant for the future detection of outbreaks of canine distemper virus...... in e.g. in farmed mink and wildlife and phocine distemper in seals. A set of primers and dual labelled probe was designed based on an alignment of distemper sequences in GenBank from various species and in-house sequences from recent outbreaks in Danish farmed mink. The assay amplifies a segment of 151...... bp in the Phosphoprotein (P) gene of the distemper virus genome. The dynamic range and PCR efficiency (E) was experimentally determined using 10-fold dilutions of a specially designed distemper DNA-oligo in addition to extracted RNA from clinical samples. E of the real-time assay was shown to range...

A Multiplex SYBR Green Real-Time PCR Assay for the Detection of Three Colistin Resistance Genes from Cultured Bacteria, Feces, and Environment Samples

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Jiyun Li

2017-10-01

Full Text Available The aim of the study was to develop a multiplex assay for rapid detection of mcr-1, mcr-2, and mcr-3, a group of genes of conferring resistance to colistin mediated by plasmid in Enterobacteriaceae. A SYBR Green based real-time PCR assay has been designed to detect the mcr genes, and applied to cultured bacteria, feces and soil samples. All three mcr genes could be detected with a lower limit of 102 cultured bacteria. This test was highly specific and sensitive, and generated no false-positive results. The assay was also conclusive when applied to feces and soil samples containing mcr-1-positive Escherichia coli, which could facilitate the screening of mcr genes not only in the bacteria, but also directly from the environment. This simple, rapid, sensitive, and specific multiplex assay will be useful for rapid screening of the colistin resistance in both clinical medicine and animal husbandry.

Detection and characterization of recombinant DNA expressing vip3A-type insecticidal gene in GMOs--standard single, multiplex and construct-specific PCR assays.

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Singh, Chandra K; Ojha, Abhishek; Bhatanagar, Raj K; Kachru, Devendra N

2008-01-01

Vegetative insecticidal protein (Vip), a unique class of insecticidal protein, is now part of transgenic plants for conferring resistance against lepidopteron pests. In order to address the imminent regulatory need for detection and labeling of vip3A carrying genetically modified (GM) products, we have developed a standard single PCR and a multiplex PCR assay. As far as we are aware, this is the first report on PCR-based detection of a vip3A-type gene (vip-s) in transgenic cotton and tobacco. Our assay involves amplification of a 284-bp region of the vip-s gene. This assay can possibly detect as many as 20 natural wild-type isolates bearing a vip3A-like gene and two synthetic genes of vip3A in transgenic plants. The limit of detection as established by our assay for GM trait (vip-s) is 0.1%. Spiking with nontarget DNA originating from diverse plant sources had no inhibitory effect on vip-s detection. Since autoclaving of vip-s bearing GM leaf samples showed no deterioration/interference in detection efficacy, the assay seems to be suitable for processed food products as well. The vip-s amplicon identity was reconfirmed by restriction endonuclease assay. The primer set for vip-s was equally effective in a multiplex PCR assay format (duplex, triplex and quadruplex), used in conjunction with the primer sets for the npt-II selectable marker gene, Cauliflower mosaic virus 35S promoter and nopaline synthetase terminator, enabling concurrent detection of the transgene, regulatory sequences and marker gene. Further, the entire transgene construct was amplified using the forward primer of the promoter and the reverse primer of the terminator. The resultant amplicon served as a template for nested PCR to confirm the construct integrity. The method is suitable for screening any vip3A-carrying GM plant and food. The availability of a reliable PCR assay method prior to commercial release of vip3A-based transgenic crops and food would facilitate rapid and efficient regulatory

Authentication of beef, carabeef, chevon, mutton and pork by a PCR-RFLP assay of mitochondrial cytb gene.

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Kumar, Deepak; Singh, S P; Karabasanavar, Nagappa S; Singh, Rashmi; Umapathi, V

2014-11-01

Authentication of meat assumes significance in view of religious, quality assurance, food safety, public health, conservation and legal concerns. Here, we describe a PCR-RFLP (Polymerase Chain Reaction- Restriction Fragment Length Polymorphism) assay targeting mitochondrial cytochrome-b gene for the identification of meats of five most common food animals namely cattle, buffalo, goat, sheep and pig. A pair of forward and reverse primers (VPH-F & VPH-R) amplifying a conserved region (168-776 bp) of mitochondrial cytochrome-b (cytb) gene for targeted species was designed which yielded a 609 bp PCR amplicon. Further, restriction enzyme digestion of the amplicons with Alu1 and Taq1 restriction enzymes resulted in a distinctive digestion pattern that was able to discriminate each species. The repeatability of the PCR-RFLP assay was validated ten times with consistent results observed. The developed assay can be used in routine diagnostic laboratories to differentiate the meats of closely related domestic livestock species namely cattle from buffalo and sheep from goat.

The accuracy of using the lytA-gene to distinguish Streptococcus pneumoniae from related species

DEFF Research Database (Denmark)

Greve, Thomas; Møller, Jens Kjølseth

2012-01-01

with primers and probes. The remaining 11 S. pneumoniae strains could be placed in a different cluster, which also contained the five S. mitis and two S. pseudopneumoniae strains. All strains had no match with primers and probes. The S. pneumoniae strains in the second cluster were all characterised by being....... The real-time PCR targeting the lytA-gene thus constitutes a sensitive and specific assay that distinguishes S. pneumoniae from its close relatives in the Mitis group....

Using reporter gene assays to identify cis regulatory differences between humans and chimpanzees.

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Chabot, Adrien; Shrit, Ralla A; Blekhman, Ran; Gilad, Yoav

2007-08-01

Most phenotypic differences between human and chimpanzee are likely to result from differences in gene regulation, rather than changes to protein-coding regions. To date, however, only a handful of human-chimpanzee nucleotide differences leading to changes in gene regulation have been identified. To hone in on differences in regulatory elements between human and chimpanzee, we focused on 10 genes that were previously found to be differentially expressed between the two species. We then designed reporter gene assays for the putative human and chimpanzee promoters of the 10 genes. Of seven promoters that we found to be active in human liver cell lines, human and chimpanzee promoters had significantly different activity in four cases, three of which recapitulated the gene expression difference seen in the microarray experiment. For these three genes, we were therefore able to demonstrate that a change in cis influences expression differences between humans and chimpanzees. Moreover, using site-directed mutagenesis on one construct, the promoter for the DDA3 gene, we were able to identify three nucleotides that together lead to a cis regulatory difference between the species. High-throughput application of this approach can provide a map of regulatory element differences between humans and our close evolutionary relatives.

Appendix 1：Upregulated genes in gene expression profile （P<0.05 ...

Indian Academy of Sciences (India)

lazi

Appendix 1: Upregulated genes in gene expression profile«P2）. Probe_s. Gene_Symbol pvalues foldchange. Probe_S. et_ID. Gene_Symbol pvalues foldchange. et_ID. 1370355. 1393751. Scd1. 1.35E-04. 25.77. Loc1009122508.06E-03. 2.55. -at at. 1398250. 1370870. Acot1. 2.43E-02. 12.18. Me1.

In planta assays involving epigenetically silenced genes reveal inhibition of cytosine methylation by genistein

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Arase Sachiko

2012-03-01

Full Text Available Abstract Background Cytosine methylation is involved in epigenetic control of gene expression in a wide range of organisms. An increasing number of examples indicate that changing the frequency of cytosine methylation in the genome is a feasible tool to engineer novel traits in plants. Although demethylating effects of compounds have been analyzed in human cultured cells in terms of suppressing cancer, their effect in plant cells has not been analyzed extensively. Here, we developed in planta assay systems to detect inhibition of cytosine methylation using plants that contain a transgene transcriptionally silenced by an epigenetic mechanism. Results Seeds of two transgenic plants were used: a petunia line that has been identified as a revertant of the co-suppression of the chalcone synthase-A (CHS-A gene and contains CHS-A transgenes whose transcription is repressed; Nicotiana benthamiana plants that contain the green fluorescent protein (GFP reporter gene whose transcription is repressed through virus-induced transcriptional gene silencing. Seeds of these plants were sown on a medium that contained a demethylating agent, either 5-azacytidine or trichostatin A, and the restoration of the transcriptionally active state of the transgene was detected in seedlings. Using these systems, we found that genistein, a major isoflavonoid compound, inhibits cytosine methylation, thus restoring transgene transcription. Genistein also restored the transcription of an epigenetically silenced endogenous gene in Arabidopsis plants. Conclusions Our assay systems allowed us to assess the inhibition of cytosine methylation, in particular of maintenance of methylation, by compounds in plant cells. These results suggest a novel role of flavonoids in plant cells and that genistein is useful for modifying the epigenetic state of plant genomes.

Loop-Mediated Isothermal Amplification Assay Targeting the MOMP Gene for Rapid Detection of Chlamydia psittaci Abortus Strain

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Guo-Zhen Lin, Fu-Ying Zheng, Ji-Zhang Zhou, Guang-Hua Wang, Xiao-An Cao, Xiao-Wei Gong and Chang-Qing Qiu*

2012-05-01

Full Text Available For rapid detection of the Chlamydia psittaci abortus strain, a loop-mediated isothermal amplification (LAMP assay was developed and evaluated in this study. The primers for the LAMP assay were designed on the basis of the main outer membrane protein (MOMP gene sequence of C. psittaci. Analysis showed that the assay could detect the abortus strain of C. psittaci with adequate specificity. The sensitivity of the test was the same as that of the nested-conventional PCR and higher than that of chick embryo isolation. Testing of 153 samples indicated that the LAMP assay could detect the genome of the C. psittaci abortus strain effectively in clinical samples. This assay is a useful tool for rapid diagnosis of C. psittaci infection in sheep, swine and cattle.

Detection of Toxoplasma gondii in Diabetic Patients Using the Nested PCR Assay via RE and B1 Genes.

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Mousavi, Mohammad; Saravani, Ramin; Jafari Modrek, Mohammad; Shahrakipour, Mahnaz; Sekandarpour, Sina

2016-02-01

Toxoplasma gondii is an obligate intracellular protozoan parasite that exists worldwide. Various techniques have been developed for T. gondii detection. The aim of this study was the detection of T. gondii in diabetic patients with RE and B1 genes and the comparison of these two genes for diagnosis using the nested-PCR assay method. DNA samples from 205 diabetic patients who had been referred to the diabetes center of Ali Asghar hospital in Zahedan, Iran, were collected and analyzed using the nested-PCR assay method. Toxoplasma antibody data gathered using the enzyme-linked immunosorbent assay (ELISA) method from a previous study was used to group patients. The data were analyzed using SPSS 18. The chi-square test was used for comparison. Of the diabetic patients selected, the following results were obtained: 53 (IgG+, IgM+); 20 (IgG-, IgM+); 72 (IgG+, IgM-); and 60 (IgG-, IgM-). The nested-PCR detected the following: in the acute group, 21/53 (39.63%), 30/53 (56.60%) (IgM+, IgG+); in the chronic group, 40/72 (55.56%), 51/72 (70.83%), (IgG+, IgM-); in the false positive group, 18/20 (90%), 17/20 (85%) (IgM+, IgG-); and sero-negative samples of 38/60 (63.33%) and 60/ 41 (77.35%) for RE and B1 genes, respectively. The prevalence of toxoplasmosis showed positive in patients with diabetes in the B1 gene 139 (67.8%) and RE gene 117 (57.1%). Our study demonstrated that the B1 gene, more so than the RE gene, showed positive samples and can be used to detect toxoplasmosis, although the B1 gene, in comparison to the RE gene, did not show any superiority of molecular diagnosing capability. Results also showed that toxoplasma molecular detection methods can be used instead of routine serological detection methods in a clinical laboratory testing.

Evaluation of a real-time PCR assay based on the single-copy SAG1 gene for the detection of Toxoplasma gondii.

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Yu, Haijie; Huang, Bin; Zhuo, Xunhui; Chen, Xueqiu; Du, Aifang

2013-11-08

Real-time PCR-based detection of Toxoplasma gondii is very sensitive and convenient for diagnosing toxoplasmosis. However, the performance of the PCR assays could be influenced by the target gene chosen. Here we evaluate a real-time PCR assay using double-stranded DNA dyes (SYBR(®) Green I assay) with a new set of primers targeting the SAG1 gene for the fast and specific detection of T. gondii. The assay showed higher sensitivity than conventional PCR protocols using T. gondii DNA as template. The detection limit of the developed real-time PCR assay was in the order of 1 tachyzoite. The assay was also assessed by experimentally infected mice and showed positive results for blood (25%), spleen (50%) and lung (50%) as early as 1 dpi. The specificity of the assay was confirmed by using DNA from Neospora caninum, Escherichia coli, Babesia bovis, Trypanosoma brucei, Cryptosporidium parvum, and Toxocara canis. Assay applicability was successfully tested in blood samples collected from slaughtered pigs. These results indicate that, based on SYBR(®) green I, the quantitative SAG1 assay may also be useful in the study of the pathogenicity, immunoprophylaxis, and treatment of T. gondii. Copyright © 2013 Elsevier B.V. All rights reserved.

Synthesis and characterization of time-resolved fluorescence probes for evaluation of competitive binding to melanocortin receptors.

Science.gov (United States)

Alleti, Ramesh; Vagner, Josef; Dehigaspitiya, Dilani Chathurika; Moberg, Valerie E; Elshan, N G R D; Tafreshi, Narges K; Brabez, Nabila; Weber, Craig S; Lynch, Ronald M; Hruby, Victor J; Gillies, Robert J; Morse, David L; Mash, Eugene A

2013-09-01

Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining Kd values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited Kd values of 27±3.9nM and 4.2±0.48nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The Ki values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the Ki values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for these

Detection of proteins using a colorimetric bio-barcode assay.

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Nam, Jwa-Min; Jang, Kyung-Jin; Groves, Jay T

2007-01-01

The colorimetric bio-barcode assay is a red-to-blue color change-based protein detection method with ultrahigh sensitivity. This assay is based on both the bio-barcode amplification method that allows for detecting miniscule amount of targets with attomolar sensitivity and gold nanoparticle-based colorimetric DNA detection method that allows for a simple and straightforward detection of biomolecules of interest (here we detect interleukin-2, an important biomarker (cytokine) for many immunodeficiency-related diseases and cancers). The protocol is composed of the following steps: (i) conjugation of target capture molecules and barcode DNA strands onto silica microparticles, (ii) target capture with probes, (iii) separation and release of barcode DNA strands from the separated probes, (iv) detection of released barcode DNA using DNA-modified gold nanoparticle probes and (v) red-to-blue color change analysis with a graphic software. Actual target detection and quantification steps with premade probes take approximately 3 h (whole protocol including probe preparations takes approximately 3 days).

Development of a Rickettsia bellii-Specific TaqMan Assay Targeting the Citrate Synthase Gene.

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Hecht, Joy A; Allerdice, Michelle E J; Krawczak, Felipe S; Labruna, Marcelo B; Paddock, Christopher D; Karpathy, Sandor E

2016-11-01

Rickettsia bellii is a rickettsial species of unknown pathogenicity that infects argasid and ixodid ticks throughout the Americas. Many molecular assays used to detect spotted fever group (SFG) Rickettsia species do not detect R. bellii, so that infection with this bacterium may be concealed in tick populations when assays are used that screen specifically for SFG rickettsiae. We describe the development and validation of a R. bellii-specific, quantitative, real-time PCR TaqMan assay that targets a segment of the citrate synthase (gltA) gene. The specificity of this assay was validated against a panel of DNA samples that included 26 species of Rickettsia, Orientia, Ehrlichia, Anaplasma, and Bartonella, five samples of tick and human DNA, and DNA from 20 isolates of R. bellii, including 11 from North America and nine from South America. A R. bellii control plasmid was constructed, and serial dilutions of the plasmid were used to determine the limit of detection of the assay to be one copy per 4 µl of template DNA. This assay can be used to better determine the role of R. bellii in the epidemiology of tick-borne rickettsioses in the Western Hemisphere. Published by Oxford University Press on behalf of Entomological Society of America 2016. This work is written by US Government employees and is in the public domain in the US.

Twin target self-amplification-based DNA machine for highly sensitive detection of cancer-related gene.

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Xu, Huo; Jiang, Yifan; Liu, Dengyou; Liu, Kai; Zhang, Yafeng; Yu, Suhong; Shen, Zhifa; Wu, Zai-Sheng

2018-06-29

The sensitive detection of cancer-related genes is of great significance for early diagnosis and treatment of human cancers, and previous isothermal amplification sensing systems were often based on the reuse of target DNA, the amplification of enzymatic products and the accumulation of reporting probes. However, no reporting probes are able to be transformed into target species and in turn initiate the signal of other probes. Herein we reported a simple, isothermal and highly sensitive homogeneous assay system for tumor suppressor p53 gene detection based on a new autonomous DNA machine, where the signaling probe, molecular beacon (MB), was able to execute the function similar to target DNA besides providing the common signal. In the presence of target p53 gene, the operation of DNA machine can be initiated, and cyclical nucleic acid strand-displacement polymerization (CNDP) and nicking/polymerization cyclical amplification (NPCA) occur, during which the MB was opened by target species and cleaved by restriction endonuclease. In turn, the cleaved fragments could activate the next signaling process as target DNA did. According to the functional similarity, the cleaved fragment was called twin target, and the corresponding fashion to amplify the signal was named twin target self-amplification. Utilizing this newly-proposed DNA machine, the target DNA could be detected down to 0.1 pM with a wide dynamic range (6 orders of magnitude) and single-base mismatched targets were discriminated, indicating a very high assay sensitivity and good specificity. In addition, the DNA machine was not only used to screen the p53 gene in complex biological matrix but also was capable of practically detecting genomic DNA p53 extracted from A549 cell line. This indicates that the proposed DNA machine holds the potential application in biomedical research and early clinical diagnosis. Copyright © 2018 Elsevier B.V. All rights reserved.

The application of reporter gene assays for the detection of endocrine disruptors in sport supplements

International Nuclear Information System (INIS)

Plotan, Monika; Elliott, Christopher T.; Scippo, Marie Louise; Muller, Marc; Antignac, Jean-Philippe; Malone, Edward; Bovee, Toine F.H.; Mitchell, Samuel; Connolly, Lisa

2011-01-01

The increasing availability and use of sports supplements is of concern as highlighted by a number of studies reporting endocrine disruptor contamination in such products. The health food supplement market, including sport supplements, is growing across the Developed World. Therefore, the need to ensure the quality and safety of sport supplements for the consumer is essential. The development and validation of two reporter gene assays coupled with solid phase sample preparation enabling the detection of estrogenic and androgenic constituents in sport supplements is reported. Both assays were shown to be of high sensitivity with the estrogen and androgen reporter gene assays having an EC 50 of 0.01 ng mL -1 and 0.16 ng mL -1 respectively. The developed assays were applied in a survey of 63 sport supplements samples obtained across the Island of Ireland with an additional seven reference samples previously investigated using LC-MS/MS. Androgen and estrogen bio-activity was found in 71% of the investigated samples. Bio-activity profiling was further broken down into agonists, partial agonists and antagonists. Supplements (13) with the strongest estrogenic bio-activity were chosen for further investigation. LC-MS/MS analysis of these samples determined the presence of phytoestrogens in seven of them. Supplements (38) with androgen bio-activity were also selected for further investigation. Androgen agonist bio-activity was detected in 12 supplements, antagonistic bio-activity was detected in 16 and partial antagonistic bio-activity was detected in 10. A further group of supplements (7) did not present androgenic bio-activity when tested alone but enhanced the androgenic agonist bio-activity of dihydrotestosterone when combined. The developed assays offer advantages in detection of known, unknown and low-level mixtures of endocrine disruptors over existing analytical screening techniques. For the detection and identification of constituent hormonally active compounds the

Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization

International Nuclear Information System (INIS)

Collomb, J.; Finance, C.; Alabouch, S.; Laporte, J.

1992-01-01

Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG 78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes , were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabelling with 32 P and enzymatic labelling through covalent linkage to peroxidase and chemiluminescence detection. The radioactive probe 174 detected as little as 1 to 3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the family Coronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in faecal samples using enzymatic probe was compared with conventional diagnostic methods. (authors)

Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization

Energy Technology Data Exchange (ETDEWEB)

Collomb, J; Finance, C; Alabouch, S [Lab. de Microbiologie Moleculaire, Faculte des Sciences Pharmaceutiques et Biologiques, Univ. de Nancy I, Nancy (France); Laporte, J [Station de Virologie et d' Immunologie Moleculaires, INRA, Jouy-en-Josas (France)

1992-01-01

Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG 78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes , were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabeled with [sup 32]P and enzymatic labeled through covalent linkage to peroxidase for chemiluminescence detection. The radioactive probe 174 detected as little as 1-3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the family Coronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in fecal samples using enzymatic probe was compared with conventional diagnostic methods. (authors).

Detection of Helicobacter Pylori Genome with an Optical Biosensor Based on Hybridization of Urease Gene with a Gold Nanoparticles-Labeled Probe

Science.gov (United States)

Shahrashoob, M.; Mohsenifar, A.; Tabatabaei, M.; Rahmani-Cherati, T.; Mobaraki, M.; Mota, A.; Shojaei, T. R.

2016-05-01

A novel optics-based nanobiosensor for sensitive determination of the Helicobacter pylori genome using a gold nanoparticles (AuNPs)-labeled probe is reported. Two specific thiol-modified capture and signal probes were designed based on a single-stranded complementary DNA (cDNA) region of the urease gene. The capture probe was immobilized on AuNPs, which were previously immobilized on an APTES-activated glass, and the signal probe was conjugated to different AuNPs as well. The presence of the cDNA in the reaction mixture led to the hybridization of the AuNPs-labeled capture probe and the signal probe with the cDNA, and consequently the optical density of the reaction mixture (AuNPs) was reduced proportionally to the cDNA concentration. The limit of detection was measured at 0.5 nM.

Testing candidate genes for attention-deficit/hyperactivity disorder in fruit flies using a high throughput assay for complex behavior

DEFF Research Database (Denmark)

Rohde, Palle Duun; Madsen, Lisbeth Strøm; Arvidson, Sandra Marie Neumann

2016-01-01

Fruit flies are important model organisms for functional testing of candidate genes in multiple disciplines, including the study of human diseases. Here we use a high-throughput locomotor activity assay to test the response on activity behavior of gene disruption in Drosophila melanogaster. The aim...

Novel MLPA procedure using self-designed probes allows comprehensive analysis for CNVs of the genes involved in Hirschsprung disease

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Antiñolo Guillermo

2010-05-01

Full Text Available Abstract Background Hirschsprung disease is characterized by the absence of intramural ganglion cells in the enteric plexuses, due to a fail during enteric nervous system formation. Hirschsprung has a complex genetic aetiology and mutations in several genes have been related to the disease. There is a clear predominance of missense/nonsense mutations in these genes whereas copy number variations (CNVs have been seldom described, probably due to the limitations of conventional techniques usually employed for mutational analysis. In this study, we have looked for CNVs in some of the genes related to Hirschsprung (EDNRB, GFRA1, NRTN and PHOX2B using the Multiple Ligation-dependent Probe Amplification (MLPA approach. Methods CNVs screening was performed in 208 HSCR patients using a self-designed set of MLPA probes, covering the coding region of those genes. Results A deletion comprising the first 4 exons in GFRA1 gene was detected in 2 sporadic HSCR patients and in silico approaches have shown that the critical translation initiation signal in the mutant gene was abolished. In this study, we have been able to validate the reliability of this technique for CNVs screening in HSCR. Conclusions The implemented MLPA based technique presented here allows CNV analysis of genes involved in HSCR that have not been not previously evaluated. Our results indicate that CNVs could be implicated in the pathogenesis of HSCR, although they seem to be an uncommon molecular cause of HSCR.

Sensitive detection of porcine DNA in processed animal proteins using a TaqMan real-time PCR assay.

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Pegels, N; González, I; Fernández, S; García, T; Martín, R

2012-01-01

A TaqMan real-time PCR method was developed for specific detection of porcine-prohibited material in industrial feeds. The assay combines the use of a porcine-specific primer pair, which amplifies a 79 bp fragment of the mitochondrial (mt) 12 S rRNA gene, and a locked nucleic acid (LNA) TaqMan probe complementary to a target sequence lying between the porcine-specific primers. The nuclear 18 S rRNA gene system, yielding a 77 bp amplicon, was employed as a positive amplification control to monitor the total content of amplifiable DNA in the samples. The specificity of the porcine primers-probe system was verified against different animal and plant species, including mammals, birds and fish. The applicability of the real-time PCR protocol to detect the presence of porcine mt DNA in feeds was determined through the analysis of 190 industrial feeds (19 known reference and 171 blind samples) subjected to stringent processing treatments. The performance of the method allows qualitative and highly sensitive detection of short fragments from porcine DNA in all the industrial feeds declared to contain porcine material. Although the method has quantitative potential, the real quantitative capability of the assay is limited by the existing variability in terms of composition and processing conditions of the feeds, which affect the amount and quality of amplifiable DNA.

Assay for mutagenesis in heterozygous diploid human lymphoblasts

Science.gov (United States)

Skopek, Thomas R.; Liber, Howard L.; Penman, Bruce W.; Thilly, William G.; Hoppe, IV, Henry

1981-01-01

An assay is disclosed for determining mutagenic damage caused by the administration of a known or suspected mutagen to diploid human lymphoblastoid cell lines. The gene locus employed for this assay is the gene for thymidine kinase, uridine kinase, or cytidine deaminase. Since human lymphoblastoid cells contain two genes for these enzymes, heterozygotes of human lymphoblastoid cells are used in this assay.

Development and validation of sensitive real-time RT-PCR assay for broad detection of rabies virus.

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Faye, Martin; Dacheux, Laurent; Weidmann, Manfred; Diop, Sylvie Audrey; Loucoubar, Cheikh; Bourhy, Hervé; Sall, Amadou Alpha; Faye, Ousmane

2017-05-01

Rabies virus (RABV) remains one of the most important global zoonotic pathogens. RABV causes rabies, an acute encephalomyelitis associated with a high rate of mortality in humans and animals and affecting different parts of the world, particularly in Asia and Africa. Confirmation of rabies diagnosis relies on laboratory diagnosis, in which molecular techniques such as detection of viral RNA by reverse transcription polymerase chain reaction (RT-PCR) are increasingly being used. In this study, two real-time quantitative RT-PCR assays were developed for large-spectrum detection of RABV, with a focus on African isolates. The primer and probe sets were targeted highly conserved regions of the nucleoprotein (N) and polymerase (L) genes. The results indicated the absence of non-specific amplification and cross-reaction with a range of other viruses belonging to the same taxonomic family, i.e. Rhabdoviridae, as well as negative brain tissues from various host species. Analytical sensitivity ranged between 100 to 10 standard RNA copies detected per reaction for N-gene and L-gene assays, respectively. Effective detection and high sensitivity of these assays on African isolates showed that they can be successfully applied in general research and used in diagnostic process and epizootic surveillance in Africa using a double-check strategy. Copyright © 2017 Elsevier B.V. All rights reserved.

The detection of HBV DNA with gold-coated iron oxide nanoparticle gene probes

International Nuclear Information System (INIS)

Xi Dong; Luo Xiaoping; Lu Qianghua; Yao Kailun; Liu Zuli; Ning Qin

2008-01-01

Gold-coated iron oxide nanoparticle Hepatitis B virus (HBV) DNA probes were prepared, and their application for HBV DNA measurement was studied. Gold-coated iron oxide nanoparticles were prepared by the citrate reduction of tetra-chloroauric acid in the presence of iron oxide nanoparticles which were added as seeds. With a fluorescence-based method, the maximal surface coverage of hexaethiol 30-mer oligonucleotides and the maximal percentage of hybridization strands on gold-coated iron oxide nanoparticles were (120 ± 8) oligonucleotides per nanoparticle, and (14 ± 2%), respectively, which were comparable with those of (132 ± 10) and (22 ± 3%) in Au nanoparticle groups. Large network aggregates were formed when gold-coated iron oxide nanoparticle HBV DNA gene probe was applied to detect HBV DNA molecules as evidenced by transmission electron microscopy and the high specificity was verified by blot hybridization. Our results further suggested that detecting DNA with iron oxide nanoparticles and magnetic separator was feasible and might be an alternative effective method

Polymerase chain reaction assay targeting cytochrome b gene for the detection of dog meat adulteration in meatball formulation.

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Rahman, Md Mahfujur; Ali, Md Eaqub; Hamid, Sharifah Bee Abd; Mustafa, Shuhaimi; Hashim, Uda; Hanapi, Ummi Kalthum

2014-08-01

A polymerase chain reaction (PCR) assay for the assessment of dog meat adulteration in meatballs was developed. The assay selectively amplified a 100-bp region of canine mitochondrial cytochrome b gene from pure, raw, processed and mixed backgrounds. The specificity of the assay was tested against 11 animals and 3 plants species, commonly available for meatball formulation. The stability of the assay was proven under extensively autoclaving conditions that breakdown target DNA. A blind test from ready to eat chicken and beef meatballs showed that the assay can repeatedly detect 0.2% canine meat tissues under complex matrices using 0.04 ng of dog DNA extracted from differentially treated meatballs. The simplicity, stability and sensitivity of the assay suggested that it could be used in halal food industry for the authentication of canine derivatives in processed foods. Copyright © 2014 Elsevier Ltd. All rights reserved.

Melt analysis of mismatch amplification mutation assays (Melt-MAMA: a functional study of a cost-effective SNP genotyping assay in bacterial models.

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Dawn N Birdsell

Full Text Available Single nucleotide polymorphisms (SNPs are abundant in genomes of all species and biologically informative markers extensively used across broad scientific disciplines. Newly identified SNP markers are publicly available at an ever-increasing rate due to advancements in sequencing technologies. Efficient, cost-effective SNP genotyping methods to screen sample populations are in great demand in well-equipped laboratories, but also in developing world situations. Dual Probe TaqMan assays are robust but can be cost-prohibitive and require specialized equipment. The Mismatch Amplification Mutation Assay, coupled with melt analysis (Melt-MAMA, is flexible, efficient and cost-effective. However, Melt-MAMA traditionally suffers from high rates of assay design failures and knowledge gaps on assay robustness and sensitivity. In this study, we identified strategies that improved the success of Melt-MAMA. We examined the performance of 185 Melt-MAMAs across eight different pathogens using various optimization parameters. We evaluated the effects of genome size and %GC content on assay development. When used collectively, specific strategies markedly improved the rate of successful assays at the first design attempt from ~50% to ~80%. We observed that Melt-MAMA accurately genotypes across a broad DNA range (~100 ng to ~0.1 pg. Genomic size and %GC content influence the rate of successful assay design in an independent manner. Finally, we demonstrated the versatility of these assays by the creation of a duplex Melt-MAMA real-time PCR (two SNPs and conversion to a size-based genotyping system, which uses agarose gel electrophoresis. Melt-MAMA is comparable to Dual Probe TaqMan assays in terms of design success rate and accuracy. Although sensitivity is less robust than Dual Probe TaqMan assays, Melt-MAMA is superior in terms of cost-effectiveness, speed of development and versatility. We detail the parameters most important for the successful application of

Validation of a PCR Assay for Chlamydophila abortus rRNA gene detection in a murine model

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Francielle Gibson da Silva-Zacarias

2009-11-01

Full Text Available Chlamydophila abortus (C. abortus is associated with reproductive problems in cattle, sheep, and goats. Diagnosis of C. abortus using embryonated chicken eggs or immortalized cell lines has a very low sensitivity. Polymerase chain reaction (PCR assays have been used to detect C. abortus infection in clinical specimens and organ fragments, such as placenta, fetal organs, vaginal secretions, and semen. The aim of this study was to develop a PCR assay for the amplification of an 856-bp fragment of the rRNA gene of the Chlamydiaceae family. The PCR assay was evaluated using organs from 15 mice experimentally infected with the S26/3 reference strain of C. abortus. The results of the rRNA PCR were compared to the results from another PCR system (Omp2 PCR that has been previously described for the Omp2 (outer major protein gene from the Chlamydiaceae family. From the 15 C. abortus-inoculated mice, 13 (K=0.84, standard error =0.20 tested positive using the rRNA PCR assay and 9 (K=0.55, standard error=0.18 tested positive using the Omp2 PCR assay. The detection limit, measured using inclusion-forming units (IFU, for C. abortus with the rRNA PCR (1.05 IFU was 100-fold lower than for the Omp2 PCR (105 IFU. The higher sensitivity of the rRNA PCR, as compared to the previously described PCR assay, and the specificity of the assay, demonstrated using different pathogenic microorganisms of the bovine reproductive system, suggest that the new PCR assay developed in this study can be used for the molecular diagnosis of C. abortus in abortion and other reproductive failures in bovines, caprines, and ovines.Chlamydophila abortus (C. abortus é frequentemente associada a distúrbios reprodutivos em bovinos, ovinos e caprinos. Para o diagnóstico, os métodos de cultivo em ovo embrionado de galinha e em células de linhagem contínua apresentam baixa sensibilidade. A reação em cadeia da polimerase (PCR tem sido utilizada em placenta, órgãos fetais, secre

Identification of a novel prophage-like gene cluster actively expressed in both virulent and avirulent strains of Leptospira interrogans serovar Lai.

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Qin, Jin-Hong; Zhang, Qing; Zhang, Zhi-Ming; Zhong, Yi; Yang, Yang; Hu, Bao-Yu; Zhao, Guo-Ping; Guo, Xiao-Kui

2008-06-01

DNA microarray analysis was used to compare the differential gene expression profiles between Leptospira interrogans serovar Lai type strain 56601 and its corresponding attenuated strain IPAV. A 22-kb genomic island covering a cluster of 34 genes (i.e., genes LA0186 to LA0219) was actively expressed in both strains but concomitantly upregulated in strain 56601 in contrast to that of IPAV. Reverse transcription-PCR assays proved that the gene cluster comprised five transcripts. Gene annotation of this cluster revealed characteristics of a putative prophage-like remnant with at least 8 of 34 sequences encoding prophage-like proteins, of which the LA0195 protein is probably a putative prophage CI-like regulator. The transcription initiation activities of putative promoter-regulatory sequences of transcripts I, II, and III, all proximal to the LA0195 gene, were further analyzed in the Escherichia coli promoter probe vector pKK232-8 by assaying the reporter chloramphenicol acetyltransferase (CAT) activities. The strong promoter activities of both transcripts I and II indicated by the E. coli CAT assay were well correlated with the in vitro sequence-specific binding of the recombinant LA0195 protein to the corresponding promoter probes detected by the electrophoresis mobility shift assay. On the other hand, the promoter activity of transcript III was very low in E. coli and failed to show active binding to the LA0195 protein in vitro. These results suggested that the LA0195 protein is likely involved in the transcription of transcripts I and II. However, the identical complete DNA sequences of this prophage remnant from these two strains strongly suggests that possible regulatory factors or signal transduction systems residing outside of this region within the genome may be responsible for the differential expression profiling in these two strains.

A multiplex PCR assay for the detection and quantification of Sclerotinia sclerotiorum and Botrytis cinerea.

Science.gov (United States)

Reich, J D; Alexander, T W; Chatterton, S

2016-05-01

Traditional culture methods for identifying the plant fungal pathogens Sclerotinia sclerotiorum (Lib.) de Bary and Botrytis cinerea Pers.:Fr. are slow and laborious. The goal of this study was to develop a multiplex real-time PCR (qPCR) assay to detect and quantify DNA from S. sclerotiorum and B. cinerea. A primer set (SsIGS_5) for S. sclerotiorum was designed that targeted the intergenic spacer (IGS) regions of the ribosomal DNA. Addition of a probe to the assay increased its specificity: when the primer/probe set was tested against 21 fungal species (35 strains), amplification was detected from all S. sclerotiorum strains and no other species. For qPCR, the SsIGS_5 primer and probe set exhibited a linear range from 7·0 ng to 0·07 pg target DNA (R(2)  = 0·99). SsIGS_5 was then multiplexed with a previously published primer/probe set for B. cinerea to develop a high-throughput method for the detection and quantification of DNA from both pathogens. When multiplexed, the sensitivity and specificity of both assays were not different from individual qPCR reactions. The multiplex assay is currently being used to detect and quantify S. sclerotiorum and B. cinerea DNA from aerosol samples collected in commercial seed alfalfa fields. A primer and probe set for the quantification of Sclerotinia sclerotiorum DNA in a PCR assay was developed. The probe-based nature of this assay signifies an improvement over previous assays for this species by allowing multiplex reactions while maintaining high sensitivity. The primer/probe set was used in a multiplex real-time PCR assay for the quantification of S. sclerotiorum and Botrytis cinerea DNA, enabling rapid analysis of environmental samples. In crops susceptible to both pathogens, this multiplex assay can be used to quickly quantify the presence of each pathogen. © 2016 Her Majesty the Queen in Right of Canada © 2016 The Society for Applied Microbiology. Reproduced with the permission of the Office of the

Development of a hydrolysis probe-based real-time assay for the detection of tropical strains of Fusarium oxysporum f. sp. cubense race 4

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Cerf-Wendling, Isabelle; Hostachy, Bruno; Viljoen, Altus; Ioos, Renaud

2017-01-01

Fusarium oxysporum f. sp. cubense (Foc) is one of the most important threats to global banana production. Strategies to control the pathogen are lacking, with plant resistance offering the only long-term solution, if sources of resistance are available. Prevention of introduction of Foc into disease-free areas thus remains a key strategy to continue sustainable banana production. In recent years, strains of Foc affecting Cavendish bananas have destroyed plantations in a number of countries in Asia and in the Middle East, and one African country. One vegetative compatibility group (VCG), 01213/16, is considered the major threat to bananas in tropical and subtropical climatic conditions. However, other genetically related VCGs, such as 0121, may potentially jeopardize banana cultures if they were introduced into disease-free areas. To prevent the introduction of these VCGs into disease-free Cavendish banana-growing countries, a real-time PCR test was developed to accurately detect both VCGs. A previously described putative virulence gene was used to develop a specific combination of hydrolysis probe/primers for the detection of tropical Foc race 4 strains. The real-time PCR parameters were optimized by following a statistical approach relying on orthogonal arrays and the Taguchi method in an attempt to enhance sensitivity and ensure high specificity of the assay. This study also assessed critical performance criteria, such as repeatability, reproducibility, robustness, and specificity, with a large including set of 136 F. oxysporum isolates, including 73 Foc pathogenic strains representing 24 VCGs. The validation data demonstrated that the new assay could be used for regulatory testing applications on banana plant material and can contribute to preventing the introduction and spread of Foc strains affecting Cavendish bananas in the tropics. PMID:28178348

Development and Application of Loop-Mediated Isothermal Amplification Assays for Rapid Visual Detection of cry2Ab and cry3A Genes in Genetically-Modified Crops

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Feiwu Li

2014-08-01

Full Text Available The cry2Ab and cry3A genes are two of the most important insect-resistant exogenous genes and had been widely used in genetically-modified crops. To develop more effective alternatives for the quick identification of genetically-modified organisms (GMOs containing these genes, a rapid and visual loop-mediated isothermal amplification (LAMP method to detect the cry2Ab and cry3A genes is described in this study. The LAMP assay can be finished within 60 min at an isothermal condition of 63 °C. The derived LAMP products can be obtained by a real-time turbidimeter via monitoring the white turbidity or directly observed by the naked eye through adding SYBR Green I dye. The specificity of the LAMP assay was determined by analyzing thirteen insect-resistant genetically-modified (GM crop events with different Bt genes. Furthermore, the sensitivity of the LAMP assay was evaluated by diluting the template genomic DNA. Results showed that the limit of detection of the established LAMP assays was approximately five copies of haploid genomic DNA, about five-fold greater than that of conventional PCR assays. All of the results indicated that this established rapid and visual LAMP assay was quick, accurate and cost effective, with high specificity and sensitivity. In addition, this method does not need specific expensive instruments or facilities, which can provide a simpler and quicker approach to detecting the cry2Ab and cry3A genes in GM crops, especially for on-site, large-scale test purposes in the field.

Development and application of loop-mediated isothermal amplification assays for rapid visual detection of cry2Ab and cry3A genes in genetically-modified crops.

Science.gov (United States)

Li, Feiwu; Yan, Wei; Long, Likun; Qi, Xing; Li, Congcong; Zhang, Shihong

2014-08-27

The cry2Ab and cry3A genes are two of the most important insect-resistant exogenous genes and had been widely used in genetically-modified crops. To develop more effective alternatives for the quick identification of genetically-modified organisms (GMOs) containing these genes, a rapid and visual loop-mediated isothermal amplification (LAMP) method to detect the cry2Ab and cry3A genes is described in this study. The LAMP assay can be finished within 60 min at an isothermal condition of 63 °C. The derived LAMP products can be obtained by a real-time turbidimeter via monitoring the white turbidity or directly observed by the naked eye through adding SYBR Green I dye. The specificity of the LAMP assay was determined by analyzing thirteen insect-resistant genetically-modified (GM) crop events with different Bt genes. Furthermore, the sensitivity of the LAMP assay was evaluated by diluting the template genomic DNA. Results showed that the limit of detection of the established LAMP assays was approximately five copies of haploid genomic DNA, about five-fold greater than that of conventional PCR assays. All of the results indicated that this established rapid and visual LAMP assay was quick, accurate and cost effective, with high specificity and sensitivity. In addition, this method does not need specific expensive instruments or facilities, which can provide a simpler and quicker approach to detecting the cry2Ab and cry3A genes in GM crops, especially for on-site, large-scale test purposes in the field.

Development and utility of an internal threshold control (ITC real-time PCR assay for exogenous DNA detection.

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Weiyi Ni

Full Text Available Sensitive and specific tests for detecting exogenous DNA molecules are useful for infectious disease diagnosis, gene therapy clinical trial safety, and gene doping surveillance. Taqman real-time PCR using specific sequence probes provides an effective approach to accurately and quantitatively detect exogenous DNA. However, one of the major challenges in these analyses is to eliminate false positive signals caused by either non-targeted exogenous or endogenous DNA sequences, or false negative signals caused by impurities that inhibit PCR. Although multiplex Taqman PCR assays have been applied to address these problems by adding extra primer-probe sets targeted to endogenous DNA sequences, the differences between targets can lead to different detection efficiencies. To avoid these complications, a Taqman PCR-based approach that incorporates an internal threshold control (ITC has been developed. In this single reaction format, the target sequence and ITC template are co-amplified by the same primers, but are detected by different probes each with a unique fluorescent dye. Sample DNA, a prescribed number of ITC template molecules set near the limit of sensitivity, a single pair of primers, target probe and ITC probe are added to one reaction. Fluorescence emission signals are obtained simultaneously to determine the cycle thresholds (Ct for amplification of the target and ITC sequences. The comparison of the target Ct with the ITC Ct indicates if a sample is a true positive for the target (i.e. Ct less than or equal to the ITC Ct or negative (i.e. Ct greater than the ITC Ct. The utility of this approach was demonstrated in a nonhuman primate model of rAAV vector mediated gene doping in vivo and in human genomic DNA spiked with plasmid DNA.

Gene expression and gene therapy imaging

International Nuclear Information System (INIS)

Rome, Claire; Couillaud, Franck; Moonen, Chrit T.W.

2007-01-01

The fast growing field of molecular imaging has achieved major advances in imaging gene expression, an important element of gene therapy. Gene expression imaging is based on specific probes or contrast agents that allow either direct or indirect spatio-temporal evaluation of gene expression. Direct evaluation is possible with, for example, contrast agents that bind directly to a specific target (e.g., receptor). Indirect evaluation may be achieved by using specific substrate probes for a target enzyme. The use of marker genes, also called reporter genes, is an essential element of MI approaches for gene expression in gene therapy. The marker gene may not have a therapeutic role itself, but by coupling the marker gene to a therapeutic gene, expression of the marker gene reports on the expression of the therapeutic gene. Nuclear medicine and optical approaches are highly sensitive (detection of probes in the picomolar range), whereas MRI and ultrasound imaging are less sensitive and require amplification techniques and/or accumulation of contrast agents in enlarged contrast particles. Recently developed MI techniques are particularly relevant for gene therapy. Amongst these are the possibility to track gene therapy vectors such as stem cells, and the techniques that allow spatiotemporal control of gene expression by non-invasive heating (with MRI guided focused ultrasound) and the use of temperature sensitive promoters. (orig.)

Sources of Blood Meals of Sylvatic Triatoma guasayana near Zurima, Bolivia, Assayed with qPCR and 12S Cloning

Science.gov (United States)

Lucero, David E.; Ribera, Wilma; Pizarro, Juan Carlos; Plaza, Carlos; Gordon, Levi W.; Peña, Reynaldo; Morrissey, Leslie A.; Rizzo, Donna M.; Stevens, Lori

2014-01-01

Background In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps). Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia. Methodology/Principal Findings We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens), five for chicken (Gallus gallus) and unicolored blackbird (Agelasticus cyanopus), and one for opossum (Monodelphis domestica). Using the qPCR assay we detected chicken (13 vectors), and human (14 vectors) blood meals as well as an additional blood meal source, Canis sp. (4 vectors). Conclusions/Significance We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors. PMID:25474154

Sources of blood meals of sylvatic Triatoma guasayana near Zurima, Bolivia, assayed with qPCR and 12S cloning.

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David E Lucero

2014-12-01

Full Text Available In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps. Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia.We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens, five for chicken (Gallus gallus and unicolored blackbird (Agelasticus cyanopus, and one for opossum (Monodelphis domestica. Using the qPCR assay we detected chicken (13 vectors, and human (14 vectors blood meals as well as an additional blood meal source, Canis sp. (4 vectors.We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors.

Quantitative Tetraplex Real-Time Polymerase Chain Reaction Assay with TaqMan Probes Discriminates Cattle, Buffalo, and Porcine Materials in Food Chain.

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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.

Value of a gene signature assay in patients with early breast cancer and intermediate risk: a single institution retrospective study.

Science.gov (United States)

Bonneterre, Jacques; Prat, Aleix; Galván, Patricia; Morel, Pascale; Giard, Sylvia

2016-05-01

Purpose In daily clinical practice, the indication for adjuvant chemotherapy (CT) is relatively easy to make in patients with early hormone-receptor-positive (HR+) breast cancer with either very poor or very good clinicopathological prognostic variables. However, this decision is much more difficult in patients with intermediate clinicopathological prognostic variables. Here, we evaluate the value of a gene-expression profile identified by the Prosigna gene signature assay in guiding treatment decision-making in patients with these intermediate features. Methods A consecutive cohort of 577 HR + breast cancer patients surgically treated in a single institution between January 2012 and December 2012 was evaluated. From this population, pre- and post-menopausal patients with intermediate prognosis clinicopathological variables were identified and indication of adjuvant CT in these patients was recorded. The gene signature assay was performed retrospectively in this intermediate risk group. Descriptive statistics are presented. Results Among 96 intermediate-risk patients, 64 postmenopausal patients underwent gene signature testing. Subtype distribution was as follows: Luminal A (N = 33; 51.6%), Luminal B (N = 31; 48.4%). Risk of recurrence (ROR) distribution was as follows: ROR-low (n = 16; 25%); ROR-intermediate (N = 26; 40.6%); and ROR-high (N = 22; 34.4%). CT was subsequently administered in 18.7%, 53.8% and 59.0% of the ROR-low, ROR-intermediate and ROR-high groups, respectively. With the use of the gene signature assay, 59.4% of the intermediate cases were re-classified to either ROR-low or ROR-high risk categories. In the ROR-intermediate group, 11/26 patients (42.3%) had Luminal A and 15/26 (57.7%) had Luminal B. Due to follow-up time constraints, no patient outcome results were evaluated. Conclusion The gene signature assay provides clinically useful information and improved treatment decision-making in patients with intermediate risk based on

Development and evaluation of a 28S rRNA gene-based nested PCR assay for P. falciparum and P. vivax

Science.gov (United States)

Pakalapati, Deepak; Garg, Shilpi; Middha, Sheetal; Acharya, Jyoti; Subudhi, Amit K; Boopathi, Arunachalam P; Saxena, Vishal; Kochar, Sanjay K; Kochar, Dhanpat K; Das, Ashis

2013-01-01

The 28S rRNA gene was amplified and sequenced from P. falciparum and P. vivax isolates collected from northwest India. Based upon the sequence diversity of the Plasmodium 28SrRNA gene in comparison with its human counterpart, various nested polymerase chain reaction (PCR) primers were designed from the 3R region of the 28SrRNA gene and evaluated on field isolates. This is the first report demonstrating the utility of this gene for species-specific diagnosis of malaria for these two species, prevalent in India. The initial evaluation on 363 clinical isolates indicated that, in comparison with microscopy, which showed sensitivity and specificity of 85.39% and 100% respectively, the sensitivity and specificity of the nested PCR assay was found to be 99.08% and 100% respectively. This assay was also successful in detecting mixed infections that are undetected by microscopy. Our results demonstrate the utility of the 28S rRNA gene as a diagnostic target for the detection of the major plasmodial species infecting humans. PMID:23816509

The application of reporter gene assays for the detection of endocrine disruptors in sport supplements

Energy Technology Data Exchange (ETDEWEB)

Plotan, Monika; Elliott, Christopher T. [Institute of Agri-Food and Land Use, School of Biological Sciences, Queen' s University Belfast, Belfast BT95AG, Northern Ireland (United Kingdom); Scippo, Marie Louise [Department of Food Sciences, University of Liege, 4000 Liege (Belgium); Muller, Marc [Molecular Biology and Genetic Engineering GIGA-R, University of Liege, 4000 Liege (Belgium); Antignac, Jean-Philippe [LABERCA, ENVN, USC INRA 2013, BP 50707, 44 307, Nantes (France); Malone, Edward [The State Laboratory, Young' s Cross, Celbridge, Co. Kildare (Ireland); Bovee, Toine F.H. [RIKILT Institute of Food Safety, P.O. Box 230, AE Wageningen 6700 (Netherlands); Mitchell, Samuel [Agri-Food and Biosciences Institute, Belfast BT9 5PX (United Kingdom); Connolly, Lisa, E-mail: l.connolly@qub.ac.uk [Institute of Agri-Food and Land Use, School of Biological Sciences, Queen' s University Belfast, Belfast BT95AG, Northern Ireland (United Kingdom)

2011-08-26

The increasing availability and use of sports supplements is of concern as highlighted by a number of studies reporting endocrine disruptor contamination in such products. The health food supplement market, including sport supplements, is growing across the Developed World. Therefore, the need to ensure the quality and safety of sport supplements for the consumer is essential. The development and validation of two reporter gene assays coupled with solid phase sample preparation enabling the detection of estrogenic and androgenic constituents in sport supplements is reported. Both assays were shown to be of high sensitivity with the estrogen and androgen reporter gene assays having an EC{sub 50} of 0.01 ng mL{sup -1} and 0.16 ng mL{sup -1} respectively. The developed assays were applied in a survey of 63 sport supplements samples obtained across the Island of Ireland with an additional seven reference samples previously investigated using LC-MS/MS. Androgen and estrogen bio-activity was found in 71% of the investigated samples. Bio-activity profiling was further broken down into agonists, partial agonists and antagonists. Supplements (13) with the strongest estrogenic bio-activity were chosen for further investigation. LC-MS/MS analysis of these samples determined the presence of phytoestrogens in seven of them. Supplements (38) with androgen bio-activity were also selected for further investigation. Androgen agonist bio-activity was detected in 12 supplements, antagonistic bio-activity was detected in 16 and partial antagonistic bio-activity was detected in 10. A further group of supplements (7) did not present androgenic bio-activity when tested alone but enhanced the androgenic agonist bio-activity of dihydrotestosterone when combined. The developed assays offer advantages in detection of known, unknown and low-level mixtures of endocrine disruptors over existing analytical screening techniques. For the detection and identification of constituent hormonally

Rapid activity-directed screening of estrogens by parallel coupling of liquid chromatography with a functional gene reporter assay and mass spectrometry

NARCIS (Netherlands)

Jonker, W.; Lamoree, M.H.; Houtman, C.J.; Hamers, T.; Somsen, G.W.; Kool, J.

2015-01-01

In this study we developed a new LC nanofractionation platform that combines a human cell (BG1.Luc) gene reporter assay with a high resolution mass spectrometer for the detection and identification of estrogenic and anti-estrogenic compounds in environmental waters. The selection of this assay was

Quantitative multiplex assay for simultaneous detection and identification of Indiana and New Jersey serotypes of vesicular stomatitis virus

DEFF Research Database (Denmark)

Rasmussen, Thomas Bruun; Uttenthal, Åse; Fernandez, Jovita

2005-01-01

In order to establish a rapid and reliable system for the detection of vesicular stomatitis virus (VSV), we developed a quantitative reverse transcription-PCR assay for the detection, quantification, and differentiation of the major serotypes, VSV Indiana and VSV New Jersey, using a closed......-tube multiplex format. The detection system is based on the recently invented primer-probe energy transfer (PriProET) system. A region of the gene encoding the RNA-dependent RNA polymerase was amplified by using VSV-specific primers in the presence of two serotype-specific fluorescent probes. By incorporating...... probes. The limits of detection ware found to be less than 10 50% tissue culture infective doses/ml for both serotypes. The diagnostic value of the new method was tested with clinical materials from experimentally infected pigs, and it is concluded that the method is a powerful tool for the rapid...

Serodiagnosis of cutaneous leishmaniasis: assessment of an enzyme-linked immunosorbent assay using a peptide sequence from gene B protein

DEFF Research Database (Denmark)

Jensen, A T; Gaafar, A; Ismail, A

1996-01-01

An enzyme-linked immunosorbent assay (ELISA) using a 28 amino acid sequence of the repetitive element of gene B protein (GBP) from Leishmania major was developed for serodiagnosis of cutaneous leishmaniasis (CL). The assay was compared to ELISAs using crude amastigote and promastigote antigens from...... samples from healthy Sudanese individuals living in an area endemic for malaria but free of leish-maniasis were negative in all the assays. Significantly higher levels of antibodies were found in the patients who had suffered from the disease for more than eight weeks than in patients with a shorter...

Rapid and Quantitative Detection of Leifsonia xyli subsp. xyli in Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan PCR Assay

Directory of Open Access Journals (Sweden)

Hua-Ying Fu

2016-01-01

Full Text Available 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.

Synthesis and detection of 3'-OH terminal biotin-labeled DNA probes

International Nuclear Information System (INIS)

Brakel, C.L.; Engelhardt, D.L.

1985-01-01

Nick translation has been used to prepare biotin-dUTP-containing DNA probes. These stable DNA probes have been identified, following hybridization to target DNA, by fluorescence using antibiotin antibodies or by enzyme reactions in which the enzyme has been linked to avidin or streptavidin. It is probable that this technology will be applicable to certain diagnostic determinations and that, with sufficient sensitivity, this technology might provide a system for obtaining rapid and specific diagnoses in situations presently requiring time-consuming growth assays. The sensitivity of this assay can be increased in two ways: (1) by increasing the amount of biotin contained in the DNA probes, and (2) by increasing the response to individual biotin molecules in the DNA probes. This report demonstrates that terminal deoxynucleotide transferase can be employed to increase the biotin content of DNA probes. We also introduce a new streptavidin-linked enzyme system that produces a greater response to biotinylated DNA probes than does streptavidin-linked horseradish peroxidase

Novel gene fusion of PRCC-MITF defines a new member of MiT family translocation renal cell carcinoma: clinicopathological analysis and detection of the gene fusion by RNA sequencing and FISH.

Science.gov (United States)

Xia, Qiu-Yuan; Wang, Xiao-Tong; Ye, Sheng-Bing; Wang, Xuan; Li, Rui; Shi, Shan-Shan; Fang, Ru; Zhang, Ru-Song; Ma, Heng-Hui; Lu, Zhen-Feng; Shen, Qin; Bao, Wei; Zhou, Xiao-Jun; Rao, Qiu

2018-04-01

MITF, TFE3, TFEB and TFEC belong to the same microphthalmia-associated transcription factor family (MiT). Two transcription factors in this family have been identified in two unusual types of renal cell carcinoma (RCC): Xp11 translocation RCC harbouring TFE3 gene fusions and t(6;11) RCC harbouring a MALAT1-TFEB gene fusion. The 2016 World Health Organisation classification of renal neoplasia grouped these two neoplasms together under the category of MiT family translocation RCC. RCCs associated with the other two MiT family members, MITF and TFEC, have rarely been reported. Herein, we identify a case of MITF translocation RCC with the novel PRCC-MITF gene fusion by RNA sequencing. Histological examination of the present tumour showed typical features of MiT family translocation RCCs, overlapping with Xp11 translocation RCC and t(6;11) RCC. However, this tumour showed negative results in TFE3 and TFEB immunochemistry and split fluorescence in-situ hybridisation (FISH) assays. The other MiT family members, MITF and TFEC, were tested further immunochemically and also showed negative results. RNA sequencing and reverse transcription-polymerase chain reaction confirmed the presence of a PRCC-MITF gene fusion: a fusion of PRCC exon 5 to MITF exon 4. We then developed FISH assays covering MITF break-apart probes and PRCC-MITF fusion probes to detect the MITF gene rearrangement. This study both proves the recurring existence of MITF translocation RCC and expands the genotype spectrum of MiT family translocation RCCs. © 2017 John Wiley & Sons Ltd.

Sequence heterogeneity in the 18S rRNA gene in Theileria equi from horses presented in Switzerland.

Science.gov (United States)

Liu, Qin; Meli, Marina L; Zhang, Yi; Meili, Theres; Stirn, Martina; Riond, Barbara; Weibel, Beatrice; Hofmann-Lehmann, Regina

2016-05-15

A reverse line blot (RLB) hybridization assay was adapted and applied for equine blood samples collected at the animal hospital of the University of Zurich to determine the presence of piroplasms in horses in Switzerland. A total of 100 equine blood samples were included in the study. The V4 hypervariable region of the 18S rRNA gene was amplified by polymerase chain reaction and analyzed using the RLB assay. Samples from seven horses hybridized to a Theileria/Babesia genus-specific and a Theileria genus-specific probe. Of these, two hybridized also to the Theileria equi-specific probe. The other five positive samples did not hybridize to any of the species-specific probes, suggesting the presence of unrecognized Theileria variants or genotypes. The 18S rRNA gene of the latter five samples were sequenced and found to be closely related to T. equi isolated from horses in Spain (AY534822) and China (KF559357) (≥98.4% identity). Four of the seven horses that tested positive had a documented travel history (France, Italy, and Spain) or lived abroad (Hungary). The present study adds new insight into the presence and sequence heterogeneity of T. equi in Switzerland. The results prompt that species-specific probes must be designed in regions of the gene unique to T. equi. Of note, none of the seven positive horses were suspected of having Theileria infection at the time of presentation to the clinic. Clinicians should be aware of the possibility of equine piroplasma infections outside of endemic areas and in horses without signs of piroplasmosis. Copyright © 2016 Elsevier B.V. All rights reserved.

The relationship between quantitative human epidermal growth factor receptor 2 gene expression by the 21-gene reverse transcriptase polymerase chain reaction assay and adjuvant trastuzumab benefit in Alliance N9831.

Science.gov (United States)

Perez, Edith A; Baehner, Frederick L; Butler, Steven M; Thompson, E Aubrey; Dueck, Amylou C; Jamshidian, Farid; Cherbavaz, Diana; Yoshizawa, Carl; Shak, Steven; Kaufman, Peter A; Davidson, Nancy E; Gralow, Julie; Asmann, Yan W; Ballman, Karla V

2015-10-01

The N9831 trial demonstrated the efficacy of adjuvant trastuzumab for patients with human epidermal growth factor receptor 2 (HER2) locally positive tumors by protein or gene analysis. We used the 21-gene assay to examine the association of quantitative HER2 messenger RNA (mRNA) gene expression and benefit from trastuzumab. N9831 tested the addition of trastuzumab to chemotherapy in stage I-III HER2-positive breast cancer. For two of the arms of the trial, doxorubicin and cyclophosphamide followed by paclitaxel (AC-T) and doxorubicin and cyclophosphamide followed by paclitaxel and trastuzumab concurrent chemotherapy-trastuzumab (AC-TH), recurrence score (RS) and HER2 mRNA expression were determined by the 21-gene assay (Oncotype DX®) (negative 10 % positive cells = positive), 91 % for RT-PCR versus central fluorescence in situ hybridization (FISH) (≥2.0 = positive) and 94 % for central IHC versus central FISH. In the primary analysis, the association of HER2 expression by 21-gene assay with trastuzumab benefit was marginally nonsignificant (nonlinear p = 0.057). In hormone receptor-positive patients (local IHC) the association was significant (p = 0.002). The association was nonlinear with the greatest estimated benefit at lower and higher HER2 expression levels. Concordance among HER2 assessments by central IHC, FISH, and RT-PCR were similar and high. Association of HER2 mRNA expression with trastuzumab benefit as measured by time to distant recurrence was nonsignificant. A consistent benefit of trastuzumab irrespective of mHER2 levels was observed in patients with either IHC-positive or FISH-positive tumors. Trend for benefit was observed also for the small groups of patients with negative results by any or all of the central assays. Clinicaltrials.gov NCT00005970 . Registered 5 July 2000.

Repression of the albumin gene in Novikoff hepatoma cells

International Nuclear Information System (INIS)

Capetanaki, Y.G.; Flytzanis, C.N.; Alonso, A.

1982-01-01

Novikoff hepatoma cells have lost their capacity to synthesize albumin. As a first approach to study the mechanisms underlying this event, in vitro translation in a reticulocyte system was performed using total polyadenylated mRNA from rat liver and Novikoff hepatoma cells. Immunoprecipitation of the in vitro translation products with albumin-specific antibody revealed a total lack of albumin synthesis in Novikoff hepatoma, suggesting the absence of functional albumin mRNA in these cells. Titration experiments using as probe albumin cDNA cloned in pBR322 plasmid demonstrated the absence of albumin-specific sequences in both polysomal and nuclear polyadenylated and total RNA from Novikoff cells. This albumin recombinant plasmid was obtained by screening a rat liver cDNA library with albumin [/sup 32/P]cDNA reverse transcribed from immuno-precipitated mRNA. The presence of an albumin-specific gene insert was documented with translation assays as well as by restriction mapping. Repression of the albumin gene at the transcriptional level was further demonstrated by RNA blotting experiments using the cloned albumin cDNA probe. Genomic DNA blots using the cloned albumin cDNA as probe did not reveal any large-scale deletions, insertions, or rearrangements in the albumin gene, suggesting that the processes involved in the suppression of albumin mRNA synthesis do not involve extensive genomic rearrangements

Recombinant phage probes for Listeria monocytogenes

Science.gov (United States)

Carnazza, S.; Gioffrè, G.; Felici, F.; Guglielmino, S.

2007-10-01

Monitoring of food and environmental samples for biological threats, such as Listeria monocytogenes, requires probes that specifically bind biological agents and ensure their immediate and efficient detection. There is a need for robust and inexpensive affinity probes as an alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we selected from two phage peptide libraries phage clones displaying peptides capable of specific and strong binding to the L. monocytogenes cell surface. The ability of isolated phage clones to interact specifically with L. monocytogenes was demonstrated using enzyme-linked immunosorbent assay (ELISA) and confirmed by co-precipitation assay. We also assessed the sensitivity of phage-bacteria binding by PCR on phage-captured Listeria cells, which could be detected at a concentration of 104 cells ml-1. In addition, as proof-of-concept, we tested the possibility of immobilizing the affinity-selected phages to a putative biosensor surface. The quality of phage deposition was monitored by ELISA and fluorescent microscopy. Phage-bacterial binding was confirmed by high power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including in food products.

Molecular beacon probes-base multiplex NASBA Real-time for detection of HIV-1 and HCV.

Science.gov (United States)

Mohammadi-Yeganeh, S; Paryan, M; Mirab Samiee, S; Kia, V; Rezvan, H

2012-06-01

Developed in 1991, nucleic acid sequence-based amplification (NASBA) has been introduced as a rapid molecular diagnostic technique, where it has been shown to give quicker results than PCR, and it can also be more sensitive. This paper describes the development of a molecular beacon-based multiplex NASBA assay for simultaneous detection of HIV-1 and HCV in plasma samples. A well-conserved region in the HIV-1 pol gene and 5'-NCR of HCV genome were used for primers and molecular beacon design. The performance features of HCV/HIV-1 multiplex NASBA assay including analytical sensitivity and specificity, clinical sensitivity and clinical specificity were evaluated. The analysis of scalar concentrations of the samples indicated that the limit of quantification of the assay was beacon probes detected all HCV genotypes and all major variants of HIV-1. This method may represent a relatively inexpensive isothermal method for detection of HIV-1/HCV co-infection in monitoring of patients.

A novel RT-qPCR assay for quantification of the MLL-MLLT3 fusion transcript in acute myeloid leukaemia

DEFF Research Database (Denmark)

Abildgaard, Lotte; Ommen, Hans Beier; Lausen, Birgitte Frederiksen

2013-01-01

OBJECTIVES: Patients with acute myeloid leukaemia (AML) of the monocytic lineage often lack molecular markers for minimal residual disease (MRD) monitoring. The MLL-MLLT3 fusion transcript found in patients with AML harbouring t(9;11) is amenable to RT-qPCR quantification but because...... of the heterogeneity of translocation break points, the MLL-MLLT3 fusion gene is a challenging target. We hypothesised that MRD monitoring using MLL-MLLT3 as a RT-qPCR marker is feasible in the majority of patients with t(9;11)-positive AML. METHODS: Using a locked nucleic acid probe, we developed a sensitive RT......-qPCR assay for quantification of the most common break point region of the MLL-MLLT3 fusion gene. Five paediatric patients with t(9;11)-positive AML were monitored using the MLL-MLLT3 assay. RESULTS: A total of 43 bone marrow (BM) and 52 Peripheral blood (PB) samples were collected from diagnosis until...

Development and Characterization of Probe-Based Real Time Quantitative RT-PCR Assays for Detection and Serotyping of Foot-And-Mouth Disease Viruses Circulating in West Eurasia

DEFF Research Database (Denmark)

Jamal, Syed M.; Belsham, Graham

2015-01-01

Asia,A-Iran05 and Asia-1 (Group-II and Group-VII (Sindh-08)). In addition, field samples from Iran and Bulgaria, containing FMDVs belonging to the O-PanAsiaANT-10 subline-agewere also tested. Each of the three primer/probe sets was designed to be specific for just one of the serotypes O, A and Asia-1 of FMDV....... Due to the heterogeneity of FMD viruses (FMDVs) in different parts of the world, region specific diagnostic tests are required. In this study, hydrolysableprobe-based real time reverse transcription quantitative polymerase chain reaction (RTqPCR) assays were developed for specific detection...... and serotyping of the FMDVs currently circulating in West Eurasia. These assays were evaluated, in parallel with pan-FMDV diagnosticassays and earlier serotype-specific assays, using field samples originating from Pakistan and Afghanistan containing FMD viruses belonging to different sublineages of OPan...

Development and application of two independent real-time PCR assays to detect clinically relevant Mucorales species.

Science.gov (United States)

Springer, Jan; Goldenberger, Daniel; Schmidt, Friderike; Weisser, Maja; Wehrle-Wieland, Elisabeth; Einsele, Hermann; Frei, Reno; Löffler, Jürgen

2016-03-01

PCR-based detection of Mucorales species could improve diagnosis of suspected invasive fungal infection, leading to a better patient outcome. This study describes two independent probe-based real-time PCR tests for detection of clinically relevant Mucorales, targeting specific fragments of the 18S and the 28S rRNA genes. Both assays have a short turnaround time, allow fast, specific and very sensitive detection of clinically relevant Mucorales and have the potential to be used as quantitative tests. They were validated on various clinical samples (fresh and formalin-fixed paraffin-embedded specimens, mainly biopsies, n = 17). The assays should be used as add-on tools to complement standard techniques; a combined approach of both real-time PCR assays has 100 % sensitivity. Genus identification by subsequent sequencing is possible for amplicons of the 18S PCR assay. In conclusion, combination of the two independent Mucorales assays described in this study, 18S and 28S, detected all clinical samples associated with proven Mucorales infection (n = 10). Reliable and specific identification of Mucorales is a prerequisite for successful antifungal therapy as these fungi show intrinsic resistance to voriconazole and caspofungin.

A Pan-GTPase Inhibitor as a Molecular Probe.

Directory of Open Access Journals (Sweden)

Lin Hong

Full Text Available Overactive GTPases have often been linked to human diseases. The available inhibitors are limited and have not progressed far in clinical trials. We report here a first-in-class small molecule pan-GTPase inhibitor discovered from a high throughput screening campaign. The compound CID1067700 inhibits multiple GTPases in biochemical, cellular protein and protein interaction, as well as cellular functional assays. In the biochemical and protein interaction assays, representative GTPases from Rho, Ras, and Rab, the three most generic subfamilies of the GTPases, were probed, while in the functional assays, physiological processes regulated by each of the three subfamilies of the GTPases were examined. The chemical functionalities essential for the activity of the compound were identified through structural derivatization. The compound is validated as a useful molecular probe upon which GTPase-targeting inhibitors with drug potentials might be developed.

A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control

Directory of Open Access Journals (Sweden)

Delogu Mauro

2006-05-01

Full Text Available Abstract Background Avian influenza viruses (AIVs are endemic in wild birds and their introduction and conversion to highly pathogenic avian influenza virus in domestic poultry is a cause of serious economic losses as well as a risk for potential transmission to humans. The ability to rapidly recognise AIVs in biological specimens is critical for limiting further spread of the disease in poultry. The advent of molecular methods such as real time polymerase chain reaction has allowed improvement of detection methods currently used in laboratories, although not all of these methods include an Internal Positive Control (IPC to monitor for false negative results. Therefore we developed a one-step reverse transcription real time PCR (RRT-PCR with a Minor Groove Binder (MGB probe for the detection of different subtypes of AIVs. This technique also includes an IPC. Methods RRT-PCR was developed using an improved TaqMan technology with a MGB probe to detect AI from reference viruses. Primers and probe were designed based on the matrix gene sequences from most animal and human A influenza virus subtypes. The specificity of RRT-PCR was assessed by detecting influenza A virus isolates belonging to subtypes from H1–H13 isolated in avian, human, swine and equine hosts. The analytical sensitivity of the RRT-PCR assay was determined using serial dilutions of in vitro transcribed matrix gene RNA. The use of a rodent RNA as an IPC in order not to reduce the efficiency of the assay was adopted. Results The RRT-PCR assay is capable to detect all tested influenza A viruses. The detection limit of the assay was shown to be between 5 and 50 RNA copies per reaction and the standard curve demonstrated a linear range from 5 to 5 × 108 copies as well as excellent reproducibility. The analytical sensitivity of the assay is 10–100 times higher than conventional RT-PCR. Conclusion The high sensitivity, rapidity, reproducibility and specificity of the AIV RRT-PCR with

A real-time PCR assay for the relative quantification of the tetrahydrocannabinolic acid (THCA) synthase gene in herbal Cannabis samples.

Science.gov (United States)

Cascini, Fidelia; Passerotti, Stella; Martello, Simona

2012-04-10

In this study, we wanted to investigate whether or not the tetrahydrocannabinolic acid (THCA) synthase gene, which codes for the enzyme involved in the biosynthesis of THCA, influences the production and storage of tetrahydrocannabinol (THC) in a dose-dependent manner. THCA is actually decarboxylated to produce THC, the main psychoactive component in the Cannabis plant. Assuming as the research hypothesis a correlation between the gene copy number and the production of THC, gene quantification could be useful in forensics in order to complement or replace chemical analysis for the identification and classification of seized Cannabis samples, thus distinguishing the drug-type from the fibre-type varieties. A real-time PCR assay for the relative quantification of the THCA synthase gene was then validated on Cannabis samples; some were seized from the illegal drug market and others were derived from experimental cultivation. In order to determine the gene copy number to compare high vs. low potency plants, we chose the ΔΔCt method for TaqMan reactions. The assay enabled single plants with zero, one, and two copies of the gene to be distinguished. As a result of this first part of the research on the THCA synthase gene (the second part will cover a study of gene expression), we found no correlation between THCA synthase gene copy number and the content of THC in the herbal Cannabis samples tested. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

A flow cytometric assay technology based on quantum dots-encoded beads

International Nuclear Information System (INIS)

Wang Haiqiao; Liu Tiancai; Cao Yuancheng; Huang Zhenli; Wang Jianhao; Li Xiuqing; Zhao Yuandi

2006-01-01

A flow cytometric detecting technology based on quantum dots (QDs)-encoded beads has been described. Using this technology, several QDs-encoded beads with different code were identified effectively, and the target molecule (DNA sequence) in solution was also detected accurately by coupling to its complementary sequence probed on QDs-encoded beads through DNA hybridization assay. The resolution of this technology for encoded beads is resulted from two longer wavelength fluorescence identification signals (yellow and red fluorescent signals of QDs), and the third shorter wavelength fluorescence signal (green reporting signal of fluorescein isothiocyanate (FITC)) for the determination of reaction between probe and target. In experiment, because of QDs' unique optical character, only one excitation light source was needed to excite the QDs and probe dye FITC synchronously comparing with other flow cytometric assay technology. The results show that this technology has present excellent repeatability and good accuracy. It will become a promising multiple assay platform in various application fields after further improvement

Efficient oligonucleotide probe selection for pan-genomic tiling arrays

Directory of Open Access Journals (Sweden)

Zhang Wei

2009-09-01

Full Text Available Abstract Background Array comparative genomic hybridization is a fast and cost-effective method for detecting, genotyping, and comparing the genomic sequence of unknown bacterial isolates. This method, as with all microarray applications, requires adequate coverage of probes targeting the regions of interest. An unbiased tiling of probes across the entire length of the genome is the most flexible design approach. However, such a whole-genome tiling requires that the genome sequence is known in advance. For the accurate analysis of uncharacterized bacteria, an array must query a fully representative set of sequences from the species' pan-genome. Prior microarrays have included only a single strain per array or the conserved sequences of gene families. These arrays omit potentially important genes and sequence variants from the pan-genome. Results This paper presents a new probe selection algorithm (PanArray that can tile multiple whole genomes using a minimal number of probes. Unlike arrays built on clustered gene families, PanArray uses an unbiased, probe-centric approach that does not rely on annotations, gene clustering, or multi-alignments. Instead, probes are evenly tiled across all sequences of the pan-genome at a consistent level of coverage. To minimize the required number of probes, probes conserved across multiple strains in the pan-genome are selected first, and additional probes are used only where necessary to span polymorphic regions of the genome. The viability of the algorithm is demonstrated by array designs for seven different bacterial pan-genomes and, in particular, the design of a 385,000 probe array that fully tiles the genomes of 20 different Listeria monocytogenes strains with overlapping probes at greater than twofold coverage. Conclusion PanArray is an oligonucleotide probe selection algorithm for tiling multiple genome sequences using a minimal number of probes. It is capable of fully tiling all genomes of a species on

A Crowdsourcing Evaluation of the NIH Chemical Probes

OpenAIRE

Oprea, Tudor I.; Bologa, Cristian G.; Boyer, Scott; Curpan, Ramona F.; Glen, Robert C.; Hopkins, Andrew L.; Lipinski, Christopher A.; Marshall, Garland R.; Martin, Yvonne C.; Ostopovici-Halip, Liliana; Rishton, Gilbert; Ursu, Oleg; Vaz, Roy J.; Waller, Chris; Waldmann, Herbert

2009-01-01

Between 2004 and 2008, the NIH molecular libraries and imaging initiative (MLI) pilot phase funded ten high-throughput Screening Centers, resulting in the deposition of 691 assays into PubChem and the nomination of 64 chemical probes. We crowdsourced the MLI output to 11 experts, who expressed medium or high levels of confidence in 48 of these 64 probes.

The KRAS Strip Assay for detection of KRAS mutation in Egyptian patients with colorectal cancer (CRC): A pilot study

International Nuclear Information System (INIS)

Abd El Kader, Y.; Safwat, E.; Kassem, H.A.; Kassem, N.M.; Emera, G.

2013-01-01

Background: Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefltinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices. Purpose: Detection of KRAS mutation in Egyptian colorectal cancer (CRC) patients by the KRAS Strip Assay. Methods: Examination of 20 colorectal cancer (CRC) patients is done to detect KRAS mutations by KRAS Strip Assay. For the Strip Assay, a mutant-enriched PCR was followed by hybridization to KRAS-specific probes bound to a nitrocellulose strip. Results: Among 20 patients, KRAS mutations were identified in 80% of patients by the KRAS Strip Assay. Conclusions: Our preliminary results suggest that KRAS Strip Assay is an alternative to protocols currently in use for KRAS mutation detection

A Highly Sensitive Telomerase Activity Assay that Eliminates False-Negative Results Caused by PCR Inhibitors

Directory of Open Access Journals (Sweden)

Hidenobu Yaku

2013-09-01

Full Text Available An assay for telomerase activity based on asymmetric polymerase chain reaction (A-PCR on magnetic beads (MBs and subsequent application of cycling probe technology (CPT is described. In this assay, the telomerase reaction products are immobilized on MBs, which are then washed to remove PCR inhibitors that are commonly found in clinical samples. The guanine-rich sequences (5'-(TTAGGGn-3' of the telomerase reaction products are then preferentially amplified by A-PCR, and the amplified products are subsequently detected via CPT, where a probe RNA with a fluorophore at the 5' end and a quencher at the 3' end is hydrolyzed by RNase H in the presence of the target DNA. The catalyst-mediated cleavage of the probe RNA enhances fluorescence from the 5' end of the probe. The assay allowed us to successfully detect HeLa cells selectively over normal human dermal fibroblast (NHDF cells. Importantly, this selectivity produced identical results with regard to detection of HeLa cells in the absence and presence of excess NHDF cells; therefore, this assay can be used for practical clinical applications. The lower limit of detection for HeLa cells was 50 cells, which is lower than that achieved with a conventional telomeric repeat amplification protocol assay. Our assay also eliminated false-negative results caused by PCR inhibitors. Furthermore, we show that this assay is appropriate for screening among G-quadruplex ligands to find those that inhibit telomerase activity.

Identification, expression profiling and fluorescence-based binding assays of a chemosensory protein gene from the Western flower thrips, Frankliniella occidentalis.

Directory of Open Access Journals (Sweden)

Zhi-Ke Zhang

Full Text Available Using RT-PCR and RACE-PCR strategies, we cloned and identified a new chemosensory protein (FoccCSP from the Western flower thrips, Frankliniella occidentalis, a species for which no chemosensory protein (CSP has yet been identified. The FoccCSP gene contains a 387 bp open-reading frame encoding a putative protein of 128 amino acids with a molecular weight of 14.51 kDa and an isoelectric point of 5.41. The deduced amino acid sequence contains a putative signal peptide of 19 amino acid residues at the N-terminus, as well as the typical four-cysteine signature found in other insect CSPs. As FoccCSP is from a different order of insect than other known CSPs, the GenBank FoccCSP homolog showed only 31-50% sequence identity with them. A neighbor-joining tree was constructed and revealed that FoccCSP is in a group with CSPs from Homopteran insects (e.g., AgosCSP4, AgosCSP10, ApisCSP, and NlugCSP9, suggesting that these genes likely developed from a common ancestral gene. The FoccCSP gene expression profile of different tissues and development stages was measured by quantitative real-time PCR. The results of this analysis revealed this gene is predominantly expressed in the antennae and also highly expressed in the first instar nymph, suggesting a function for FoccCSP in olfactory reception and in particular life activities during the first instar nymph stage. We expressed recombinant FoccCSP protein in a prokaryotic expression system and purified FoccCSP protein by affinity chromatography using a Ni-NTA-Sepharose column. Using N-phenyl-1-naphthylamine (1-NPN as a fluorescent probe in fluorescence-based competitive binding assay, we determined the binding affinities of 19 volatile substances for FoccCSP protein. This analysis revealed that anisic aldehyde, geraniol and methyl salicylate have high binding affinities for FoccCSP, with KD values of 10.50, 15.35 and 35.24 μM, respectively. Thus, our study indicates that FoccCSP may play an important role in

Comparison of multiplex RT-PCR and real-time HybProbe assay for serotyping of dengue virus using reference strains and clinical samples from India

Directory of Open Access Journals (Sweden)

Anita Chakravarti

2016-01-01

Full Text Available Background: Dengue virus serotyping is crucial from clinical management and epidemiological point of view. Aims: To compare efficacy of two molecular detection and typing methods, namely, multiplex reverse transcription polymerase chain reaction (RT-PCR and real-time Hybprobe assay using a panel of known dilution of four reference Dengue virus strains and a panel of sera collected from clinically suspected dengue patients. Settings: This study was conducted at a tertiary-care teaching hospital in Delhi, India. Materials and Methods: Dengue serotype specific virus strains were used as prototypes for serotyping assays. Viral load was quantified by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR. Acute phase serum samples were collected from 79 patients with clinically suspected Dengue fever on their first day of presentation during September-October 2012. Viral RNA from serum and cell culture supernatant was extracted. Reverse transcription was carried out. Quantitative detection of DENV RNA from reference strain culture supernatants and each of the 79 patient samples by real-time PCR was performed using light cycler Taqman master mix kit. Serotyping was done by multiplex RT-PCR assay and Hybprobe assay. Results: The multiplex RT-PCR assay, though found to be 100% specific, couldn't serotype either patient or reference strains with viral load less than 1000 RNA copies/ml. The Hybprobe assay was found to have 100% specificity and had a lower limit of serotype detection of merely 3.54 RNA copies/ml. Conclusions: HybProbe assay has an important role especially in situations where serotyping is to be performed in clinical samples with low viral load.

Far Western: probing membranes.

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Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

2007-08-01

INTRODUCTIONThe far-Western technique described in this protocol is fundamentally similar to Western blotting. In Western blots, an antibody is used to detect a query protein on a membrane. In contrast, in a far-Western blot (also known as an overlay assay) the antibody is replaced by a recombinant GST fusion protein (produced and purified from bacteria), and the assay detects the interaction of this protein with target proteins on a membrane. The membranes are washed and blocked, incubated with probe protein, washed again, and subjected to autoradiography. The GST fusion (probe) proteins are often labeled with (32)P; alternatively, the membrane can be probed with unlabeled GST fusion protein, followed by detection using commercially available GST antibodies. The nonradioactive approach is substantially more expensive (due to the purchase of antibody and detection reagents) than using radioactively labeled proteins. In addition, care must be taken to control for nonspecific interactions with GST alone and a signal resulting from antibody cross-reactivity. In some instances, proteins on the membrane are not able to interact after transfer. This may be due to improper folding, particularly in the case of proteins expressed from a phage expression library. This protocol describes a way to overcome this by washing the membrane in denaturation buffer, which is then serially diluted to permit slow renaturation of the proteins.

Introduction of a hydrolysis probe PCR assay for high-throughput screening of methicillin-resistant Staphylococcus aureus with the ability to include or exclude detection of Staphylococcus argenteus.

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Bogestam, Katja; Vondracek, Martin; Karlsson, Mattias; Fang, Hong; Giske, Christian G

2018-01-01

Many countries using sensitive screening methods for detection of carriage of methicillin-resistant Staphylococcus aureus (MRSA) have a sustained low incidence of MRSA infections. For diagnostic laboratories with high sample volumes, MRSA screening requires stability, low maintenance and high performance at a low cost. Herein we designed oligonucleotides for a new nuc targeted hydrolysis probe PCR to replace the standard in-house nuc SybrGreen PCR assay. This new, more time-efficient, PCR assay resulted in a 40% increase in daily sample capacity, with maintained high specificity and sensitivity. The assay was also able to detect Staphylococcus aureus clonal cluster 75 (CC75) lineage strains, recently re-classified as Staphylococcus argenteus, with a sensitivity considerably increased compared to our previous assay. While awaiting consensus if the CC75 lineage of S. aureus should be considered as S. argenteus, and whether methicillin-resistant S. argenteus should be included in the MRSA definition, many diagnostic laboratories need to update their MRSA assay sensitivity/specificity towards this lineage/species. The MRSA screening assay presented in this manuscript is comprised of nuc oligonucleotides separately targeting S. aureus and CC75 lineage strains/S. argenteus, thus providing high user flexibility for the detection of CC75 lineage strains/S. argenteus.

The fluorometric microculture cytotoxicity assay.

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Lindhagen, Elin; Nygren, Peter; Larsson, Rolf

2008-01-01

The fluorometric microculture cytotoxicity assay (FMCA) is a nonclonogenic microplate-based cell viability assay used for measurement of the cytotoxic and/or cytostatic effect of different compounds in vitro. The assay is based on hydrolysis of the probe, fluorescein diacetate (FDA) by esterases in cells with intact plasma membranes. The assay is available as both a semiautomated 96-well plate setup and a 384-well plate version fully adaptable to robotics. Experimental plates are prepared with a small amount of drug solution and can be stored frozen. Cells are seeded on the plates and cell viability is evaluated after 72 h. The protocol described here is applicable both for cell lines and freshly prepared tumor cells from patients and is suitable both for screening in drug development and as a basis for a predictive test for individualization of anticancer drug therapy.

Comparison of in-house and commercial real time-PCR based carbapenemase gene detection methods in Enterobacteriaceae and non-fermenting gram-negative bacterial isolates.

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Smiljanic, M; Kaase, M; Ahmad-Nejad, P; Ghebremedhin, B

2017-07-10

Carbapenemase-producing gram-negative bacteria are increasing globally and have been associated with outbreaks in hospital settings. Thus, the accurate detection of these bacteria in infections is mandatory for administering the adequate therapy and infection control measures. This study aimed to establish and evaluate a multiplex real-time PCR assay for the simultaneous detection of carbapenemase gene variants in gram-negative rods and to compare the performance with a commercial RT-PCR assay (Check-Direct CPE). 116 carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii isolates were genotyped for carbapenemase genes by PCR and sequencing. The defined isolates were used for the validation of the in-house RT-PCR by use of designed primer pairs and probes. Among the carbapenem-resistant isolates the genes bla KPC , bla VIM , bla NDM or bla OXA were detected. Both RT-PCR assays detected all bla KPC , bla VIM and bla NDM in the isolates. The in-house RT-PCR detected 53 of 67 (79.0%) whereas the commercial assay detected only 29 (43.3%) of the OXA genes. The in-house sufficiently distinguished the most prevalent OXA types (23-like and 48-like) in the melting curve analysis and direct detection of the genes from positive blood culture vials. The Check-Direct CPE and the in-house RT-PCR assay detected the carbapenem resistance from solid culture isolates. Moreover, the in-house assay enabled the identification of carbapenemase genes directly from positive blood-culture vials. However, we observed insufficient detection of various OXA genes in both assays. Nevertheless, the in-house RT-PCR detected the majority of the OXA type genes in Enterobacteriaceae and A. baumannii.

GeneXpert HIV-1 quant assay, a new tool for scale up of viral load monitoring in the success of ART programme in India.

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Kulkarni, Smita; Jadhav, Sushama; Khopkar, Priyanka; Sane, Suvarna; Londhe, Rajkumar; Chimanpure, Vaishali; Dhilpe, Veronica; Ghate, Manisha; Yelagate, Rajendra; Panchal, Narayan; Rahane, Girish; Kadam, Dilip; Gaikwad, Nitin; Rewari, Bharat; Gangakhedkar, Raman

2017-07-21

Recent WHO guidelines identify virologic monitoring for diagnosing and confirming ART failure. In view of this, validation and scale up of point of care viral load technologies is essential in resource limited settings. A systematic validation of the GeneXpert® HIV-1 Quant assay (a point-of-care technology) in view of scaling up HIV-1 viral load in India to monitor the success of national ART programme was carried out. Two hundred nineteen plasma specimens falling in nine viral load ranges (5 L copies/ml) were tested by the Abbott m2000rt Real Time and GeneXpert HIV-1 Quant assays. Additionally, 20 seronegative; 16 stored specimens and 10 spiked controls were also tested. Statistical analysis was done using Stata/IC and sensitivity, specificity, PPV, NPV and %misclassification rates were calculated as per DHSs/AISs, WHO, NACO cut-offs for virological failure. The GeneXpert assay compared well with the Abbott assay with a higher sensitivity (97%), specificity (97-100%) and concordance (91.32%). The correlation between two assays (r = 0.886) was statistically significant (p performance and rapidity will aid in timely diagnosis of ART failures, integrated HIV-TB management and will facilitate the UNAIDS 90-90-90 target.

Assaying the reporter gene chloramphenicol acetyltransferase

International Nuclear Information System (INIS)

Crabb, D.W.; Minth, C.D.; Dixon, J.E.

1989-01-01

These experiments document the presence of enzymatic activities in extracts of commonly used cell lines which interfere with the determination of CAT activity. We suspect that the deacetylase activity is the most important, as the extract of the H4IIE C3 cells was capable of completely deacetylating the mono- and diacetylchloramphenicol formed during a 2-hr incubation of CAT with chloramphenicol and acetyl-CoA. The results of the inhibitor experiments are consistent with the presence of proteases which degrade CAT, or a serine carboxylesterase. The interference was also reduced by about half by EDTA; a metalloenzyme (either a protease or esterase) may therefore be involved. This interference appears to be a common phenomenon. We have surveyed 23 different cell types for the presence of the interfering activity and found it in 15. The interference was particularly prominent in several neuroendocrine and hepatoma cells. We took advantage of the effect of EDTA and the heat stability of CAT to eliminate the interference. Addition of 5 mM EDTA and a 10-min incubation of the sonicated cell suspension at 60 degrees prior to centrifugation abolished the interference in all cell lines tested. It is important to note that in order to reveal any CAT activity in some of the extracts (e.g., PC-12 or Hep3B), it was necessary to run the CAT assay for 2 hr. The control assays were therefore run almost to completion, and were well beyond the linear range of the assay. Therefore, the small differences which we observed between the heat-treated and control samples in some instances (e.g., rice, corn, or HeLa cells) will be dramatically amplified when the CAT assay is performed under conditions in which only a small percentage of the substrate is converted to product

Modeling Hybridization Kinetics of Gene Probes in a DNA Biochip Using FEMLAB

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Munir, Ahsan; Waseem, Hassan; Williams, Maggie R.; Stedtfeld, Robert D.; Gulari, Erdogan; Tiedje, James M.; Hashsham, Syed A.

2017-01-01

Microfluidic DNA biochips capable of detecting specific DNA sequences are useful in medical diagnostics, drug discovery, food safety monitoring and agriculture. They are used as miniaturized platforms for analysis of nucleic acids-based biomarkers. Binding kinetics between immobilized single stranded DNA on the surface and its complementary strand present in the sample are of interest. To achieve optimal sensitivity with minimum sample size and rapid hybridization, ability to predict the kinetics of hybridization based on the thermodynamic characteristics of the probe is crucial. In this study, a computer aided numerical model for the design and optimization of a flow-through biochip was developed using a finite element technique packaged software tool (FEMLAB; package included in COMSOL Multiphysics) to simulate the transport of DNA through a microfluidic chamber to the reaction surface. The model accounts for fluid flow, convection and diffusion in the channel and on the reaction surface. Concentration, association rate constant, dissociation rate constant, recirculation flow rate, and temperature were key parameters affecting the rate of hybridization. The model predicted the kinetic profile and signal intensities of eighteen 20-mer probes targeting vancomycin resistance genes (VRGs). Predicted signal intensities and hybridization kinetics strongly correlated with experimental data in the biochip (R2 = 0.8131). PMID:28555058

Modeling Hybridization Kinetics of Gene Probes in a DNA Biochip Using FEMLAB

Directory of Open Access Journals (Sweden)

Ahsan Munir

2017-05-01

Full Text Available Microfluidic DNA biochips capable of detecting specific DNA sequences are useful in medical diagnostics, drug discovery, food safety monitoring and agriculture. They are used as miniaturized platforms for analysis of nucleic acids-based biomarkers. Binding kinetics between immobilized single stranded DNA on the surface and its complementary strand present in the sample are of interest. To achieve optimal sensitivity with minimum sample size and rapid hybridization, ability to predict the kinetics of hybridization based on the thermodynamic characteristics of the probe is crucial. In this study, a computer aided numerical model for the design and optimization of a flow-through biochip was developed using a finite element technique packaged software tool (FEMLAB; package included in COMSOL Multiphysics to simulate the transport of DNA through a microfluidic chamber to the reaction surface. The model accounts for fluid flow, convection and diffusion in the channel and on the reaction surface. Concentration, association rate constant, dissociation rate constant, recirculation flow rate, and temperature were key parameters affecting the rate of hybridization. The model predicted the kinetic profile and signal intensities of eighteen 20-mer probes targeting vancomycin resistance genes (VRGs. Predicted signal intensities and hybridization kinetics strongly correlated with experimental data in the biochip (R2 = 0.8131.

Mismatch discrimination of lipidated DNA and LNA-probes (LiNAs) in hybridization-controlled liposome assembly

DEFF Research Database (Denmark)

Jakobsen, Ulla; Vogel, Stefan

2016-01-01

Assays for mismatch discrimination and detection of single nucleotide variations by hybridization-controlled assembly of liposomes, which do not require tedious surface chemistry, are versatile for both DNA and RNA targets. We report herein a comprehensive study on different DNA and LNA (locked...... assay in the context of mismatch discrimination and SNP detection are presented. The advantages of membrane-anchored LiNA-probes compared to chemically attached probes on solid nanoparticles (e.g. gold nanoparticles) are described. Key functionalities such as non-covalent attachment of LiNA probes...... without the need for long spacers and the inherent mobility of membrane-anchored probes in lipid-bilayer membranes will be described for several different probe designs....

Recombinant phage probes for Listeria monocytogenes

Energy Technology Data Exchange (ETDEWEB)

Carnazza, S; Gioffre, G; Felici, F; Guglielmino, S [Department of Microbiological, Genetic and Molecular Sciences, University of Messina, Messina (Italy)

2007-10-03

Monitoring of food and environmental samples for biological threats, such as Listeria monocytogenes, requires probes that specifically bind biological agents and ensure their immediate and efficient detection. There is a need for robust and inexpensive affinity probes as an alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we selected from two phage peptide libraries phage clones displaying peptides capable of specific and strong binding to the L. monocytogenes cell surface. The ability of isolated phage clones to interact specifically with L. monocytogenes was demonstrated using enzyme-linked immunosorbent assay (ELISA) and confirmed by co-precipitation assay. We also assessed the sensitivity of phage-bacteria binding by PCR on phage-captured Listeria cells, which could be detected at a concentration of 10{sup 4} cells ml{sup -1}. In addition, as proof-of-concept, we tested the possibility of immobilizing the affinity-selected phages to a putative biosensor surface. The quality of phage deposition was monitored by ELISA and fluorescent microscopy. Phage-bacterial binding was confirmed by high power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including in food products.

A Crowdsourcing Evaluation of the NIH Chemical Probes

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Oprea, Tudor I.; Bologa, Cristian G.; Boyer, Scott; Curpan, Ramona F.; Glen, Robert C.; Hopkins, Andrew L.; Lipinski, Christopher A.; Marshall, Garland R.; Martin, Yvonne C.; Ostopovici-Halip, Liliana; Rishton, Gilbert; Ursu, Oleg; Vaz, Roy J.; Waller, Chris; Waldmann, Herbert; Sklar, Larry A.

2013-01-01

Between 2004 and 2008, the NIH molecular libraries and imaging initiative (MLI) pilot phase funded ten high-throughput Screening Centers, resulting in the deposition of 691 assays into PubChem and the nomination of 64 chemical probes. We crowdsourced the MLI output to 11 experts, who expressed medium or high levels of confidence in 48 of these 64 probes. PMID:19536101

The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells.

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Jayakumar, Sundarraj; Bhilwade, Hari N; Pandey, Badri N; Sandur, Santosh K; Chaubey, Ramesh C

2012-10-09

The assessment of tumor radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. Therefore, the degree of correlation between radiation-induced DNA damage, as measured by the alkaline and the neutral comet assays, and the clonogenic survival of different human tumor cells was studied. Further, tumor radiosensitivity was compared with the expression of genes associated with the cellular response to radiation damage. Five different human tumor cell lines were chosen and the radiosensitivity of these cells was established by clonogenic assay. Alkaline and neutral comet assays were performed in γ-irradiated cells (2-8Gy; either acute or fractionated). Quantitative PCR was performed to evaluate the expression of DNA damage response genes in control and irradiated cells. The relative radiosensitivity of the cell lines assessed by the extent of DNA damage (neutral comet assay) immediately after irradiation (4Gy or 6Gy) was in agreement with radiosensitivity pattern obtained by the clonogenic assay. The survival fraction of irradiated cells showed a better correlation with the magnitude of DNA damage measured by the neutral comet assay (r=-0.9; Pcomet assay (r=-0.73; Pcomet assay was better than alkaline comet assay for assessment of radiosensitivities of tumor cells after acute or fractionated doses of irradiation. © 2012 Elsevier B.V. All rights reserved.

Evaluating the genotoxic effects of workers exposed to lead using micronucleus assay, comet assay and TCR gene mutation test

International Nuclear Information System (INIS)

Chen Zhijian; Lou Jianlin; Chen Shijie; Zheng Wei; Wu Wei; Jin Lifen; Deng Hongping; He Jiliang

2006-01-01

To evaluate the genotoxic effects of lead (Pb) exposure, 25 workers in a workplace producing storage battery were monitored for three genetic end-points using micronucleus (MN) assay, comet assay and TCR gene mutation test. Twenty-five controls were matched with workers according to age, gender and smoking. The air Pb concentration in the workplace was 1.26 mg/m 3 . All subjects were measured for Pb concentration of blood by atom absorption spectrophotometry. The mean Pb concentration of blood in workers (0.32 mg/l) was significantly higher than that in controls (0.02 mg/l). The results of MN test showed that the mean micronuclei rate (MNR) and mean micronucleated cells rate (MCR) in workers were 9.04 ± 1.51 per mille and 7.76 ± 1.23 per mille , respectively, which were significantly higher than those (2.36 ± 0.42 per mille and 1.92 ± 0.31 per mille ) in controls (P -4 and 1.74 ± 0.17 x 10 -4 , respectively, there was no significant difference between workers and controls (P > 0.05). The results of our study indicated that the genetic damage was detectable in 25 workers occupationally exposed to lead

Analyzing Multiple-Probe Microarray: Estimation and Application of Gene Expression Indexes

KAUST Repository

Maadooliat, Mehdi

2012-07-26

Gene expression index estimation is an essential step in analyzing multiple probe microarray data. Various modeling methods have been proposed in this area. Amidst all, a popular method proposed in Li and Wong (2001) is based on a multiplicative model, which is similar to the additive model discussed in Irizarry et al. (2003a) at the logarithm scale. Along this line, Hu et al. (2006) proposed data transformation to improve expression index estimation based on an ad hoc entropy criteria and naive grid search approach. In this work, we re-examined this problem using a new profile likelihood-based transformation estimation approach that is more statistically elegant and computationally efficient. We demonstrate the applicability of the proposed method using a benchmark Affymetrix U95A spiked-in experiment. Moreover, We introduced a new multivariate expression index and used the empirical study to shows its promise in terms of improving model fitting and power of detecting differential expression over the commonly used univariate expression index. As the other important content of the work, we discussed two generally encountered practical issues in application of gene expression index: normalization and summary statistic used for detecting differential expression. Our empirical study shows somewhat different findings from the MAQC project (MAQC, 2006).

A TaqMan real-time PCR-based assay for the identification of Fasciola spp.

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Alasaad, Samer; Soriguer, Ramón C; Abu-Madi, Marawan; El Behairy, Ahmed; Jowers, Michael J; Baños, Pablo Díez; Píriz, Ana; Fickel, Joerns; Zhu, Xing-Quan

2011-06-30

Real time quantitative PCR (qPCR) is one of the key technologies of the post-genome era, with clear advantages compared to normal end-point PCR. In this paper, we report the first qPCR-based assay for the identification of Fasciola spp. Based on sequences of the second internal transcribed spacers (ITS-2) of the ribosomal rRNA gene, we used a set of genus-specific primers for Fasciola ITS-2 amplification, and we designed species-specific internal TaqMan probes to identify F. hepatica and F. gigantica, as well as the hybrid 'intermediate'Fasciola. These primers and probes were used for the highly specific, sensitive, and simple identification of Fasciola species collected from different animal host from China, Spain, Niger and Egypt. The novel qPCR-based technique for the identification of Fasciola spp. may provide a useful tool for the epidemiological investigation of Fasciola infection, including their intermediate snail hosts. Copyright © 2011 Elsevier B.V. All rights reserved.

In situ detection of the Clostridium botulinum type C1 toxin gene in wetland sediments with a nested PCR assay

Science.gov (United States)

Williamson, Judy L.; Rocke, Tonie E.; Aiken, Judd M.

1999-01-01

A nested PCR was developed for detection of the Clostridium botulinum type C1 toxin gene in sediments collected from wetlands where avian botulism outbreaks had or had not occurred. The C1 toxin gene was detected in 16 of 18 sites, demonstrating both the ubiquitous distribution of C. botulinum type C in wetland sediments and the sensitivity of the detection assay.

Prospective Validation of a 21-Gene Expression Assay in Breast Cancer.

Science.gov (United States)

Sparano, Joseph A; Gray, Robert J; Makower, Della F; Pritchard, Kathleen I; Albain, Kathy S; Hayes, Daniel F; Geyer, Charles E; Dees, Elizabeth C; Perez, Edith A; Olson, John A; Zujewski, JoAnne; Lively, Tracy; Badve, Sunil S; Saphner, Thomas J; Wagner, Lynne I; Whelan, Timothy J; Ellis, Matthew J; Paik, Soonmyung; Wood, William C; Ravdin, Peter; Keane, Maccon M; Gomez Moreno, Henry L; Reddy, Pavan S; Goggins, Timothy F; Mayer, Ingrid A; Brufsky, Adam M; Toppmeyer, Deborah L; Kaklamani, Virginia G; Atkins, James N; Berenberg, Jeffrey L; Sledge, George W

2015-11-19

Prior studies with the use of a prospective-retrospective design including archival tumor samples have shown that gene-expression assays provide clinically useful prognostic information. However, a prospectively conducted study in a uniformly treated population provides the highest level of evidence supporting the clinical validity and usefulness of a biomarker. We performed a prospective trial involving women with hormone-receptor-positive, human epidermal growth factor receptor type 2 (HER2)-negative, axillary node-negative breast cancer with tumors of 1.1 to 5.0 cm in the greatest dimension (or 0.6 to 1.0 cm in the greatest dimension and intermediate or high tumor grade) who met established guidelines for the consideration of adjuvant chemotherapy on the basis of clinicopathologic features. A reverse-transcriptase-polymerase-chain-reaction assay of 21 genes was performed on the paraffin-embedded tumor tissue, and the results were used to calculate a score indicating the risk of breast-cancer recurrence; patients were assigned to receive endocrine therapy without chemotherapy if they had a recurrence score of 0 to 10, indicating a very low risk of recurrence (on a scale of 0 to 100, with higher scores indicating a greater risk of recurrence). Of the 10,253 eligible women enrolled, 1626 women (15.9%) who had a recurrence score of 0 to 10 were assigned to receive endocrine therapy alone without chemotherapy. At 5 years, in this patient population, the rate of invasive disease-free survival was 93.8% (95% confidence interval [CI], 92.4 to 94.9), the rate of freedom from recurrence of breast cancer at a distant site was 99.3% (95% CI, 98.7 to 99.6), the rate of freedom from recurrence of breast cancer at a distant or local-regional site was 98.7% (95% CI, 97.9 to 99.2), and the rate of overall survival was 98.0% (95% CI, 97.1 to 98.6). Among patients with hormone-receptor-positive, HER2-negative, axillary node-negative breast cancer who met established guidelines for

Probing chromatin structure with nuclease sensitivity assays.

Science.gov (United States)

Gregory, R I; Khosla, S; Feil, R

2001-01-01

To further our understanding of genomic imprinting it will be essential to identify key control elements, and to investigate their regulation by both epigenetic modifications (such as DNA methylation) and trans-acting factors. So far, sequence elements that regulate parental allele-specific gene expression have been identified in a number of imprinted loci, either because of their differential DNA methylation or through functional studies in transgenic mice (1,2). A systematic search for allele-specific chromatin features constitutes an alternative strategy to identify elements that regulate imprinting. The validity of such an in vivo chromatin approach derives from the fact that in several known imprinting control-elements, a specialized organization of chromatin characterized by nuclease hypersensitivity is present on only one of the two parental chromosome (3). For example, the differentially methylated 5 -portion of the human SNRPN gene-a sequence element that controls imprinting in the Prader-Willi and Angelman syndromes' domain on chromosome 15q11- q13-has strong DNase-I hypersensitive sites on the unmethylated paternal chromosome (4). A differentially methylated region that regulates the imprinting of H19 and that of the neighboring insulin-like growth factor-2 gene on mouse chromosome 7 was also found to have parental chromosome-specific hypersensitive sites (5,6). The precise nature of the allelic nuclease hypersensitivity in these and other imprinted loci remains to be determined in more detail, for example, by applying complementary chromatin methodologies (7,8). However, it is commonly observed that a nuclease hypersensitive site corresponds to a small region where nucleosomes are absent or partially disrupted.

A label-free, fluorescence based assay for microarray

Science.gov (United States)

Niu, Sanjun

DNA chip technology has drawn tremendous attention since it emerged in the mid 90's as a method that expedites gene sequencing by over 100-fold. DNA chip, also called DNA microarray, is a combinatorial technology in which different single-stranded DNA (ssDNA) molecules of known sequences are immobilized at specific spots. The immobilized ssDNA strands are called probes. In application, the chip is exposed to a solution containing ssDNA of unknown sequence, called targets, which are labeled with fluorescent dyes. Due to specific molecular recognition among the base pairs in the DNA, the binding or hybridization occurs only when the probe and target sequences are complementary. The nucleotide sequence of the target is determined by imaging the fluorescence from the spots. The uncertainty of background in signal detection and statistical error in data analysis, primarily due to the error in the DNA amplification process and statistical distribution of the tags in the target DNA, have become the fundamental barriers in bringing the technology into application for clinical diagnostics. Furthermore, the dye and tagging process are expensive, making the cost of DNA chips inhibitive for clinical testing. These limitations and challenges make it difficult to implement DNA chip methods as a diagnostic tool in a pathology laboratory. The objective of this dissertation research is to provide an alternative approach that will address the above challenges. In this research, a label-free assay is designed and studied. Polystyrene (PS), a commonly used polymeric material, serves as the fluorescence agent. Probe ssDNA is covalently immobilized on polystyrene thin film that is supported by a reflecting substrate. When this chip is exposed to excitation light, fluorescence light intensity from PS is detected as the signal. Since the optical constants and conformations of ssDNA and dsDNA (double stranded DNA) are different, the measured fluorescence from PS changes for the same

Different approaches in the molecular analysis of the SHOX gene dysfunctions.

Science.gov (United States)

Stuppia, L; Gatta, V; Antonucci, I; Giuliani, R; Palka, G

2010-06-01

Deficit of the short stature homeobox containing gene (SHOX) accounts for 2.15% of cases of idiopathic short stature (ISS) and 50-100% of cases of Leri-Weill dyschondrosteosis (LWD). It has been demonstrated that patients with SHOX deficit show a good response to treatment with GH. Thus, the early identification of SHOX alterations is a crucial point in order to choose the best treatment for ISS and LWD patients. In this study, we analyze the most commonly used molecular techniques for the detection of SHOX gene alterations. multiple ligation-dependent probe amplification analysis appears to represent the gold standard for the detection of deletion involving the SHOX gene or the enhancer region, being able to show both alterations in a single assay.

Radioactive probes as diagnostic tools for rice tungro viruses

International Nuclear Information System (INIS)

Azzam, O.; Arboleda, M.; Reyes. J. de los

1996-01-01

Rice tungro bacilliform (RTBV) and rice tungro spherical viruses (RTSV) are the two viral components responsible for rice tungro disease which has seriously affected the irrigated rice ecosystem in Southeast Asia for the last 30 years. RTBV has an 8 Kb double-stranded DNA circular genome, and it is primarily responsible for induction of symptoms in infected plants. RTSV has a 12 kb single-stranded RNA genome. It does not induce any apparent symptoms in the infected plant, and it is transmitted by greenleafhopper. RTBV depends upon RTSV for its own transmission. The two viruses are limited to the vascular tissue of the rice plant and are present at a low titer. Most of the detection methods used for the identification of these viruses have relied on the virus protein properties and therefore, early detection of the virus activity was not possible. We were interested in evaluating tissue printing, dot blot, and southern techniques for early detection of virus nucleic acids in rice plant using radioactive and non radioactive probes. 32 P-labeled T7 or SP6 RNA polymerase transcripts complementary to the RTBV genome and RTSV coat protein genes were used as probes of the positive stand of both viruses. For nonradioactive probes, RTBV DNA genome was labeled using the ECL detection kit (Amersham). Preliminary results show that viral nucleic acids of RTBV and RTSV could be detected using both labelling systems. Non radioactive probes were comparable in their sensitivity to the radioactive probes. Less than 100 pg of viral DNA was detected in the dot-blot assays. More data will be presented to compare the efficiency and reliability of these two techniques in detecting early virus activity in the rice plant. (author)

Isolation of probes specific to human chromosomal region 6p21 from immunoselected irradiation-fusion gene transfer hybrids

International Nuclear Information System (INIS)

Ragoussis, J.; Jones, T.A.; Sheer, D.; Shrimpton, A.E.; Goodfellow, P.N.; Trowsdale, J.; Ziegler, A.

1991-01-01

A hybrid cell line (R21/B1) containing a truncated human chromosome 6 (6pter-6q21) and a human Y chromosome on a hamster background was irradiated and fused to A23 (TK-) or W3GH (HPRT-) hamster cells. Clones containing expressed HLA class I genes (4/40) were selected using monoclonal antibodies. These clones were recloned and analyzed with a panel of probes from the HLA region. One hybrid (4G6) contained the entire HLA complex. Two other hybrids (4J4 and 4H2) contained only the HLA class I region, while the fourth hybrid (5P9) contained HLA class I and III genes in addition to other genes located in the 6p21 chromosomal region. In situ hybridization showed that the hybrid cells contained more than one fragment of human DNA. Alu and LINE PCR products were derived from these cells and compared to each other as well as to products from two somatic cell hybrids having the 6p21 region in common. The PCR fragments were then screened on conventional Southern blots of the somatic cell hybrids to select a panel of novel probes encompassing the 6p21 region. In addition, the origin of the human DNA fragments in hybrid 4J4 was determined by regional mapping of PCR products

Prevalence of H63D, S65C and C282Y hereditary hemochromatosis gene mutations in Slovenian population by an improved high-throughput genotyping assay

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Rupreht Ruth

2007-11-01

Full Text Available Abstract Background Hereditary hemochromatosis (HH is a common genetic disease characterized by excessive iron overload that leads to multi-organ failure. Although the most prevalent genotype in HH is homozygosity for C282Y mutation of the HFE gene, two additional mutations, H63D and S65C, appear to be associated with a milder form of HH. The aim of this study was to develop a high-throughput assay for HFE mutations screening based on TaqMan technology and to determine the frequencies of HFE mutations in the Slovenian population. Methods Altogether, 1282 randomly selected blood donors from different Slovenian regions and 21 HH patients were analyzed for the presence of HFE mutations by an in-house developed real-time PCR assay based on TaqMan technology using shorter non-interfering fluorescent single nucleotide polymorphism (SNP-specific MGB probes. The assay was validated by RFLP analysis and DNA sequencing. Results The genotyping assay of the H63D, S65C and C282Y mutations in the HFE gene, based on TaqMan technology proved to be fast, reliable, with a high-throughput capability and 100% concordant with genotypes obtained by RFLP and DNA sequencing. The observed frequency of C282Y homozygotes in the group of HH patients was only 48%, others were of the heterogeneous HFE genotype. Among 1282 blood donors tested, the observed H63D, S65C and C282Y allele frequency were 12.8% (95% confidence interval (CI 11.5 – 14.2%, 1.8% (95% CI 1.4 – 2.5% and 3.6% (95% CI 3.0 – 4.5%, respectively. Approximately 33% of the tested subjects had at least one of the three HH mutations, and 1% of them were C282Y homozygotes or compound heterozygotes C282Y/H63D or C282Y/S65C, presenting an increased risk for iron overload disease. A significant variation in H63D allele frequency was observed for one of the Slovenian regions. Conclusion The improved real-time PCR assay for H63D, S65C and C282Y mutations detection is accurate, fast, cost-efficient and ready for

Label-Free, LC-MS-Based Assays to Quantitate Small-Molecule Antagonist Binding to the Mammalian BLT1 Receptor.

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Chen, Xun; Stout, Steven; Mueller, Uwe; Boykow, George; Visconti, Richard; Siliphaivanh, Phieng; Spencer, Kerrie; Presland, Jeremy; Kavana, Michael; Basso, Andrea D; McLaren, David G; Myers, Robert W

2017-08-01

We have developed and validated label-free, liquid chromatography-mass spectrometry (LC-MS)-based equilibrium direct and competition binding assays to quantitate small-molecule antagonist binding to recombinant human and mouse BLT1 receptors expressed in HEK 293 cell membranes. Procedurally, these binding assays involve (1) equilibration of the BLT1 receptor and probe ligand, with or without a competitor; (2) vacuum filtration through cationic glass fiber filters to separate receptor-bound from free probe ligand; and (3) LC-MS analysis in selected reaction monitoring mode for bound probe ligand quantitation. Two novel, optimized probe ligands, compounds 1 and 2, were identified by screening 20 unlabeled BLT1 antagonists for direct binding. Saturation direct binding studies confirmed the high affinity, and dissociation studies established the rapid binding kinetics of probe ligands 1 and 2. Competition binding assays were established using both probe ligands, and the affinities of structurally diverse BLT1 antagonists were measured. Both binding assay formats can be executed with high specificity and sensitivity and moderate throughput (96-well plate format) using these approaches. This highly versatile, label-free method for studying ligand binding to membrane-associated receptors should find broad application as an alternative to traditional methods using labeled ligands.

Development and validation of cell-based luciferase reporter gene assays for measuring neutralizing anti-drug antibodies against interferon beta

DEFF Research Database (Denmark)

Hermanrud, Christina; Ryner, Malin; Luft, Thomas

2016-01-01

a normal distribution for the majority of runs, allowing a parametric approach for cut-point calculation to be used, where NAb positive samples could be identified with 95% confidence. An analysis of means and variances indicated that a floating cut-point should be used for all assays. The assays......Neutralizing anti-drug antibodies (NAbs) against therapeutic interferon beta (IFNβ) in people with multiple sclerosis (MS) are measured with cell-based bioassays. The aim of this study was to redevelop and validate two luciferase reporter-gene bioassays, LUC and iLite, using a cut-point approach...... to identify NAb positive samples. Such an approach is favored by the pharmaceutical industry and governmental regulatory agencies as it has a clear statistical basis and overcomes the limitations of the current assays based on the Kawade principle. The work was conducted following the latest assay guidelines...

Determination of gene expression patterns using high-throughput RNA in situ hybridizaion to whole-mount Drosophila embryos

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Weiszmann, R.; Hammonds, A.S.; Celniker, S.E.

2009-04-09

We describe a high-throughput protocol for RNA in situ hybridization (ISH) to Drosophila embryos in a 96-well format. cDNA or genomic DNA templates are amplified by PCR and then digoxigenin-labeled ribonucleotides are incorporated into antisense RNA probes by in vitro transcription. The quality of each probe is evaluated before ISH using a RNA probe quantification (dot blot) assay. RNA probes are hybridized to fixed, mixed-staged Drosophila embryos in 96-well plates. The resulting stained embryos can be examined and photographed immediately or stored at 4oC for later analysis. Starting with fixed, staged embryos, the protocol takes 6 d from probe template production through hybridization. Preparation of fixed embryos requires a minimum of 2 weeks to collect embryos representing all stages. The method has been used to determine the expression patterns of over 6,000 genes throughout embryogenesis.

Allele specific hybridization using oligonucleotide probes of very high specific activity: Discrimination of the human β/sup A/ and β/sup S/-globin genes

International Nuclear Information System (INIS)

Studencki, A.B.; Wallace, R.B.

1984-01-01

The repair activity of E. coli DNA polymerase I (Klenow fragment) was used to prepare nonadecanucleotide hybridization probes which were complementary either to the normal human β-globin (β/sup A/) or to the sickle cell human β-globin (β/sup S/) gene. Template directed polymerization of highly radiolabeled α-/sup 32/P-deoxyribonucleoside triphosphates (3200, 5000 and/or 7800 Ci/mmol) onto nonamer and decamer primers produced probes with specific activities ranging from 1.0 - 2.0 x 10/sup 10/ dpm/μg. The extremely high specific activities of these probes made it possible to detect the β/sup A/ and β/sup S/ single copy gene sequences in as little as 1 μg of total human genomic DNA as well as to discriminate between the homozygous and heterozygous states. This means that it was possible to detect 0.5 - 1.0 x 10/sup -18/ moles of a given single copy sequence

Combining portable Raman probes with nanotubes for theranostic applications.

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Bhirde, Ashwinkumar A; Liu, Gang; Jin, Albert; Iglesias-Bartolome, Ramiro; Sousa, Alioscka A; Leapman, Richard D; Gutkind, J Silvio; Lee, Seulki; Chen, Xiaoyuan

2011-01-01

Recently portable Raman probes have emerged along with a variety of applications, including carbon nanotube (CNT) characterization. Aqueous dispersed CNTs have shown promise for biomedical applications such as drug/gene delivery vectors, photo-thermal therapy, and photoacoustic imaging. In this study we report the simultaneous detection and irradiation of carbon nanotubes in 2D monolayers of cancer cells and in 3D spheroids using a portable Raman probe. A portable handheld Raman instrument was utilized for dual purposes: as a CNT detector and as an irradiating laser source. Single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) were dispersed aqueously using a lipid-polymer (LP) coating, which formed highly stable dispersions both in buffer and cell media. The LP coated SWCNT and MWCNT aqueous dispersions were characterized by atomic force microscopy, transmission electron microscopy, dynamic light scattering, Fourier transform infrared spectroscopy and Raman spectroscopy. The cellular uptake of the LP-dispersed SWCNTs and MWCNTs was observed using confocal microscopy, and fluorescein isothiocyanate (FITC)-nanotube conjugates were found to be internalized by ovarian cancer cells by using Z-stack fluorescence confocal imaging. Biocompatibility of SWCNTs and MWCNTs was assessed using a cell viability MTT assay, which showed that the nanotube dispersions did not hinder the proliferation of ovarian cancer cells at the dosage tested. Ovarian cancer cells treated with SWCNTs and MWCNTs were simultaneously detected and irradiated live in 2D layers of cancer cells and in 3D environments using the portable Raman probe. An apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay carried out after laser irradiation confirmed that cell death occurred only in the presence of nanotube dispersions. We show for the first time that both SWCNTs and MWCNTs can be selectively irradiated and detected in cancer cells using a simple

Validation of Performance of the Gen-Probe Human Immunodeficiency Virus Type 1 Viral Load Assay with Genital Swabs and Breast Milk Samples

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DeVange Panteleeff, Dana; Emery, Sandra; Richardson, Barbra A.; Rousseau, Christine; Benki, Sarah; Bodrug, Sharon; Kreiss, Joan K.; Overbaugh, Julie

2002-01-01

Human immunodeficiency type 1 (HIV-1) continues to spread at an alarming rate. The virus may be transmitted through blood, genital secretions, and breast milk, and higher levels of systemic virus in the index case, as measured by plasma RNA viral load, have been shown to correlate with increased risk of transmitting HIV-1 both vertically and sexually. Less is known about the correlation between transmission and HIV-1 levels in breast milk or genital secretions, in part because reliable quantitative assays to detect HIV-1 in these fluids are not available. Here we show that the Gen-Probe HIV-1 viral load assay can be used to accurately quantify viral load in expressed breast milk and in cervical and vaginal samples collected on swabs. Virus could be quantified from breast milk and swab samples spiked with known amounts of virus, including HIV-1 subtypes A, C, and D. As few as 10 copies of HIV-1 RNA could be detected above background threshold levels in ≥77% of assays performed with spiked breast milk supernatants and mock swabs. In genital swab samples from HIV-1-infected women, similar levels of HIV-1 RNA were consistently detected in duplicate swabs taken from the same woman on the same clinic visit, suggesting that the RNA values from a single swab sample can be used to measure genital viral load. PMID:12409354

Equivalent Gene Expression Profiles between Glatopa™ and Copaxone®.

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Josephine S D'Alessandro

Full Text Available Glatopa™ is a generic glatiramer acetate recently approved for the treatment of patients with relapsing forms of multiple sclerosis. Gene expression profiling was performed as a means to evaluate equivalence of Glatopa and Copaxone®. Microarray analysis containing 39,429 unique probes across the entire genome was performed in murine glatiramer acetate--responsive Th2-polarized T cells, a test system highly relevant to the biology of glatiramer acetate. A closely related but nonequivalent glatiramoid molecule was used as a control to establish assay sensitivity. Multiple probe-level (Student's t-test and sample-level (principal component analysis, multidimensional scaling, and hierarchical clustering statistical analyses were utilized to look for differences in gene expression induced by the test articles. The analyses were conducted across all genes measured, as well as across a subset of genes that were shown to be modulated by Copaxone. The following observations were made across multiple statistical analyses: the expression of numerous genes was significantly changed by treatment with Copaxone when compared against media-only control; gene expression profiles induced by Copaxone and Glatopa were not significantly different; and gene expression profiles induced by Copaxone and the nonequivalent glatiramoid were significantly different, underscoring the sensitivity of the test system and the multiple analysis methods. Comparative analysis was also performed on sets of transcripts relevant to T-cell biology and antigen presentation, among others that are known to be modulated by glatiramer acetate. No statistically significant differences were observed between Copaxone and Glatopa in the expression levels (magnitude and direction of these glatiramer acetate-regulated genes. In conclusion, multiple methods consistently supported equivalent gene expression profiles between Copaxone and Glatopa.

Investigation of histone H4 hyperacetylation dynamics in the 5S rRNA genes family by chromatin immunoprecipitation assay.

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Burlibașa, Liliana; Suciu, Ilinca

2015-12-01

Oogenesis is a critical event in the formation of female gamete, whose role in development is to transfer genomic information to the next generation. During this process, the gene expression pattern changes dramatically concomitant with genome remodelling, while genomic information is stably maintained. The aim of the present study was to investigate the presence of H4 acetylation of the oocyte and somatic 5S rRNA genes in Triturus cristatus, using chromatin immunoprecipitation assay (ChIP). Our findings suggest that some epigenetic mechanisms such as histone acetylation could be involved in the transcriptional regulation of 5S rRNA gene families.

Improved precision and accuracy for microarrays using updated probe set definitions

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Larsson Ola

2007-02-01

Full Text Available Abstract Background Microarrays enable high throughput detection of transcript expression levels. Different investigators have recently introduced updated probe set definitions to more accurately map probes to our current knowledge of genes and transcripts. Results We demonstrate that updated probe set definitions provide both better precision and accuracy in probe set estimates compared to the original Affymetrix definitions. We show that the improved precision mainly depends on the increased number of probes that are integrated into each probe set, but we also demonstrate an improvement when the same number of probes is used. Conclusion Updated probe set definitions does not only offer expression levels that are more accurately associated to genes and transcripts but also improvements in the estimated transcript expression levels. These results give support for the use of updated probe set definitions for analysis and meta-analysis of microarray data.

Automated design of genomic Southern blot probes

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Komiyama Noboru H

2010-01-01

Full Text Available Abstract Background Sothern blotting is a DNA analysis technique that has found widespread application in molecular biology. It has been used for gene discovery and mapping and has diagnostic and forensic applications, including mutation detection in patient samples and DNA fingerprinting in criminal investigations. Southern blotting has been employed as the definitive method for detecting transgene integration, and successful homologous recombination in gene targeting experiments. The technique employs a labeled DNA probe to detect a specific DNA sequence in a complex DNA sample that has been separated by restriction-digest and gel electrophoresis. Critically for the technique to succeed the probe must be unique to the target locus so as not to cross-hybridize to other endogenous DNA within the sample. Investigators routinely employ a manual approach to probe design. A genome browser is used to extract DNA sequence from the locus of interest, which is searched against the target genome using a BLAST-like tool. Ideally a single perfect match is obtained to the target, with little cross-reactivity caused by homologous DNA sequence present in the genome and/or repetitive and low-complexity elements in the candidate probe. This is a labor intensive process often requiring several attempts to find a suitable probe for laboratory testing. Results We have written an informatic pipeline to automatically design genomic Sothern blot probes that specifically attempts to optimize the resultant probe, employing a brute-force strategy of generating many candidate probes of acceptable length in the user-specified design window, searching all against the target genome, then scoring and ranking the candidates by uniqueness and repetitive DNA element content. Using these in silico measures we can automatically design probes that we predict to perform as well, or better, than our previous manual designs, while considerably reducing design time. We went on to

Advances in study of reporter gene imaging for monitoring gene therapy

International Nuclear Information System (INIS)

Mu Chuanjie; Zhou Jiwen

2003-01-01

To evaluate the efficiency of gene therapy, it is requisite to monitor localization and expression of the therapeutic gene in vivo. Monitoring expression of reporter gene using radionuclide reporter gene technique is the best method. Adenoviral vectors expressing reporter gene are constructed using gene fusion, bicistronic, double promoter or bidirectional transcriptional recombination techniques, and transferred into target cells and tissues, then injected radiolabeled reporter probes which couple to the reporter genes. The reporter genes can be imaged invasively, repeatedly, quantitatively with γ-camera, PET and SPECT. Recently, several reporter gene and reporter probe systems have been used in studies of gene therapy. The part of them has been used for clinic trials

A luminescence-based probe for sensitive detection of hydrogen peroxide in seconds

International Nuclear Information System (INIS)

Zscharnack, Kristin; Kreisig, Thomas; Prasse, Agneta A.; Zuchner, Thole

2014-01-01

Highlights: • We describe a novel probe for the sensitive detection of H 2 O 2 . • H 2 O 2 quenches the luminescence of a complex consisting of phthalic acid and terbium ions. • A stable fluorescence signal is generated immediately after mixing probe and sample. • The PATb probe detects H 2 O 2 over four orders of magnitude. - Abstract: Here, we present a fast and simple hydrogen peroxide assay that is based on time-resolved fluorescence. The emission intensity of a complex consisting of terbium ions (Tb 3+ ) and phthalic acid (PA) in HEPES buffer is quenched in the presence of H 2 O 2 and this quenching is concentration-dependent. The novel PATb assay detects hydrogen peroxide at a pH range from 7.5 to 8.5 and with a detection limit of 150 nmol L −1 at pH 8.5. The total assay time is less than 1 min. The linear range of the assay can be adapted by a pH adjustment of the aqueous buffer and covers a concentration range from 310 nmol L −1 to 2.56 mmol L −1 in total which encompasses four orders of magnitude. The assay is compatible with high concentrations of all 47 tested inorganic and organic compounds. The PATb assay was applied to quantify H 2 O 2 in polluted river water samples. In conclusion, this fast and easy-to-use assay detects H 2 O 2 with high sensitivity and precision

Evaluation of a novel Dot-ELISA assay utilizing a recombinant protein for the effective diagnosis of Taenia pisiformis larval infections.

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Chen, Lin; Yang, Deying; Gu, Xiaobin; Peng, Xuerong; Yang, Guangyou

2014-08-29

Cysticercosis, caused by the larvae of Taenia pisiformis, is a common disease in domestic breeds of the rabbit Oryctolagus cuniculus that results in economic losses. At present, there is no convenient and effective method for the rapid detection of T. pisiformis larvae. Here, we developed and tested the efficacy of a Dot-ELISA assay for the diagnosis of T. pisiformis larval infections in rabbits, based on the expression of the recombinant fusion protein (rTp1) from the Tp1 gene. Rapid amplification of cDNA ends (RACE) was used to amplify the 3' ends of the Tp1 gene, based on the unigene similar to Ts1 gene (EU009656.1) which comes from transcriptome sequencing of T. pisiformis. The Tp1 gene was successfully amplified, cloned and expressed in BL21 (DE3). Western blot analysis revealed that the recombinant Tp1 protein is specifically recognized by rabbit T. pisiformis cysticercosis antisera. This purified recombinant fusion protein, rTp1, was probed by Dot-ELISA with sera from rabbits infected with T. pisiformis larvae and with other parasitic infections. Results showed that this Dot-ELISA assay had both high sensitivity (92.9-97.6%) and specificity (95.2-98.4%) to detect T. pisiformis larval infections. We also found very low levels of cross-reaction with other parasitic infections. This study has revealed that our novel Dot-ELISA assay utilizing the recombinant fusion protein, rTp1, has a strong potential for the effective diagnosis of T. pisiformis infections in rabbits. Copyright © 2014 Elsevier B.V. All rights reserved.

Evaluation of the Gen-Probe DNA probe for the detection of legionellae in culture

International Nuclear Information System (INIS)

Edelstein, P.H.

1986-01-01

A commercial DNA probe kit designed to detect rRNA from legionellae was evaluated for its ability to correctly discriminate between legionellae and non-legionellae taken from culture plates. The probe kit, made by the Gen-Probe Corp. (San Diego, Calif.), was radiolabeled with 125 I, and probe bacterial RNA hybridization, detected in a simple one-tube system hybridization assay, was quantitated with a gamma counter. A total of 156 Legionella sp. strains were tested, of which 125 were Legionella pneumophila and the remainder were strains from 21 other Legionella spp. A total of 106 gram-negative non-legionellae, isolated from human respiratory tract (81%) and other body site (19%) specimens, were also tested; 14 genera and 28 species were represented. The probe easily distinguished all of the legionellae from the non-legionellae. The average legionellae/non-legionellae hybridization ratio was 42:1, and the lowest ratio was 2:1; a minor modification in the procedure increased the lowest ratio to 5:1. In addition to correctly identifying all Legionella species, the probe was able to separate some of the various species of Legionella. L. pneumophila strains hybridized more completely to the probe than did the other Legionella spp.; L. wadsworthii and L. oakridgensis hybridized only about 25% of the probe relative to L. pneumophila. Some strains of phenotypically identified L. pneumophila had much lower hybridization to the probe than other members of the species and may represent a new Legionella species. The simplicity of the technique and specificity of the probe make it a good candidate for confirming the identity of legionellae in culture

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

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Meng Shuang

2010-06-01

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

Differentiation of the virulence potential of Campylobacter jejuni strains by use of gene transcription analysis and a caco-2 assay

DEFF Research Database (Denmark)

Poli, Vanessa Fadanelli Schoenardie; Thorsen, Line; Olesen, Inger

2012-01-01

properties were evaluated by analyzing transcriptions of the virulence genes cdtB, ciaB, cadF and the stress associated genes clpP, htrB using reverse transcription quantitative PCR (RT-qPCR) and by the ability of the C. jejuni strains to adhere to and invade Caco-2 cells. Similar cell survival and no growth...... gene, cipA between DFVF1099 and NCTC11168 resulting in a 14 amino acid deletion and 28 amino acid addition at the N and C terminal ends respectively of the CipA protein of DFVF1099. In contrast to DFVF1099, strains NCTC1168 and TB1048 were able to invade Caco-2 cells. Invasion ability was not affected...... expression of C. jejuni. The clinical strains appeared to be more virulent than the chicken isolate as measured by the Caco-2 invasion assay which could be due to differences in CipA functionality. The RT-qPCR analysis and Caco-2 assay showed to be useful tools for differentiating virulence potentials...

Tissue-specific methylation of human insulin gene and PCR assay for monitoring beta cell death.

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Mohamed I Husseiny

Full Text Available The onset of metabolic dysregulation in type 1 diabetes (T1D occurs after autoimmune destruction of the majority of pancreatic insulin-producing beta cells. We previously demonstrated that the DNA encoding the insulin gene is uniquely unmethylated in these cells and then developed a methylation-specific PCR (MSP assay to identify circulating beta cell DNA in streptozotocin-treated mice prior to the rise in blood glucose. The current study extends to autoimmune non-obese diabetic (NOD mice and humans, showing in NOD mice that beta cell death occurs six weeks before the rise in blood sugar and coincides with the onset of islet infiltration by immune cells, demonstrating the utility of MSP for monitoring T1D. We previously reported unique patterns of methylation of the human insulin gene, and now extend this to other human tissues. The methylation patterns of the human insulin promoter, intron 1, exon 2, and intron 2 were determined in several normal human tissues. Similar to our previous report, the human insulin promoter was unmethylated in beta cells, but methylated in all other tissues tested. In contrast, intron 1, exon 2 and intron 2 did not exhibit any tissue-specific DNA methylation pattern. Subsequently, a human MSP assay was developed based on the methylation pattern of the insulin promoter and human islet DNA was successfully detected in circulation of T1D patients after islet transplantation therapy. Signal levels of normal controls and pre-transplant samples were shown to be similar, but increased dramatically after islet transplantation. In plasma the signal declines with time but in whole blood remains elevated for at least two weeks, indicating that association of beta cell DNA with blood cells prolongs the signal. This assay provides an effective method to monitor beta cell destruction in early T1D and in islet transplantation therapy.

A Real-Time PCR Assay to Identify and Discriminate Among Wild-Type and Vaccine Strains of Varicella-Zoster Virus and Herpes Simplex Virus in Clinical Specimens, and Comparison With the Clinical Diagnoses

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Harbecke, Ruth; Oxman, Michael N.; Arnold, Beth A.; Ip, Charlotte; Johnson, Gary R.; Levin, Myron J.; Gelb, Lawrence D.; Schmader, Kenneth E.; Straus, Stephen E.; Wang, Hui; Wright, Peter F.; Pachucki, Constance T.; Gershon, Anne A.; Arbeit, Robert D.; Davis, Larry E.; Simberkoff, Michael S.; Weinberg, Adriana; Williams, Heather M.; Cheney, Carol; Petrukhin, Luba; Abraham, Katalin G.; Shaw, Alan; Manoff, Susan; Antonello, Joseph M.; Green, Tina; Wang, Yue; Tan, Charles; Keller, Paul M.

2014-01-01

A real-time PCR assay was developed to identify varicella-zoster virus (VZV) and herpes simplex virus (HSV) DNA in clinical specimens from subjects with suspected herpes zoster (HZ; shingles). Three sets of primers and probes were used in separate PCR reactions to detect and discriminate among wild-type VZV (VZV-WT), Oka vaccine strain VZV (VZV-Oka), and HSV DNA, and the reaction for each virus DNA was multiplexed with primers and probe specific for the human β-globin gene to assess specimen adequacy. Discrimination of all VZV-WT strains, including Japanese isolates and the Oka parent strain, from VZV-Oka was based upon a single nucleotide polymorphism at position 106262 in ORF 62, resulting in preferential amplification by the homologous primer pair. The assay was highly sensitive and specific for the target virus DNA, and no cross-reactions were detected with any other infectious agent. With the PCR assay as the gold standard, the sensitivity of virus culture was 53% for VZV and 77% for HSV. There was 92% agreement between the clinical diagnosis of HZ by the Clinical Evaluation Committee and the PCR assay results. PMID:19475609

Biobarcode assay for the oral anticoagulant acenocoumarol.

Science.gov (United States)

Broto, Marta; Salvador, J Pablo; Galve, Roger; Marco, M Pilar

2018-02-01

A novel approach for therapeutic drug monitoring of oral anticoagulants (OA) in clinical samples is reported, based on a NP-based biobarcode assay. The proposed strategy uses specific antibodies for acenocumarol (ACL) covalently bound to magnetic particles (pAb236-MP) and a bioconjugate competitor (hACL-BSA) linked to encoded polystyrene probes (hACL-BSA-ePSP) on a classical competitive immunochemical format. By using this scheme ACL can be detected in low nM range (LOD, 0.96 ± 0.26, N = 3, in buffer) even in complex samples such as serum or plasma (LOD 4 ± 1). The assay shows a high reproducibility (%CV 1.1 day-to-day) and is robust, as it is demonstrated by the fact that ACL can be quantified in complex biological samples with a very good accuracy (slope = 0.97 and R 2 = 0.91, of the linear regression obtained when analyzing spiked vs measured values). Moreover, we have demonstrated that the biobarcode approach has the potential to overcome one of the main challenges of the multiplexed diagnostic, which is the possibility to measure in a single run biomarker targets present at different concentration ranges. Thus, it has been proven that the signal and the detectability can be modulated by just modifying the oligonucleotide load of the encoded probes. This fact opens the door for combining in the same assay encoded probes with the necessary oligonucleotide load to achieve the detectability required for each biomarker target. Copyright © 2017 Elsevier B.V. All rights reserved.

Prognostic Utility of the 21-Gene Assay in Hormone Receptor–Positive Operable Breast Cancer Compared With Classical Clinicopathologic Features

Science.gov (United States)

Goldstein, Lori J.; Gray, Robert; Badve, Sunil; Childs, Barrett H.; Yoshizawa, Carl; Rowley, Steve; Shak, Steven; Baehner, Frederick L.; Ravdin, Peter M.; Davidson, Nancy E.; Sledge, George W.; Perez, Edith A.; Shulman, Lawrence N.; Martino, Silvana; Sparano, Joseph A.

2008-01-01

Purpose Adjuvant! is a standardized validated decision aid that projects outcomes in operable breast cancer based on classical clinicopathologic features and therapy. Genomic classifiers offer the potential to more accurately identify individuals who benefit from chemotherapy than clinicopathologic features. Patients and Methods A sample of 465 patients with hormone receptor (HR) –positive breast cancer with zero to three positive axillary nodes who did (n = 99) or did not have recurrence after chemohormonal therapy had tumor tissue evaluated using a 21-gene assay. Histologic grade and HR expression were evaluated locally and in a central laboratory. Results Recurrence Score (RS) was a highly significant predictor of recurrence, including node-negative and node-positive disease (P < .001 for both) and when adjusted for other clinical variables. RS also predicted recurrence more accurately than clinical variables when integrated by an algorithm modeled after Adjuvant! that was adjusted to 5-year outcomes. The 5-year recurrence rate was only 5% or less for the estimated 46% of patients who have a low RS (< 18). Conclusion The 21-gene assay was a more accurate predictor of relapse than standard clinical features for individual patients with HR-positive operable breast cancer treated with chemohormonal therapy and provides information that is complementary to features typically used in anatomic staging, such as tumor size and lymph node involvement. The 21-gene assay may be used to select low-risk patients for abbreviated chemotherapy regimens similar to those used in our study or high-risk patients for more aggressive regimens or clinical trials evaluating novel treatments. PMID:18678838

Prognostic utility of the 21-gene assay in hormone receptor-positive operable breast cancer compared with classical clinicopathologic features.

Science.gov (United States)

Goldstein, Lori J; Gray, Robert; Badve, Sunil; Childs, Barrett H; Yoshizawa, Carl; Rowley, Steve; Shak, Steven; Baehner, Frederick L; Ravdin, Peter M; Davidson, Nancy E; Sledge, George W; Perez, Edith A; Shulman, Lawrence N; Martino, Silvana; Sparano, Joseph A

2008-09-01

Adjuvant! is a standardized validated decision aid that projects outcomes in operable breast cancer based on classical clinicopathologic features and therapy. Genomic classifiers offer the potential to more accurately identify individuals who benefit from chemotherapy than clinicopathologic features. A sample of 465 patients with hormone receptor (HR) -positive breast cancer with zero to three positive axillary nodes who did (n = 99) or did not have recurrence after chemohormonal therapy had tumor tissue evaluated using a 21-gene assay. Histologic grade and HR expression were evaluated locally and in a central laboratory. Recurrence Score (RS) was a highly significant predictor of recurrence, including node-negative and node-positive disease (P < .001 for both) and when adjusted for other clinical variables. RS also predicted recurrence more accurately than clinical variables when integrated by an algorithm modeled after Adjuvant! that was adjusted to 5-year outcomes. The 5-year recurrence rate was only 5% or less for the estimated 46% of patients who have a low RS (< 18). The 21-gene assay was a more accurate predictor of relapse than standard clinical features for individual patients with HR-positive operable breast cancer treated with chemohormonal therapy and provides information that is complementary to features typically used in anatomic staging, such as tumor size and lymph node involvement. The 21-gene assay may be used to select low-risk patients for abbreviated chemotherapy regimens similar to those used in our study or high-risk patients for more aggressive regimens or clinical trials evaluating novel treatments.

A molecular-beacon-based asymmetric PCR assay for easy visualization of amplicons in the diagnosis of trichomoniasis.

Science.gov (United States)

Sonkar, Subash C; Sachdev, Divya; Mishra, Prashant K; Kumar, Anita; Mittal, Pratima; Saluja, Daman

2016-12-15

The currently available nucleic acid amplification tests (NAATs) for trichomoniasis are accurate, quick and confirmative with superior sensitivity than traditional culture-based microbiology assays. However, these assays are associated with problems of carry over contamination, false positive results, requirement of technical expertise for performance and detection of end product. Hence, a diagnostic assay with easy visualization of the amplified product will be profitable. An in-house, rapid, sensitive, specific molecular-beacon-based PCR assay, using primers against pfoB gene of Trichomonas vaginalis, was developed and evaluated using dry ectocervical swabs (n=392) from symptomatic females with vaginal discharge. Total DNA was isolated and used as template for the PCR assays. The performance and reproducibility of PCR assay was evaluated by composite reference standard (CRS). For easy visualization of the amplified product, molecular-beacon was designed and amplicons were visualized directly using fluorescent handheld dark reader or by Micro-Plate Reader. Molecular-beacons are single-stranded hairpin shaped nucleic acid probes composed of a stem, with fluorophore/quencher pair and a loop region complementary to the desired DNA. The beacon-based PCR assay designed in the present study is highly specific as confirmed by competition experiments and extremely sensitive with detection limit of 20fg of genomic DNA (3-4 pathogens). The minimum infrastructure requirement and ease to perform the assay makes this method highly useful for resource poor countries for better disease management. Copyright © 2016 Elsevier B.V. All rights reserved.

Simultaneous detection of Legionella species and L. anisa, L. bozemanii, L. longbeachae and L. micdadei using conserved primers and multiple probes in a multiplex real-time PCR assay.

Science.gov (United States)

Cross, Kristen E; Mercante, Jeffrey W; Benitez, Alvaro J; Brown, Ellen W; Diaz, Maureen H; Winchell, Jonas M

2016-07-01

Legionnaires' disease is a severe respiratory disease that is estimated to cause between 8,000 and 18,000 hospitalizations each year, though the exact burden is unknown due to under-utilization of diagnostic testing. Although Legionella pneumophila is the most common species detected in clinical cases (80-90%), other species have also been reported to cause disease. However, little is known about Legionnaires' disease caused by these non-pneumophila species. We designed a multiplex real-time PCR assay for detection of all Legionella spp. and simultaneous specific identification of four clinically-relevant Legionella species, L. anisa, L. bozemanii, L. longbeachae, and L. micdadei, using 5'-hydrolysis probe real-time PCR. The analytical sensitivity for detection of nucleic acid from each target species was ≤50fg per reaction. We demonstrated the utility of this assay in spiked human sputum specimens. This assay could serve as a tool for understanding the scope and impact of non-pneumophila Legionella species in human disease. Published by Elsevier Inc.

Probing intracellular motor protein activity using an inducible cargo trafficking assay

NARCIS (Netherlands)

L.C. Kapitein (Lukas); M.A. Schlager (Max); W.A. van der Zwan (Wouter); P. Wulf (Phebe); N. Keijzer (Nanda); C.C. Hoogenraad (Casper)

2010-01-01

textabstractAlthough purified cytoskeletal motor proteins have been studied extensively with the use of in vitro approaches, a generic approach to selectively probe actin and microtubule-based motor protein activity inside living cells is lacking. To examine specific motor activity inside living

Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify Ebola Virus VP24 Inhibition of IFN Signaling

Directory of Open Access Journals (Sweden)

Elisa Fanunza

2018-02-01

Full Text Available The interferon (IFN system is the first line of defense against viral infections. Evasion of IFN signaling by Ebola viral protein 24 (VP24 is a critical event in the pathogenesis of the infection and, hence, VP24 is a potential target for drug development. Since no drugs target VP24, the identification of molecules able to inhibit VP24, restoring and possibly enhancing the IFN response, is a goal of concern. Accordingly, we developed a dual signal firefly and Renilla luciferase cell-based drug screening assay able to quantify IFN-mediated induction of Interferon Stimulated Genes (ISGs and its inhibition by VP24. Human Embryonic Kidney 293T (HEK293T cells were transiently transfected with a luciferase reporter gene construct driven by the promoter of ISGs, Interferon-Stimulated Response Element (ISRE. Stimulation of cells with IFN-α activated the IFN cascade leading to the expression of ISRE. Cotransfection of cells with a plasmid expressing VP24 cloned from a virus isolated during the last 2014 outbreak led to the inhibition of ISRE transcription, quantified by a luminescent signal. To adapt this system to test a large number of compounds, we performed it in 96-well plates; optimized the assay analyzing different parameters; and validated the system by calculating the Z′- and Z-factor, which showed values of 0.62 and 0.53 for IFN-α stimulation assay and VP24 inhibition assay, respectively, indicative of robust assay performance.

Conservation of the fourth gene among rotaviruses recovered from asymptomatic newborn infants and its possible role in attenuation

International Nuclear Information System (INIS)

Flores, J.; Midthun, K.; Hoshino, Y.; Green, K.; Gorziglia, M.; Kapikian, A.Z.; Chanock, R.M.

1986-01-01

RNA-RNA hybridization was performed to assess the extent of genetic relatedness among human rotaviruses isolated from children with gastroenteritis and from asymptomatic newborn infants. 32 P-labeled single-stranded RNAs produced by in vitro transcription from viral cores of the different strains tested were used as probes in two different hybridization assays: (1) undenatured genomic RNAs were resolved by polyacrylamide gel electrophoresis, denatured in situ, electrophoretically transferred to diazobenzyloxymethyl-paper (Northern blots), and then hybridized to the probes under two different conditions of stringency; and (ii) denatured genomic double-stranded RNAs were hybridized to the probes in solution and the hybrids which formed were identified by polyacrylamide gel electrophoresis. When analyzed by Northern blot hybridization at a low level of stringency, all genes from the strains tested cross-hybridized, providing evidence for some sequence homology in each of the corresponding genes. However, when hybridization stringency was increased, a difference in gene 4 sequence was detected between strains recovered from asymptomatic newborn infants (nursery strains) and strains recovered from infants and young children with diarrhea. Although the nursery strains exhibited serotypic diversity, the fourth gene appeared to be highly conversed. These results were confirmed and extended during experiments in which the RNA-RNA hybridization was carried out in solution and the resulting hybrids were analyzed by polyacrylamide gel electrophoresis. Full-length hybrids did not form between the fourth genes from the nursery strains and the corresponding genes from the strains recovered from symptomatic infants and young children

Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors.

Science.gov (United States)

Ikten, Cengiz; Ustun, Rustem; Catal, Mursel; Yol, Engin; Uzun, Bulent

2016-01-01

Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.). Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease.

Analytical Performance Characteristics of the Cepheid GeneXpert Ebola Assay for the Detection of Ebola Virus

Science.gov (United States)

Pinsky, Benjamin A.; Sahoo, Malaya K.; Sandlund, Johanna; Kleman, Marika; Kulkarni, Medha; Grufman, Per; Nygren, Malin; Kwiatkowski, Robert; Baron, Ellen Jo; Tenover, Fred; Denison, Blake; Higuchi, Russell; Van Atta, Reuel; Beer, Neil Reginald; Carrillo, Alda Celena; Naraghi-Arani, Pejman; Mire, Chad E.; Ranadheera, Charlene; Grolla, Allen; Lagerqvist, Nina; Persing, David H.

2015-01-01

Background The recently developed Xpert® Ebola Assay is a novel nucleic acid amplification test for simplified detection of Ebola virus (EBOV) in whole blood and buccal swab samples. The assay targets sequences in two EBOV genes, lowering the risk for new variants to escape detection in the test. The objective of this report is to present analytical characteristics of the Xpert® Ebola Assay on whole blood samples. Methods and Findings This study evaluated the assay’s analytical sensitivity, analytical specificity, inclusivity and exclusivity performance in whole blood specimens. EBOV RNA, inactivated EBOV, and infectious EBOV were used as targets. The dynamic range of the assay, the inactivation of virus, and specimen stability were also evaluated. The lower limit of detection (LoD) for the assay using inactivated virus was estimated to be 73 copies/mL (95% CI: 51–97 copies/mL). The LoD for infectious virus was estimated to be 1 plaque-forming unit/mL, and for RNA to be 232 copies/mL (95% CI 163–302 copies/mL). The assay correctly identified five different Ebola viruses, Yambuku-Mayinga, Makona-C07, Yambuku-Ecran, Gabon-Ilembe, and Kikwit-956210, and correctly excluded all non-EBOV isolates tested. The conditions used by Xpert® Ebola for inactivation of infectious virus reduced EBOV titer by ≥6 logs. Conclusion In summary, we found the Xpert® Ebola Assay to have high analytical sensitivity and specificity for the detection of EBOV in whole blood. It offers ease of use, fast turnaround time, and remote monitoring. The test has an efficient viral inactivation protocol, fulfills inclusivity and exclusivity criteria, and has specimen stability characteristics consistent with the need for decentralized testing. The simplicity of the assay should enable testing in a wide variety of laboratory settings, including remote laboratories that are not capable of performing highly complex nucleic acid amplification tests, and during outbreaks where time to detection

Sandwich hybridization probes for the detection of Pseudo-nitzschia (Bacillariophyceae) species: An update to existing probes and a description of new probes.

Science.gov (United States)

Bowers, Holly A; Marin, Roman; Birch, James M; Scholin, Christopher A

2017-12-01

New sandwich hybridization assay (SHA) probes for detecting Pseudo-nitzschia species (P. arenysensis, P. fraudulenta, P. hasleana, P. pungens) are presented, along with updated cross-reactivity information on historical probes (SHA and FISH; fluorescence in situ hybridization) targeting P. australis and P. multiseries. Pseudo-nitzschia species are a cosmopolitan group of diatoms that produce varying levels of domoic acid (DA), a neurotoxin that can accumulate in finfish and shellfish and transfer throughout the food web. Consumption of infected food sources can lead to illness in humans (amnesic shellfish poisoning; ASP) and marine wildlife (domoic acid poisoning; DAP). The threat of human illness, along with economic loss from fishery closures has resulted in the implementation of monitoring protocols and intensive ecological studies. SHA probes have been instrumental in some of these efforts, as the technique performs well in complex heterogeneous sample matrices and has been adapted to benchtop and deployable (Environmental Sample Processor) platforms. The expanded probe set will enhance future efforts towards understanding spatial, temporal and successional patterns in species during bloom and non-bloom periods. Copyright © 2017 Elsevier B.V. All rights reserved.

Detection of the Light Organ Symbiont, Vibrio fischeri, in Hawaiian Seawater by Using lux Gene Probes †

OpenAIRE

Lee, Kyu-Ho; Ruby, Edward G.

1992-01-01

Symbiotic bacteria that inhabit the light-emitting organ of the Hawaiian squid Euprymna scolopes are distinctive from typical Vibrio fischeri organisms in that they are not visibly luminous when grown in laboratory culture. Therefore, the abundance of these bacteria in seawater samples cannot be estimated simply by identifying them among luminous colonies that arise on nutrient agar plates. Instead, we have used luxR and polymerase chain reaction generated luxA gene probes to identify both lu...

Comparative Evaluation of the Diagnostic Performance of the Prototype Cepheid GeneXpert Ebola Assay

Science.gov (United States)

Jansen van Vuren, Petrus; Grobbelaar, Antoinette; Storm, Nadia; Conteh, Ousman; Konneh, Kelfala; Kamara, Abdul; Sanne, Ian

2015-01-01

The Ebola virus disease (EVD) outbreak in West Africa has highlighted an urgent need for point-of-care (POC) assays for the diagnosis of this devastating disease in resource-limited African countries. The diagnostic performance characteristics of a prototype Cepheid GeneXpert Ebola POC used to detect Ebola virus (EBOV) in stored serum and plasma samples collected from suspected EVD cases in Sierra Leone in 2014 and 2015 was evaluated. The GeneXpert Ebola POC is a self-contained single-cartridge automated system that targets the glycoprotein (GP) and nucleoprotein (NP) genes of EBOV and yields results within 90 min. Results from 281 patient samples were compared to the results of a TaqMan real-time reverse transcription-PCR (RT-PCR) targeting the polymerase gene and performed on two real-time PCR machines. Agreement between the three platforms was 100% at cycle threshold (CT) values of ≤34.99, but discordant results were noted between CT values of 35 and 45.The diagnostic sensitivity of the three platforms was 100% in 91 patient samples that were confirmed to be infectious by virus isolation. All three molecular platforms detected viral EBOV RNA in additional samples that did not contain viable EBOV. The analytical sensitivity of the GeneXpert Ebola POC for the detection of NP was higher, and comparable to that of polymerase gene detection, than that for the detection of GP when using a titrated laboratory stock of EBOV. There was no detectable cross-reactivity with other hemorrhagic fever viruses or arboviruses. The GeneXpert Ebola POC offers an easy to operate and sensitive diagnostic tool that can be used for the rapid screening of suspected EVD cases in treatment or in holding centers during EVD outbreaks. PMID:26637383

Polymerase chain reaction-hybridization method using urease gene sequences for high-throughput Ureaplasma urealyticum and Ureaplasma parvum detection and differentiation.

Science.gov (United States)

Xu, Chen; Zhang, Nan; Huo, Qianyu; Chen, Minghui; Wang, Rengfeng; Liu, Zhili; Li, Xue; Liu, Yunde; Bao, Huijing

2016-04-15

In this article, we discuss the polymerase chain reaction (PCR)-hybridization assay that we developed for high-throughput simultaneous detection and differentiation of Ureaplasma urealyticum and Ureaplasma parvum using one set of primers and two specific DNA probes based on urease gene nucleotide sequence differences. First, U. urealyticum and U. parvum DNA samples were specifically amplified using one set of biotin-labeled primers. Furthermore, amine-modified DNA probes, which can specifically react with U. urealyticum or U. parvum DNA, were covalently immobilized to a DNA-BIND plate surface. The plate was then incubated with the PCR products to facilitate sequence-specific DNA binding. Horseradish peroxidase-streptavidin conjugation and a colorimetric assay were used. Based on the results, the PCR-hybridization assay we developed can specifically differentiate U. urealyticum and U. parvum with high sensitivity (95%) compared with cultivation (72.5%). Hence, this study demonstrates a new method for high-throughput simultaneous differentiation and detection of U. urealyticum and U. parvum with high sensitivity. Based on these observations, the PCR-hybridization assay developed in this study is ideal for detecting and discriminating U. urealyticum and U. parvum in clinical applications. Copyright © 2016 Elsevier Inc. All rights reserved.

A luciferase reporter gene assay and aryl hydrocarbon receptor 1 genotype predict the LD50 of polychlorinated biphenyls in avian species

International Nuclear Information System (INIS)

Manning, Gillian E.; Farmahin, Reza; Crump, Doug; Jones, Stephanie P.; Klein, Jeff; Konstantinov, Alex; Potter, Dave; Kennedy, Sean W.

2012-01-01

Birds differ in sensitivity to the embryotoxic effects of polychlorinated biphenyls (PCBs), which complicates environmental risk assessments for these chemicals. Recent research has shown that the identities of amino acid residues 324 and 380 in the avian aryl hydrocarbon receptor 1 (AHR1) ligand binding domain (LBD) are primarily responsible for differences in avian species sensitivity to selected dibenzo-p-dioxins and furans. A luciferase reporter gene (LRG) assay was developed in our laboratory to measure AHR1-mediated induction of a cytochrome P450 1A5 reporter gene in COS-7 cells transfected with different avian AHR1 constructs. In the present study, the LRG assay was used to measure the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and PCBs 126, 77, 105 and 118 on luciferase activity in COS-7 cells transfected with AHR1 constructs representative of 86 avian species in order to predict their sensitivity to PCB-induced embryolethality and the relative potency of PCBs in these species. The results of the LRG assay indicate that the identity of amino acid residues 324 and 380 in the AHR1 LBD are the major determinants of avian species sensitivity to PCBs. The relative potency of PCBs did not differ greatly among AHR1 constructs. Luciferase activity was significantly correlated with embryolethality data obtained from the literature (R 2 ≥ 0.87, p < 0.0001). Thus, the LRG assay in combination with the knowledge of a species' AHR1 LBD sequence can be used to predict PCB-induced embryolethality in potentially any avian species of interest without the use of lethal methods on a large number of individuals. -- Highlights: ► PCB embryolethality in birds can be predicted from a species' AHR1 genotype. ► The reporter gene assay is useful for predicting species sensitivity to PCBs. ► The relative potency of PCBs does not appear to differ between AHR1 genotypes. ► Contamination of PCB 105 and PCB 118 did not affect their relative

Evaluation of a duplex reverse-transcription real-time PCR assay for the detection of encephalomyocarditis virus.

Science.gov (United States)

Qin, Shaomin; Underwood, Darren; Driver, Luke; Kistler, Carol; Diallo, Ibrahim; Kirkland, Peter D

2018-06-01

We evaluated a fluorogenic probe-based assay for the detection of encephalomyocarditis virus (EMCV) by comparing a set of published primers and probe to a new set of primers and probe. The published reagents failed to amplify a range of Australian isolates and an Italian reference strain of EMCV. In contrast, an assay based on 2 new sets of primers and probes that were run in a duplex reverse-transcription real-time PCR (RT-rtPCR) worked well, with high amplification efficiency. The analytical sensitivity was ~100-fold higher than virus isolation in cell culture. The intra-assay variation was 0.21-4.90%. No cross-reactivity was observed with a range of other porcine viruses. One hundred and twenty-two clinical specimens were tested simultaneously by RT-rtPCR and virus isolation in cell culture; 72 specimens gave positive results by RT-rtPCR, and 63 of these were also positive by virus isolation. Of 245 archived cell culture isolates of EMCV that were tested in the RT-rtPCR, 242 samples were positive. The new duplex RT-rtPCR assay is a reliable tool for the detection of EMCV in clinical specimens and for use in epidemiologic investigations.

Gene expression

International Nuclear Information System (INIS)

Hildebrand, C.E.; Crawford, B.D.; Walters, R.A.; Enger, M.D.

1983-01-01

We prepared probes for isolating functional pieces of the metallothionein locus. The probes enabled a variety of experiments, eventually revealing two mechanisms for metallothionein gene expression, the order of the DNA coding units at the locus, and the location of the gene site in its chromosome. Once the switch regulating metallothionein synthesis was located, it could be joined by recombinant DNA methods to other, unrelated genes, then reintroduced into cells by gene-transfer techniques. The expression of these recombinant genes could then be induced by exposing the cells to Zn 2+ or Cd 2+ . We would thus take advantage of the clearly defined switching properties of the metallothionein gene to manipulate the expression of other, perhaps normally constitutive, genes. Already, despite an incomplete understanding of how the regulatory switch of the metallothionein locus operates, such experiments have been performed successfully

Fluorescent Probes for Analysis and Imaging of Monoamine Oxidase Activity

Energy Technology Data Exchange (ETDEWEB)

Kim, Dokyoung; Jun, Yong Woong; Ahn, Kyo Han [POSTECH, Pohang (Korea, Republic of)

2014-05-15

Monoamine oxidases catalyze the oxidative deamination of dietary amines and amine neurotransmitters, and assist in maintaining the homeostasis of the amine neurotransmitters in the brain. Dysfunctions of these enzymes can cause neurological and behavioral disorders including Parkinson's and Alzheimer's diseases. To understand their physiological roles, efficient assay methods for monoamine oxidases are essential. Reviewed in this Perspective are the recent progress in the development of fluorescent probes for monoamine oxidases and their applications to enzyme assays in cells and tissues. It is evident that still there is strong need for a fluorescent probe with desirable substrate selectivity and photophysical properties to challenge the much unsolved issues associated with the enzymes and the diseases.

Field Validation of a Transcriptional Assay for the Prediction of Age of Uncaged Aedes aegypti Mosquitoes in Northern Australia

Science.gov (United States)

Hugo, Leon E.; Cook, Peter E.; Johnson, Petrina H.; Rapley, Luke P.; Kay, Brian H.; Ryan, Peter A.; Ritchie, Scott A.; O'Neill, Scott L.

2010-01-01

Background New strategies to eliminate dengue have been proposed that specifically target older Aedes aegypti mosquitoes, the proportion of the vector population that is potentially capable of transmitting dengue viruses. Evaluation of these strategies will require accurate and high-throughput methods of predicting mosquito age. We previously developed an age prediction assay for individual Ae. aegypti females based on the transcriptional profiles of a selection of age responsive genes. Here we conducted field testing of the method on Ae. aegypti that were entirely uncaged and free to engage in natural behavior. Methodology/Principal Findings We produced “free-range” test specimens by releasing 8007 adult Ae. aegypti inside and around an isolated homestead in north Queensland, Australia, and recapturing females at two day intervals. We applied a TaqMan probe-based assay design that enabled high-throughput quantitative RT-PCR of four transcripts from three age-responsive genes and a reference gene. An age prediction model was calibrated on mosquitoes maintained in small sentinel cages, in which 68.8% of the variance in gene transcription measures was explained by age. The model was then used to predict the ages of the free-range females. The relationship between the predicted and actual ages achieved an R2 value of 0.62 for predictions of females up to 29 days old. Transcriptional profiles and age predictions were not affected by physiological variation associated with the blood feeding/egg development cycle and we show that the age grading method could be applied to differentiate between two populations of mosquitoes having a two-fold difference in mean life expectancy. Conclusions/Significance The transcriptional profiles of age responsive genes facilitated age estimates of near-wild Ae. aegypti females. Our age prediction assay for Ae. aegypti provides a useful tool for the evaluation of mosquito control interventions against dengue where mosquito

Field validation of a transcriptional assay for the prediction of age of uncaged Aedes aegypti mosquitoes in Northern Australia.

Directory of Open Access Journals (Sweden)

Leon E Hugo

Full Text Available BACKGROUND: New strategies to eliminate dengue have been proposed that specifically target older Aedes aegypti mosquitoes, the proportion of the vector population that is potentially capable of transmitting dengue viruses. Evaluation of these strategies will require accurate and high-throughput methods of predicting mosquito age. We previously developed an age prediction assay for individual Ae. aegypti females based on the transcriptional profiles of a selection of age responsive genes. Here we conducted field testing of the method on Ae. aegypti that were entirely uncaged and free to engage in natural behavior. METHODOLOGY/PRINCIPAL FINDINGS: We produced "free-range" test specimens by releasing 8007 adult Ae. aegypti inside and around an isolated homestead in north Queensland, Australia, and recapturing females at two day intervals. We applied a TaqMan probe-based assay design that enabled high-throughput quantitative RT-PCR of four transcripts from three age-responsive genes and a reference gene. An age prediction model was calibrated on mosquitoes maintained in small sentinel cages, in which 68.8% of the variance in gene transcription measures was explained by age. The model was then used to predict the ages of the free-range females. The relationship between the predicted and actual ages achieved an R(2 value of 0.62 for predictions of females up to 29 days old. Transcriptional profiles and age predictions were not affected by physiological variation associated with the blood feeding/egg development cycle and we show that the age grading method could be applied to differentiate between two populations of mosquitoes having a two-fold difference in mean life expectancy. CONCLUSIONS/SIGNIFICANCE: The transcriptional profiles of age responsive genes facilitated age estimates of near-wild Ae. aegypti females. Our age prediction assay for Ae. aegypti provides a useful tool for the evaluation of mosquito control interventions against dengue where

The performance of the SEPT9 gene methylation assay and a comparison with other CRC screening tests: A meta-analysis.

Science.gov (United States)

Song, Lele; Jia, Jia; Peng, Xiumei; Xiao, Wenhua; Li, Yuemin

2017-06-08

The SEPT9 gene methylation assay is the first FDA-approved blood assay for colorectal cancer (CRC) screening. Fecal immunochemical test (FIT), FIT-DNA test and CEA assay are also in vitro diagnostic (IVD) tests used in CRC screening. This meta-analysis aims to review the SEPT9 assay performance and compare it with other IVD CRC screening tests. By searching the Ovid MEDLINE, EMBASE, CBMdisc and CJFD database, 25 out of 180 studies were identified to report the SEPT9 assay performance. 2613 CRC cases and 6030 controls were included, and sensitivity and specificity were used to evaluate its performance at various algorithms. 1/3 algorithm exhibited the best sensitivity while 2/3 and 1/1 algorithm exhibited the best balance between sensitivity and specificity. The performance of the blood SEPT9 assay is superior to that of the serum protein markers and the FIT test in symptomatic population, while appeared to be less potent than FIT and FIT-DNA tests in asymptomatic population. In conclusion, 1/3 algorithm is recommended for CRC screening, and 2/3 or 1/1 algorithms are suitable for early detection for diagnostic purpose. The SEPT9 assay exhibited better performance in symptomatic population than in asymptomatic population.

A novel data mining method to identify assay-specific signatures in functional genomic studies

Directory of Open Access Journals (Sweden)

Guidarelli Jack W

2006-08-01

Full Text Available Abstract Background: The highly dimensional data produced by functional genomic (FG studies makes it difficult to visualize relationships between gene products and experimental conditions (i.e., assays. Although dimensionality reduction methods such as principal component analysis (PCA have been very useful, their application to identify assay-specific signatures has been limited by the lack of appropriate methodologies. This article proposes a new and powerful PCA-based method for the identification of assay-specific gene signatures in FG studies. Results: The proposed method (PM is unique for several reasons. First, it is the only one, to our knowledge, that uses gene contribution, a product of the loading and expression level, to obtain assay signatures. The PM develops and exploits two types of assay-specific contribution plots, which are new to the application of PCA in the FG area. The first type plots the assay-specific gene contribution against the given order of the genes and reveals variations in distribution between assay-specific gene signatures as well as outliers within assay groups indicating the degree of importance of the most dominant genes. The second type plots the contribution of each gene in ascending or descending order against a constantly increasing index. This type of plots reveals assay-specific gene signatures defined by the inflection points in the curve. In addition, sharp regions within the signature define the genes that contribute the most to the signature. We proposed and used the curvature as an appropriate metric to characterize these sharp regions, thus identifying the subset of genes contributing the most to the signature. Finally, the PM uses the full dataset to determine the final gene signature, thus eliminating the chance of gene exclusion by poor screening in earlier steps. The strengths of the PM are demonstrated using a simulation study, and two studies of real DNA microarray data – a study of

Differentiating Botulinum Neurotoxin-Producing Clostridia with a Simple, Multiplex PCR Assay.

Science.gov (United States)

Williamson, Charles H D; Vazquez, Adam J; Hill, Karen; Smith, Theresa J; Nottingham, Roxanne; Stone, Nathan E; Sobek, Colin J; Cocking, Jill H; Fernández, Rafael A; Caballero, Patricia A; Leiser, Owen P; Keim, Paul; Sahl, Jason W

2017-09-15

Diverse members of the genus Clostridium produce botulinum neurotoxins (BoNTs), which cause a flaccid paralysis known as botulism. While multiple species of clostridia produce BoNTs, the majority of human botulism cases have been attributed to Clostridium botulinum groups I and II. Recent comparative genomic studies have demonstrated the genomic diversity within these BoNT-producing species. This report introduces a multiplex PCR assay for differentiating members of C. botulinum group I, C. sporogenes , and two major subgroups within C. botulinum group II. Coding region sequences unique to each of the four species/subgroups were identified by in silico analyses of thousands of genome assemblies, and PCR primers were designed to amplify each marker. The resulting multiplex PCR assay correctly assigned 41 tested isolates to the appropriate species or subgroup. A separate PCR assay to determine the presence of the ntnh gene (a gene associated with the botulinum neurotoxin gene cluster) was developed and validated. The ntnh gene PCR assay provides information about the presence or absence of the botulinum neurotoxin gene cluster and the type of gene cluster present ( ha positive [ ha + ] or orfX + ). The increased availability of whole-genome sequence data and comparative genomic tools enabled the design of these assays, which provide valuable information for characterizing BoNT-producing clostridia. The PCR assays are rapid, inexpensive tests that can be applied to a variety of sample types to assign isolates to species/subgroups and to detect clostridia with botulinum neurotoxin gene ( bont ) clusters. IMPORTANCE Diverse clostridia produce the botulinum neurotoxin, one of the most potent known neurotoxins. In this study, a multiplex PCR assay was developed to differentiate clostridia that are most commonly isolated in connection with human botulism cases: C. botulinum group I, C. sporogenes , and two major subgroups within C. botulinum group II. Since Bo

Development and inter-laboratory validation of unlabeled probe melting curve analysis for detection of JAK2 V617F mutation in polycythemia vera.

Science.gov (United States)

Wu, Zhiyuan; Yuan, Hong; Zhang, Xinju; Liu, Weiwei; Xu, Jinhua; Zhang, Wei; Guan, Ming

2011-01-01

JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described. Asymmetric PCR for detection of JAK2 V617F with a 3'-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex) in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type) covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments. With a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.

Development and inter-laboratory validation of unlabeled probe melting curve analysis for detection of JAK2 V617F mutation in polycythemia vera.

Directory of Open Access Journals (Sweden)

Zhiyuan Wu

Full Text Available BACKGROUND: JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described. METHODOLOGY/PRINCIPAL FINDINGS: Asymmetric PCR for detection of JAK2 V617F with a 3'-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments. CONCLUSIONS: With a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.

Screening of molecular cell targets for carcinogenic heterocyclic aromatic amines by using CALUX® reporter gene assays.

Science.gov (United States)

Steinberg, Pablo; Behnisch, Peter A; Besselink, Harrie; Brouwer, Abraham A

2017-06-01

Heterocyclic aromatic amines (HCAs) are compounds formed when meat or fish are cooked at high temperatures for a long time or over an open fire. To determine which pathways of toxicity are activated by HCAs, nine out of the ten HCAs known to be carcinogenic in rodents (2-amino-9H-pyrido[2,3-b]indole (AαC), 2-aminodipyrido[1,2-a:3',2-d]imidazole (Glu-P-2), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2)) were tested in the estrogen receptor α (ERα), androgen receptor (AR), glucocorticoid receptor (GR), peroxisome proliferator-activated receptor γ2 (PPARγ2), polycyclic aromatic hydrocarbons (PAH), Nrf2, and p53 CALUX® reporter gene assays. Trp-P-1 was the only HCA that led to a positive response in the ERα, PPARγ2, and Nrf2 CALUX® assays. In the PAH CALUX® assay, Trp-P-2, MeAαC, and AαC induced luciferase activity to a greater extent than MeIQ and PhIP. In the p53 CALUX® assay without a coupled metabolic activation, only Trp-P-1 and Trp-P-2 enhanced luciferase expression; when a metabolic activation step was coupled to the p53 CALUX® assay, Trp-P-1, Glu-P-2, MeIQ, MeIQx, and PhIP induced a positive response. No HCA was positive in the AR and GR CALUX® assays. Taken together, the results obtained show that the battery of CALUX® assays performed in the present study can successfully be used to screen for molecular cell targets of carcinogenic compounds such as HCAs.

Neutron-neutron probe for uranium exploration

International Nuclear Information System (INIS)

Smith, R.C.

1979-01-01

A neutron activation probe for assaying the amount of fissionable isotopes in an ore body is described which comprises a casing which is movable through a borehole in the ore body, a neutron source and a number of delayed neutron detectors arranged colinearly in the casing below the neutron source for detecting delayed neutrons

Tetraplex PCR assay involving double gene-sites discriminates beef and buffalo in Malaysian meat curry and burger products.

Science.gov (United States)

Hossain, M A Motalib; Ali, Md Eaqub; Hamid, Sharifah Bee Abd; Hossain, S M Azad; Asing; Nizar, Nina Naquiah Ahmad; Uddin, Mohammad Nasir; Ali, Lokman; Asaduzzaman, Md; Akanda, Md Jahurul Haque

2017-06-01

Replacement of beef by buffalo and vice versa is frequent in global markets, but their authentication is challenging in processed foods due to the fragmentation of most biomarkers including DNA. The shortening of target sequences through use of two target sites might ameliorate assay reliability because it is highly unlikely that both targets will be lost during food processing. For the first time, we report a tetraplex polymerase chain reaction (PCR) assay targeting two different DNA regions in beef (106 and 120-bp) and buffalo (90 and 138-bp) mitochondrial genes to discriminate beef and buffalo in processed foods. All targets were stable under boiling, autoclaving and microwave cooking conditions. A survey in Malaysian markets revealed 71% beef curries contained buffalo but there was no buffalo in beef burgers. The assay detected down to 0.01ng DNA and 1% meat in admixed and burger products. Copyright © 2016 Elsevier Ltd. All rights reserved.

Real-time PCR genotyping assay for canine progressive rod-cone degeneration and mutant allele frequency in Toy Poodles, Chihuahuas and Miniature Dachshunds in Japan

OpenAIRE

KOHYAMA, Moeko; TADA, Naomi; MITSUI, Hiroko; TOMIOKA, Hitomi; TSUTSUI, Toshihiko; YABUKI, Akira; RAHMAN, Mohammad Mahbubur; KUSHIDA, Kazuya; MIZUKAMI, Keijiro; YAMATO, Osamu

2015-01-01

Canine progressive rod-cone degeneration (PRCD) is a middle- to late-onset, autosomal recessive, inherited retinal disorder caused by a substitution (c.5G>A) in the canine PRCD gene that has been identified in 29 or more purebred dogs. In the present study, a TaqMan probe-based real-time PCR assay was developed and evaluated for rapid genotyping and large-scale screening of the mutation. Furthermore, a genotyping survey was carried out in a population of the three most popular breeds in Japan...

Multiplex real-time PCR assays for detection of eight Shiga toxin-producing Escherichia coli in food samples by melting curve analysis.

Science.gov (United States)

Singh, Prashant; Mustapha, Azlin

2015-12-23

Shiga toxin-producing Escherichia coli (STEC) are pathogenic strains of E. coli that can cause bloody diarrhea and kidney failure. Seven STEC serogroups, O157, O26, O45, O103, O111, O121 and O145 are responsible for more than 71% of the total infections caused by this group of pathogens. All seven serogroups are currently considered as adulterants in non-intact beef products in the U.S. In this study, two multiplex melt curve real-time PCR assays with internal amplification controls (IACs) were standardized for the detection of eight STEC serogroups. The first multiplex assay targeted E. coli serogroups O145, O121, O104, and O157; while the second set detected E. coli serogroups O26, O45, O103 and O111. The applicability of the assays was tested using 11 different meat and produce samples. For food samples spiked with a cocktail of four STEC serogroups with a combined count of 10 CFU/25 g food, all targets of the multiplex assays were detected after an enrichment period of 6h. The assays also worked efficiently when 325 g of food samples were spiked with 10 CFU of STECs. The assays are not dependent on fluorescent-labeled probes or immunomagnetic beads, and can be used for the detection of eight STEC serogroups in less than 11h. Routine preliminary screening of STECs in food samples is performed by testing for the presence of STEC virulence genes. The assays developed in this study can be useful as a first- or second-tier test for the identification of the eight O serogroup-specific genes in suspected food samples. Copyright © 2015. Published by Elsevier B.V.

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

Science.gov (United States)

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

2017-05-12

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

Simultaneous detection of mutations and copy number variation of NPM1 in the acute myeloid leukemia using multiplex ligation-dependent probe amplification

International Nuclear Information System (INIS)

Marcinkowska-Swojak, Malgorzata; Handschuh, Luiza; Wojciechowski, Pawel; Goralski, Michal; Tomaszewski, Kamil; Kazmierczak, Maciej; Lewandowski, Krzysztof; Komarnicki, Mieczyslaw

2016-01-01

Highlights: • The NPM1 mutations were detected exclusively in AML accounting for 25% of cases. • The NPM1 gene did not reveal any copy number alterations. • The NPM1mut+ assay is a reliable test for the analysis of mutations and CNA in NPM1. - Abstract: The NPM1 gene encodes nucleophosmin, a protein involved in multiple cell functions and carcinogenesis. Mutation of the NPM1 gene, causing delocalization of the protein, is the most frequent genetic lesion in acute myeloid leukemia (AML); it is considered a founder event in AML pathogenesis and serves as a favorable prognostic marker. Moreover, in solid tumors and some leukemia cell lines, overexpression of the NPM1 gene is commonly observed. Therefore, the purpose of this study was to develop a new method for the detection of NPM1 mutations and the simultaneous analysis of copy number alterations (CNAs), which may underlie NPM1 gene expression deregulation. To address both of the issues, we applied a strategy based on multiplex ligation-dependent probe amplification (MLPA). A designed NPM1mut+ assay enables the detection of three of the most frequent NPM1 mutations: A, B and D. The accuracy of the assay was tested using a group of 83 samples from Polish patients with AML and other blood-proliferative disorders. To verify the results, we employed traditional Sanger sequencing and next-generation transcriptome sequencing. With the use of the NPM1mut+ assay, we detected mutations A, D and B in 14, 1 and 0 of the analyzed samples, respectively. All of these mutations were confirmed by complementary sequencing approaches, proving the 100% specificity and sensitivity of the proposed test. The performed sequencing analysis allowed the identification of two additional rare mutations (I and ZE). All of the mutations were identified exclusively in AML cases, accounting for 25% of those cases. We did not observe any CNAs (amplifications) of the NPM1 gene in the studied samples, either with or without the mutation. The

Simultaneous detection of mutations and copy number variation of NPM1 in the acute myeloid leukemia using multiplex ligation-dependent probe amplification

Energy Technology Data Exchange (ETDEWEB)

Marcinkowska-Swojak, Malgorzata, E-mail: m-marcinkowska@o2.pl [European Center of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan (Poland); Handschuh, Luiza, E-mail: luizahan@ibch.poznan.pl [European Center of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan (Poland); Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Szamarzewskiego 82/84, 60-569 Poznan (Poland); Wojciechowski, Pawel, E-mail: Pawel.Wojciechowski@cs.put.poznan.pl [European Center of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan (Poland); Institute of Computing Science, Poznan University of Technology, Piotrowo 2, 60-965 Poznan (Poland); Goralski, Michal, E-mail: mgoralsk@ibch.poznan.pl [European Center of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan (Poland); Tomaszewski, Kamil, E-mail: kamil.tomaszewsky@gmail.com [European Center of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan (Poland); Kazmierczak, Maciej, E-mail: maciej.kazmierczak@onet.eu [Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Szamarzewskiego 82/84, 60-569 Poznan (Poland); Lewandowski, Krzysztof, E-mail: krzysztof.lewandowski@skpp.edu.pl [Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Szamarzewskiego 82/84, 60-569 Poznan (Poland); Komarnicki, Mieczyslaw, E-mail: mak7@pro.onet.pl [Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Szamarzewskiego 82/84, 60-569 Poznan (Poland); and others

2016-04-15

Highlights: • The NPM1 mutations were detected exclusively in AML accounting for 25% of cases. • The NPM1 gene did not reveal any copy number alterations. • The NPM1mut+ assay is a reliable test for the analysis of mutations and CNA in NPM1. - Abstract: The NPM1 gene encodes nucleophosmin, a protein involved in multiple cell functions and carcinogenesis. Mutation of the NPM1 gene, causing delocalization of the protein, is the most frequent genetic lesion in acute myeloid leukemia (AML); it is considered a founder event in AML pathogenesis and serves as a favorable prognostic marker. Moreover, in solid tumors and some leukemia cell lines, overexpression of the NPM1 gene is commonly observed. Therefore, the purpose of this study was to develop a new method for the detection of NPM1 mutations and the simultaneous analysis of copy number alterations (CNAs), which may underlie NPM1 gene expression deregulation. To address both of the issues, we applied a strategy based on multiplex ligation-dependent probe amplification (MLPA). A designed NPM1mut+ assay enables the detection of three of the most frequent NPM1 mutations: A, B and D. The accuracy of the assay was tested using a group of 83 samples from Polish patients with AML and other blood-proliferative disorders. To verify the results, we employed traditional Sanger sequencing and next-generation transcriptome sequencing. With the use of the NPM1mut+ assay, we detected mutations A, D and B in 14, 1 and 0 of the analyzed samples, respectively. All of these mutations were confirmed by complementary sequencing approaches, proving the 100% specificity and sensitivity of the proposed test. The performed sequencing analysis allowed the identification of two additional rare mutations (I and ZE). All of the mutations were identified exclusively in AML cases, accounting for 25% of those cases. We did not observe any CNAs (amplifications) of the NPM1 gene in the studied samples, either with or without the mutation. The

A luciferase reporter gene assay and aryl hydrocarbon receptor 1 genotype predict the LD{sub 50} of polychlorinated biphenyls in avian species

Energy Technology Data Exchange (ETDEWEB)

Manning, Gillian E., E-mail: gmann017@uottawa.ca [Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, Canada K1N 6N5 (Canada); Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Farmahin, Reza, E-mail: mfarm070@uottawa.ca [Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, Canada K1N 6N5 (Canada); Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Crump, Doug, E-mail: doug.crump@ec.gc.ca [Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Jones, Stephanie P., E-mail: stephanie.jones@ec.gc.ca [Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Klein, Jeff, E-mail: jeffery@well-labs.com [Wellington Laboratories Inc., Research Division, Guelph, ON, Canada N1G 3M5 (Canada); Konstantinov, Alex, E-mail: alex@well-labs.com [Wellington Laboratories Inc., Research Division, Guelph, ON, Canada N1G 3M5 (Canada); Potter, Dave, E-mail: dpotter@well-labs.com [Wellington Laboratories Inc., Research Division, Guelph, ON, Canada N1G 3M5 (Canada); Kennedy, Sean W., E-mail: sean.kennedy@ec.gc.ca [Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, Canada K1N 6N5 (Canada); Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada)

2012-09-15

Birds differ in sensitivity to the embryotoxic effects of polychlorinated biphenyls (PCBs), which complicates environmental risk assessments for these chemicals. Recent research has shown that the identities of amino acid residues 324 and 380 in the avian aryl hydrocarbon receptor 1 (AHR1) ligand binding domain (LBD) are primarily responsible for differences in avian species sensitivity to selected dibenzo-p-dioxins and furans. A luciferase reporter gene (LRG) assay was developed in our laboratory to measure AHR1-mediated induction of a cytochrome P450 1A5 reporter gene in COS-7 cells transfected with different avian AHR1 constructs. In the present study, the LRG assay was used to measure the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and PCBs 126, 77, 105 and 118 on luciferase activity in COS-7 cells transfected with AHR1 constructs representative of 86 avian species in order to predict their sensitivity to PCB-induced embryolethality and the relative potency of PCBs in these species. The results of the LRG assay indicate that the identity of amino acid residues 324 and 380 in the AHR1 LBD are the major determinants of avian species sensitivity to PCBs. The relative potency of PCBs did not differ greatly among AHR1 constructs. Luciferase activity was significantly correlated with embryolethality data obtained from the literature (R{sup 2} ≥ 0.87, p < 0.0001). Thus, the LRG assay in combination with the knowledge of a species' AHR1 LBD sequence can be used to predict PCB-induced embryolethality in potentially any avian species of interest without the use of lethal methods on a large number of individuals. -- Highlights: ► PCB embryolethality in birds can be predicted from a species' AHR1 genotype. ► The reporter gene assay is useful for predicting species sensitivity to PCBs. ► The relative potency of PCBs does not appear to differ between AHR1 genotypes. ► Contamination of PCB 105 and PCB 118 did not affect

Telemetric Technologies for the Assay of Gene Expression

Science.gov (United States)

Paul, Anna-Lisa; Bamsey, Matthew; Berinstain, Alain; Neron, Philip; Graham, Thomas; Ferl, Robert

Telemetric data collection has been widely used in spaceflight applications where human participation is limited (orbital mission payloads) or unfeasible (planetary landers, satellites, and probes). The transmission of digital data from electronic sensors of typical environmental parameters, growth patterns and physical properties of materials is routine telemetry, and even the collection and transmission of deep space images is a standard tool of astrophysics. But telemetric imaging for current biological payloads has thus far been limited to the collection of standard white-light photography that is largely confined to reporting the surface characteristics of the specimens involved. Advances in imaging technologies that facilitate the collection of a variety of light wavelengths will expand the science return on biological payloads to include evaluations of the molecular genetic response of organisms to the spaceflight or extraterrestrial environment, with minimal or no human intervention. Advanced imaging technology in combination with biologically engineered sensor organisms can create a system that can report via telemetry on the patterns of gene expression required to adapt to a novel environment. The utilization of genetically engineered plants as biosensors has made elegant strides in the recent years, providing keen insights into the health of plants in general and particularly in the nature and cellular location of stress responses. Moreover, molecular responses to gravitational vectors have been elegantly analyzed with fluorescent tools. Green Fluorescence Protein (GFP) and other fluorophores have made it possible for analyses of gene expression and biological responses to occur telemetrically, with the information potentially delivered to the investigator over large distances as simple, preprocessed fluorescence images. Having previously deployed transgenic plant biosensors to evaluate responses to orbital spaceflight, we wish to develop both the plants

Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors.

Directory of Open Access Journals (Sweden)

Cengiz Ikten

Full Text Available Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.. Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease.

Evolving BioAssay Ontology (BAO): modularization, integration and applications.

Science.gov (United States)

Abeyruwan, Saminda; Vempati, Uma D; Küçük-McGinty, Hande; Visser, Ubbo; Koleti, Amar; Mir, Ahsan; Sakurai, Kunie; Chung, Caty; Bittker, Joshua A; Clemons, Paul A; Brudz, Steve; Siripala, Anosha; Morales, Arturo J; Romacker, Martin; Twomey, David; Bureeva, Svetlana; Lemmon, Vance; Schürer, Stephan C

2014-01-01

The lack of established standards to describe and annotate biological assays and screening outcomes in the domain of drug and chemical probe discovery is a severe limitation to utilize public and proprietary drug screening data to their maximum potential. We have created the BioAssay Ontology (BAO) project (http://bioassayontology.org) to develop common reference metadata terms and definitions required for describing relevant information of low-and high-throughput drug and probe screening assays and results. The main objectives of BAO are to enable effective integration, aggregation, retrieval, and analyses of drug screening data. Since we first released BAO on the BioPortal in 2010 we have considerably expanded and enhanced BAO and we have applied the ontology in several internal and external collaborative projects, for example the BioAssay Research Database (BARD). We describe the evolution of BAO with a design that enables modeling complex assays including profile and panel assays such as those in the Library of Integrated Network-based Cellular Signatures (LINCS). One of the critical questions in evolving BAO is the following: how can we provide a way to efficiently reuse and share among various research projects specific parts of our ontologies without violating the integrity of the ontology and without creating redundancies. This paper provides a comprehensive answer to this question with a description of a methodology for ontology modularization using a layered architecture. Our modularization approach defines several distinct BAO components and separates internal from external modules and domain-level from structural components. This approach facilitates the generation/extraction of derived ontologies (or perspectives) that can suit particular use cases or software applications. We describe the evolution of BAO related to its formal structures, engineering approaches, and content to enable modeling of complex assays and integration with other ontologies and

A paper-based resonance energy transfer nucleic acid hybridization assay using upconversion nanoparticles as donors and quantum dots as acceptors.

Science.gov (United States)

Doughan, Samer; Uddayasankar, Uvaraj; Krull, Ulrich J

2015-06-09

Monodisperse aqueous upconverting nanoparticles (UCNPs) were covalently immobilized on aldehyde modified cellulose paper via reduction amination to develop a luminescence resonance energy transfer (LRET)-based nucleic acid hybridization assay. This first account of covalent immobilization of UCNPs on paper for a bioassay reports an optically responsive method that is sensitive, reproducible and robust. The immobilized UCNPs were decorated with oligonucleotide probes to capture HPRT1 housekeeping gene fragments, which in turn brought reporter conjugated quantum dots (QDs) in close proximity to the UCNPs for LRET. This sandwich assay could detect unlabeled oligonucleotide target, and had a limit of detection of 13 fmol and a dynamic range spanning nearly 3 orders of magnitude. The use of QDs, which are excellent LRET acceptors, demonstrated improved sensitivity, limit of detection, dynamic range and selectivity compared to similar assays that have used molecular fluorophores as acceptors. The selectivity of the assay was attributed to the decoration of the QDs with polyethylene glycol to eliminate non-specific adsorption. The kinetics of hybridization were determined to be diffusion limited and full signal development occurred within 3 min. Copyright © 2015 Elsevier B.V. All rights reserved.

Rapid Identification of Seven Waterborne Exophiala Species by RCA DNA Padlock Probes.

Science.gov (United States)

Najafzadeh, M J; Vicente, V A; Feng, Peiying; Naseri, A; Sun, Jiufeng; Rezaei-Matehkolaei, A; de Hoog, G S

2018-03-05

The black yeast genus Exophiala includes numerous potential opportunistic species that potentially cause systematic and disseminated infections in immunocompetent individuals. Species causing systemic disease have ability to grow at 37-40 °C, while others consistently lack thermotolerance and are involved in diseases of cold-blooded, waterborne vertebrates and occasionally invertebrates. We explain a fast and sensitive assay for recognition and identification of waterborne Exophiala species without sequencing. The ITS rDNA region of seven Exophiala species (E. equina, E. salmonis, E. opportunistica, E. pisciphila, E. aquamarina, E. angulospora and E. castellanii) along with the close relative Veronaea botryosa was sequenced and aligned for the design of specific padlock probes for the detection of characteristic single-nucleotide polymorphisms. The assay demonstrated to successfully amplify DNA of target fungi, allowing detection at the species level. Amplification products were visualized on 1% agarose gels to confirm specificity of probe-template binding. Amounts of reagents were reduced to prevent the generation of false positive results. The simplicity, tenderness, robustness and low expenses provide padlock probe assay (RCA) a definite place as a very practical method among isothermal approaches for DNA diagnostics.

Improving probe set selection for microbial community analysis by leveraging taxonomic information of training sequences

Directory of Open Access Journals (Sweden)

Jiang Tao

2011-10-01

Full Text Available Abstract Background Population levels of microbial phylotypes can be examined using a hybridization-based method that utilizes a small set of computationally-designed DNA probes targeted to a gene common to all. Our previous algorithm attempts to select a set of probes such that each training sequence manifests a unique theoretical hybridization pattern (a binary fingerprint to a probe set. It does so without taking into account similarity between training gene sequences or their putative taxonomic classifications, however. We present an improved algorithm for probe set selection that utilizes the available taxonomic information of training gene sequences and attempts to choose probes such that the resultant binary fingerprints cluster into real taxonomic groups. Results Gene sequences manifesting identical fingerprints with probes chosen by the new algorithm are more likely to be from the same taxonomic group than probes chosen by the previous algorithm. In cases where they are from different taxonomic groups, underlying DNA sequences of identical fingerprints are more similar to each other in probe sets made with the new versus the previous algorithm. Complete removal of large taxonomic groups from training data does not greatly decrease the ability of probe sets to distinguish those groups. Conclusions Probe sets made from the new algorithm create fingerprints that more reliably cluster into biologically meaningful groups. The method can readily distinguish microbial phylotypes that were excluded from the training sequences, suggesting novel microbes can also be detected.

Improving probe set selection for microbial community analysis by leveraging taxonomic information of training sequences.

Science.gov (United States)

Ruegger, Paul M; Della Vedova, Gianluca; Jiang, Tao; Borneman, James

2011-10-10

Population levels of microbial phylotypes can be examined using a hybridization-based method that utilizes a small set of computationally-designed DNA probes targeted to a gene common to all. Our previous algorithm attempts to select a set of probes such that each training sequence manifests a unique theoretical hybridization pattern (a binary fingerprint) to a probe set. It does so without taking into account similarity between training gene sequences or their putative taxonomic classifications, however. We present an improved algorithm for probe set selection that utilizes the available taxonomic information of training gene sequences and attempts to choose probes such that the resultant binary fingerprints cluster into real taxonomic groups. Gene sequences manifesting identical fingerprints with probes chosen by the new algorithm are more likely to be from the same taxonomic group than probes chosen by the previous algorithm. In cases where they are from different taxonomic groups, underlying DNA sequences of identical fingerprints are more similar to each other in probe sets made with the new versus the previous algorithm. Complete removal of large taxonomic groups from training data does not greatly decrease the ability of probe sets to distinguish those groups. Probe sets made from the new algorithm create fingerprints that more reliably cluster into biologically meaningful groups. The method can readily distinguish microbial phylotypes that were excluded from the training sequences, suggesting novel microbes can also be detected.

Developing high throughput quantitative PCR assays for diagnosing Ikeda and other Theileria orientalis types common to New Zealand in bovine blood samples.

Science.gov (United States)

Pulford, D J; Gias, E; Bueno, I M; McFadden, Amj

2016-01-01

To develop rapid, quantitative PCR (qPCR) assays using high resolution melt (HRM) analysis and type-specific TaqMan assays for identifying the prevalent types of Theileria orientalis found in New Zealand cattle; and to evaluate their analytical and diagnostic characteristics compared with other assays for T. orientalis. Nucleotide sequences aligned with T. orientalis Buffeli, Chitose and Ikeda types, obtained from DNA extracted from blood samples from infected cattle, were used to design HRM and type-specific probe-based qPCR assays. The three type-specific assays were also incorporated into a single-tube multiplex qPCR assay. These assays were validated using DNA extracted from blood samples from cattle in herds with or without clinical signs of T. orientalis infection, other veterinary laboratory samples, as well as plasmids containing T. orientalis type-specific sequences. Diagnostic specificity (DSp) and sensitivity (DSe) estimates for the qPCR assays were compared to blood smear piroplasm results, and other PCR assays for T. orientalis. Copy number estimates of Ikeda DNA in blood were determined from cattle exhibiting anaemia using the Ikeda-specific qPCR assay. The T. orientalis type-specific and the HRM qPCR assays displayed 100% analytical specificity. The Ikeda-specific qPCR assay exhibited linearity (R(2) = 0.997) with an efficiency of 94.3%. Intra-assay CV were ≤0.08 and inter-assay CV were ≤0.095. For blood samples from cows with signs of infection with T. orientalis, the DSp and DSe of the multiplex probe qPCR assay were 93 and 96%, respectively compared with blood smears, and 97 and 100%, respectively compared with conventional PCR assays. For the Ikeda-specific qPCR assay, the number of positive samples (n=66) was slightly higher than a conventional PCR assay (n=64). The concentration of Ikeda genomes in blood samples from 41 dairy cows with signs of infection with T. orientalis ranged between 5.6 × 10(4) and 3.3 × 10(6) genomes per

Development of an androgen reporter gene assay (AR-LUX) utilizing a human cell line with an endogenously regulated androgen receptor

NARCIS (Netherlands)

Blankvoort, B.M.G.; Groene, E.M. de; Meeteren-Kreikamp, A.P. van; Witkamp, R.F.; Rodenburg, R.J.T.; Aarts, J.M.M.J.G.

2001-01-01

The aim of the work described in this report is to develop and characterize a cell-based androgen reporter assay. For this purpose, the androgen receptor (AR) expressing human breast cancer cell line T47D was stably transfected with a luciferase gene under transcriptional control of the PB-ARE-2

Sex genes for genomic analysis in human brain: internal controls for comparison of probe level data extraction.

Directory of Open Access Journals (Sweden)

Ellis Steven P

2003-09-01

Full Text Available Abstract Background Genomic studies of complex tissues pose unique analytical challenges for assessment of data quality, performance of statistical methods used for data extraction, and detection of differentially expressed genes. Ideally, to assess the accuracy of gene expression analysis methods, one needs a set of genes which are known to be differentially expressed in the samples and which can be used as a "gold standard". We introduce the idea of using sex-chromosome genes as an alternative to spiked-in control genes or simulations for assessment of microarray data and analysis methods. Results Expression of sex-chromosome genes were used as true internal biological controls to compare alternate probe-level data extraction algorithms (Microarray Suite 5.0 [MAS5.0], Model Based Expression Index [MBEI] and Robust Multi-array Average [RMA], to assess microarray data quality and to establish some statistical guidelines for analyzing large-scale gene expression. These approaches were implemented on a large new dataset of human brain samples. RMA-generated gene expression values were markedly less variable and more reliable than MAS5.0 and MBEI-derived values. A statistical technique controlling the false discovery rate was applied to adjust for multiple testing, as an alternative to the Bonferroni method, and showed no evidence of false negative results. Fourteen probesets, representing nine Y- and two X-chromosome linked genes, displayed significant sex differences in brain prefrontal cortex gene expression. Conclusion In this study, we have demonstrated the use of sex genes as true biological internal controls for genomic analysis of complex tissues, and suggested analytical guidelines for testing alternate oligonucleotide microarray data extraction protocols and for adjusting multiple statistical analysis of differentially expressed genes. Our results also provided evidence for sex differences in gene expression in the brain prefrontal cortex

Analysis of conditional gene deletion using probe based Real-Time PCR

Directory of Open Access Journals (Sweden)

Lyko Frank

2010-12-01

Full Text Available Abstract Following publication of this article 1 the authors noticed that an incorrect probe reference was cited on page 3, 4, 5 and 6 ("UP #69, Roche Applied Science". The correct probe that was used for the 1lox/2lox allele ratio analysis in the paper is as follows Probe for 1lox/2lox allele quantification: 5'-6-FAM-atAaCtTCgtatagCATaCattatac-BHQ-1 -3' (uppercase letters = LNA bases Manufacturer: EUROGENTEC, Seraing, Belgium All other information and reaction conditions in the paper are correct as stated.

Development and validation of cell-based luciferase reporter gene assays for measuring neutralizing anti-drug antibodies against interferon beta.

Science.gov (United States)

Hermanrud, Christina; Ryner, Malin; Luft, Thomas; Jensen, Poul Erik; Ingenhoven, Kathleen; Rat, Dorothea; Deisenhammer, Florian; Sørensen, Per Soelberg; Pallardy, Marc; Sikkema, Dan; Bertotti, Elisa; Kramer, Daniel; Creeke, Paul; Fogdell-Hahn, Anna

2016-03-01

Neutralizing anti-drug antibodies (NAbs) against therapeutic interferon beta (IFNβ) in people with multiple sclerosis (MS) are measured with cell-based bioassays. The aim of this study was to redevelop and validate two luciferase reporter-gene bioassays, LUC and iLite, using a cut-point approach to identify NAb positive samples. Such an approach is favored by the pharmaceutical industry and governmental regulatory agencies as it has a clear statistical basis and overcomes the limitations of the current assays based on the Kawade principle. The work was conducted following the latest assay guidelines. The assays were re-developed and validated as part of the "Anti-Biopharmaceutical Immunization: Prediction and analysis of clinical relevance to minimize the risk" (ABIRISK) consortium and involved a joint collaboration between four academic laboratories and two pharmaceutical companies. The LUC assay was validated at Innsbruck Medical University (LUCIMU) and at Rigshospitalet (LUCRH) Copenhagen, and the iLite assay at Karolinska Institutet, Stockholm. For both assays, the optimal serum sample concentration in relation to sensitivity and recovery was 2.5% (v/v) in assay media. A Shapiro-Wilk test indicated a normal distribution for the majority of runs, allowing a parametric approach for cut-point calculation to be used, where NAb positive samples could be identified with 95% confidence. An analysis of means and variances indicated that a floating cut-point should be used for all assays. The assays demonstrated acceptable sensitivity for being cell-based assays, with a confirmed limit of detection in neat serum of 1519 ng/mL for LUCIMU, 814 ng/mL for LUCRH, and 320 ng/mL for iLite. Use of the validated cut-point assay, in comparison with the previously used Kawade method, identified 14% more NAb positive samples. In conclusion, implementation of the cut-point design resulted in increased sensitivity to detect NAbs. However, the clinical significance of these low

Comparison of Five Assays for Detection of Clostridium difficile Toxin

Science.gov (United States)

Chapin, Kimberle C.; Dickenson, Roberta A.; Wu, Fongman; Andrea, Sarah B.

2011-01-01

Performance characteristics of five assays for detection of Clostridium difficile toxin were compared using fresh stool samples from patients with C. difficile infection (CDI). Assays were performed simultaneously and according to the manufacturers' instructions. Patients were included in the study if they exhibited clinical symptoms consistent with CDI. Nonmolecular assays included glutamate dehydrogenase antigen tests, with positive findings followed by the Premier Toxin A and B Enzyme Immunoassay (GDH/EIA), and the C. Diff Quik Chek Complete test. Molecular assays (PCR) included the BD GeneOhm Cdiff Assay, the Xpert C. difficile test, and the ProGastro Cd assay. Specimens were considered true positive if results were positive in two or more assays. For each method, the Youden index was calculated and cost-effectiveness was analyzed. Of 81 patients evaluated, 26 (32.1%) were positive for CDI. Sensitivity of the BD GeneOhm Cdiff assay, the Xpert C. difficile test, the ProGastro Cd assay, C. Diff Quik Chek Complete test, and two-step GDH/EIA was 96.2%, 96.2%, 88.5%, 61.5%, and 42.3%, respectively. Specificity of the Xpert C. difficile test was 96.4%, and for the other four assays was 100%. Compared with nonmolecular methods, molecular methods detected 34.7% more positive specimens. Assessment of performance characteristics and cost-effectiveness demonstrated that the BD GeneOhm Cdiff assay yielded the best results. While costly, the Xpert C. difficile test required limited processing and yielded rapid results. Because of discordant results, specimen processing, and extraction equipment requirements, the ProGastro Cd assay was the least favored molecular assay. The GDH/EIA method lacked sufficient sensitivity to be recommended. PMID:21704273

Fluorescent porous silicon biological probes with high quantum efficiency and stability.

Science.gov (United States)

Tu, Chang-Ching; Chou, Ying-Nien; Hung, Hsiang-Chieh; Wu, Jingda; Jiang, Shaoyi; Lin, Lih Y

2014-12-01

We demonstrate porous silicon biological probes as a stable and non-toxic alternative to organic dyes or cadmium-containing quantum dots for imaging and sensing applications. The fluorescent silicon quantum dots which are embedded on the porous silicon surface are passivated with carboxyl-terminated ligands through stable Si-C covalent bonds. The porous silicon bio-probes have shown photoluminescence quantum yield around 50% under near-UV excitation, with high photochemical and thermal stability. The bio-probes can be efficiently conjugated with antibodies, which is confirmed by a standard enzyme-linked immunosorbent assay (ELISA) method.

Authentication of beef, carabeef, chevon, mutton and pork by a PCR-RFLP assay of mitochondrial cytb gene

OpenAIRE

Kumar, Deepak; Singh, S. P.; Karabasanavar, Nagappa S.; Singh, Rashmi; Umapathi, V.

2012-01-01

Authentication of meat assumes significance in view of religious, quality assurance, food safety, public health, conservation and legal concerns. Here, we describe a PCR-RFLP (Polymerase Chain Reaction- Restriction Fragment Length Polymorphism) assay targeting mitochondrial cytochrome-b gene for the identification of meats of five most common food animals namely cattle, buffalo, goat, sheep and pig. A pair of forward and reverse primers (VPH-F & VPH-R) amplifying a conserved region (168–776 b...

New generation pharmacogenomic tools: a SNP linkage disequilibrium Map, validated SNP assay resource, and high-throughput instrumentation system for large-scale genetic studies.

Science.gov (United States)

De La Vega, Francisco M; Dailey, David; Ziegle, Janet; Williams, Julie; Madden, Dawn; Gilbert, Dennis A

2002-06-01

Since public and private efforts announced the first draft of the human genome last year, researchers have reported great numbers of single nucleotide polymorphisms (SNPs). We believe that the availability of well-mapped, quality SNP markers constitutes the gateway to a revolution in genetics and personalized medicine that will lead to better diagnosis and treatment of common complex disorders. A new generation of tools and public SNP resources for pharmacogenomic and genetic studies--specifically for candidate-gene, candidate-region, and whole-genome association studies--will form part of the new scientific landscape. This will only be possible through the greater accessibility of SNP resources and superior high-throughput instrumentation-assay systems that enable affordable, highly productive large-scale genetic studies. We are contributing to this effort by developing a high-quality linkage disequilibrium SNP marker map and an accompanying set of ready-to-use, validated SNP assays across every gene in the human genome. This effort incorporates both the public sequence and SNP data sources, and Celera Genomics' human genome assembly and enormous resource ofphysically mapped SNPs (approximately 4,000,000 unique records). This article discusses our approach and methodology for designing the map, choosing quality SNPs, designing and validating these assays, and obtaining population frequency ofthe polymorphisms. We also discuss an advanced, high-performance SNP assay chemisty--a new generation of the TaqMan probe-based, 5' nuclease assay-and high-throughput instrumentation-software system for large-scale genotyping. We provide the new SNP map and validation information, validated SNP assays and reagents, and instrumentation systems as a novel resource for genetic discoveries.

A micromachined membrane-based active probe for biomolecular mechanics measurement

Science.gov (United States)

Torun, H.; Sutanto, J.; Sarangapani, K. K.; Joseph, P.; Degertekin, F. L.; Zhu, C.

2007-04-01

A novel micromachined, membrane-based probe has been developed and fabricated as assays to enable parallel measurements. Each probe in the array can be individually actuated, and the membrane displacement can be measured with high resolution using an integrated diffraction-based optical interferometer. To illustrate its application in single-molecule mechanics experiments, this membrane probe was used to measure unbinding forces between L-selectin reconstituted in a polymer-cushioned lipid bilayer on the probe membrane and an antibody adsorbed on an atomic force microscope cantilever. Piconewton range forces between single pairs of interacting molecules were measured from the cantilever bending while using the membrane probe as an actuator. The integrated diffraction-based optical interferometer of the probe was demonstrated to have floor for frequencies as low as 3 Hz with a differential readout scheme. With soft probe membranes, this low noise level would be suitable for direct force measurements without the need for a cantilever. Furthermore, the probe membranes were shown to have 0.5 µm actuation range with a flat response up to 100 kHz, enabling measurements at fast speeds.

Colorimetric detection of Cucumber green mottle mosaic virus using unmodified gold nanoparticles as colorimetric probes.

Science.gov (United States)

Wang, Lin; Liu, Zhanmin; Xia, Xueying; Yang, Cuiyun; Huang, Junyi; Wan, Sibao

2017-05-01

Cucumber green mottle mosaic virus (CGMMV)causes a severe mosaic symptom of watermelon and cucumber, and can be transmitted via infected cucumber seeds, leaves and soil. It remains a challenge to detect this virus to prevent its introduction and infection and spread in fields. For this purpose, a simple and sensitive label-free colorimetric detection method for CGMMV has been developed with unmodified gold nanoparticles (AuNPs) as colorimetric probes. The method is based on the finding that the presence of RT-PCR target products of CGMMV and species-specific probes results in color change of AuNPs from red to blue after NaCl induction. Normally, species-specific probes attach to the surface of AuNPs and thereby increasing their resistance to NaCl-induced aggregation. The concentration of sodium, probes in the reaction system and evaluation of specificity and sensitivity of a novel assay, visual detection of Cucumber green mottle mosaic virus using unmodified AuNPs has been carried out with simple preparation of samples in our study. Through this assay, as low as 30pg/μL of CGMMV RNA was thus detected visually, by the naked eye, without the need for any sophisticated, expensive instrumentation and biochemical reagents. The specificity was 100% and exhibited good reproducibility in our assays. The results note that this assay is highly species-specific, simple, low-cost, and visual for easy detection of CGMMV in plant tissues. Therefore, visual assay is a potentially useful tool for middle or small-scales corporations and entry-exit inspection and quarantine bureau to detect CGMMV in cucumber seeds or plant tissues. Copyright © 2017 Elsevier B.V. All rights reserved.

Detection of Balamuthia mandrillaris DNA by real-time PCR targeting the RNase P gene

Directory of Open Access Journals (Sweden)

Lewin Astrid

2008-12-01

Full Text Available Abstract Background The free-living amoeba Balamuthia mandrillaris may cause fatal encephalitis both in immunocompromised and in – apparently – immunocompetent humans and other mammalian species. Rapid, specific, sensitive, and reliable detection requiring little pathogen-specific expertise is an absolute prerequisite for a successful therapy and a welcome tool for both experimental and epidemiological research. Results A real-time polymerase chain reaction assay using TaqMan® probes (real-time PCR was established specifically targeting the RNase P gene of B. mandrillaris amoebae. The assay detected at least 2 (down to 0.5 genomes of B. mandrillaris grown in axenic culture. It did not react with DNA from closely related Acanthamoeba (3 species, nor with DNA from Toxoplasma gondii, Leishmania major, Pneumocystis murina, Mycobacterium bovis (BCG, human brain, various mouse organs, or from human and murine cell lines. The assay efficiently detected B. mandrillaris DNA in spiked cell cultures, spiked murine organ homogenates, B. mandrillaris-infected mice, and CNS tissue-DNA preparations from 2 patients with proven cerebral balamuthiasis. This novel primer set was successfully combined with a published set that targets the B. mandrillaris 18S rRNA gene in a duplex real-time PCR assay to ensure maximum specificity and as a precaution against false negative results. Conclusion A real-time PCR assay for B. mandrillaris amoebae is presented, that is highly specific, sensitive, and reliable and thus suited both for diagnosis and for research.

Genetic diagnosis of Duchenne and Becker muscular dystrophy using multiplex ligation-dependent probe amplification in Rwandan patients.

Science.gov (United States)

Uwineza, Annette; Hitayezu, Janvier; Murorunkwere, Seraphine; Ndinkabandi, Janvier; Kalala Malu, Celestin Kaputu; Caberg, Jean Hubert; Dideberg, Vinciane; Bours, Vincent; Mutesa, Leon

2014-04-01

Duchenne and Becker muscular dystrophies are the most common clinical forms of muscular dystrophies. They are genetically X-linked diseases caused by a mutation in the dystrophin (DMD) gene. A genetic diagnosis was carried out in six Rwandan patients presenting a phenotype of Duchenne and Becker muscular dystrophies and six asymptomatic female carrier relatives using multiplex ligation-dependent probe amplification (MLPA). Our results revealed deletion of the exons 48-51 in one patient, an inherited deletion of the exons 8-21 in two brothers and a de novo deletion of the exons 46-50 in the fourth patient. No copy number variation was found in two patients. Only one female carrier presented exon deletion in the DMD gene. This is the first cohort of genetic analysis in Rwandan patients affected by Duchenne and Becker muscular dystrophies. This report confirmed that MLPA assay can be easily implemented in low-income countries.

Design of a dual-function peptide probe as a binder of angiotensin II and an inducer of silver nanoparticle aggregation for use in label-free colorimetric assays.

Science.gov (United States)

Okochi, Mina; Kuboyama, Masashi; Tanaka, Masayoshi; Honda, Hiroyuki

2015-09-01

Label-free colorimetric assays using metallic nanoparticles have received much recent attention, for their application in simple and sensitive methods for detection of biomolecules. Short peptide probes that can bind to analyte biomolecules are attractive ligands in molecular nanotechnology; however, identification of biological recognition motifs is usually based on trial-and-error experiments. Herein, a peptide probe was screened for colorimetric detection of angiotensin II (Ang II) using a mechanism for non-crosslinking aggregation of silver nanoparticles (AgNPs). The dual-function peptides, which bind to the analyte and induce AgNP aggregation, were identified using a two-step strategy: (1) screening of an Ang II-binding peptide from an Ang II receptor sequence library, using SPOT technology, which enable peptides synthesis on cellulose membranes via an Fmoc method and (2) selection of peptide probes that effectively induce aggregation of AgNPs using a photolinker modified peptide array. Using the identified peptide probe, KGKNKRRR, aggregation of AgNPs was detected by observation of a pink color in the absence of Ang II, whereas AgNPs remained dispersed in the presence of Ang II (yellow). The color changes were not observed in the presence of other hormone molecules. Ang II could be detected within 15 min, with a detection limit of 10 µM, by measuring the ratio of absorbance at 400 nm and 568 nm; the signal could also be observed with the naked eye. These data suggest that the peptide identified here could be used as a probe for simple and rapid colorimetric detection of Ang II. This strategy for the identification of functional peptides shows promise for the development of colorimetric detection of various diagnostically important biomolecules. Copyright © 2015 Elsevier B.V. All rights reserved.

Development and accuracy of quantitative real-time polymerase chain reaction assays for detection and quantification of enterotoxigenic Escherichia coli (ETEC) heat labile and heat stable toxin genes in travelers' diarrhea samples.

Science.gov (United States)

Youmans, Bonnie P; Ajami, Nadim J; Jiang, Zhi-Dong; Petrosino, Joseph F; DuPont, Herbert L; Highlander, Sarah K

2014-01-01

Enterotoxigenic Escherichia coli (ETEC), the leading bacterial pathogen of travelers' diarrhea, is routinely detected by an established DNA hybridization protocol that is neither sensitive nor quantitative. Quantitative real-time polymerase chain reaction (qPCR) assays that detect the ETEC toxin genes eltA, sta1, and sta2 in clinical stool samples were developed and tested using donor stool inoculated with known quantities of ETEC bacteria. The sensitivity of the qPCR assays is 89%, compared with 22% for the DNA hybridization assay, and the limits of detection are 10,000-fold lower than the DNA hybridization assays performed in parallel. Ninety-three clinical stool samples, previously characterized by DNA hybridization, were tested using the new ETEC qPCR assays. Discordant toxin profiles were observed for 22 samples, notably, four samples originally typed as ETEC negative were ETEC positive. The qPCR assays are unique in their sensitivity and ability to quantify the three toxin genes in clinical stool samples.

The loss-of-allele assay for ES cell screening and mouse genotyping.

Science.gov (United States)

Frendewey, David; Chernomorsky, Rostislav; Esau, Lakeisha; Om, Jinsop; Xue, Yingzi; Murphy, Andrew J; Yancopoulos, George D; Valenzuela, David M

2010-01-01

Targeting vectors used to create directed mutations in mouse embryonic stem (ES) cells consist, in their simplest form, of a gene for drug selection flanked by mouse genomic sequences, the so-called homology arms that promote site-directed homologous recombination between the vector and the target gene. The VelociGene method for the creation of targeted mutations in ES cells employs targeting vectors, called BACVecs, that are based on bacterial artificial chromosomes. Compared with conventional short targeting vectors, BacVecs provide two major advantages: (1) their much larger homology arms promote high targeting efficiencies without the need for isogenicity or negative selection strategies; and (2) they enable deletions and insertions of up to 100kb in a single targeting event, making possible gene-ablating definitive null alleles and other large-scale genomic modifications. Because of their large arm sizes, however, BACVecs do not permit screening by conventional assays, such as long-range PCR or Southern blotting, that link the inserted targeting vector to the targeted locus. To exploit the advantages of BACVecs for gene targeting, we inverted the conventional screening logic in developing the loss-of-allele (LOA) assay, which quantifies the number of copies of the native locus to which the mutation was directed. In a correctly targeted ES cell clone, the LOA assay detects one of the two native alleles (for genes not on the X or Y chromosome), the other allele being disrupted by the targeted modification. We apply the same principle in reverse as a gain-of-allele assay to quantify the copy number of the inserted targeting vector. The LOA assay reveals a correctly targeted clone as having lost one copy of the native target gene and gained one copy of the drug resistance gene or other inserted marker. The combination of these quantitative assays makes LOA genotyping unequivocal and amenable to automated scoring. We use the quantitative polymerase chain reaction

Comparison of five assays for detection of Clostridium difficile toxin.

Science.gov (United States)

Chapin, Kimberle C; Dickenson, Roberta A; Wu, Fongman; Andrea, Sarah B

2011-07-01

Performance characteristics of five assays for detection of Clostridium difficile toxin were compared using fresh stool samples from patients with C. difficile infection (CDI). Assays were performed simultaneously and according to the manufacturers' instructions. Patients were included in the study if they exhibited clinical symptoms consistent with CDI. Nonmolecular assays included glutamate dehydrogenase antigen tests, with positive findings followed by the Premier Toxin A and B Enzyme Immunoassay (GDH/EIA), and the C. Diff Quik Chek Complete test. Molecular assays (PCR) included the BD GeneOhm Cdiff Assay, the Xpert C. difficile test, and the ProGastro Cd assay. Specimens were considered true positive if results were positive in two or more assays. For each method, the Youden index was calculated and cost-effectiveness was analyzed. Of 81 patients evaluated, 26 (32.1%) were positive for CDI. Sensitivity of the BD GeneOhm Cdiff assay, the Xpert C. difficile test, the ProGastro Cd assay, C. Diff Quik Chek Complete test, and two-step GDH/EIA was 96.2%, 96.2%, 88.5%, 61.5%, and 42.3%, respectively. Specificity of the Xpert C. difficile test was 96.4%, and for the other four assays was 100%. Compared with nonmolecular methods, molecular methods detected 34.7% more positive specimens. Assessment of performance characteristics and cost-effectiveness demonstrated that the BD GeneOhm Cdiff assay yielded the best results. While costly, the Xpert C. difficile test required limited processing and yielded rapid results. Because of discordant results, specimen processing, and extraction equipment requirements, the ProGastro Cd assay was the least favored molecular assay. The GDH/EIA method lacked sufficient sensitivity to be recommended. Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Progress in hprt mutation assay and its application in radiation biology

International Nuclear Information System (INIS)

He Jing; Li Qiang

2008-01-01

hprt gene is an X-linked locus that has been well studied and widely used as a bio-marker in mutation detection, hprt mutation assay is a gene mutation test system in mammalian cells in vitro which has been used as a biological dosimeter. In this paper, the biological characteristics of hprt gene, hprt mutation detection methodology and the application of hprt mutation assay in radiation biology are comprehensively reviewed. (authors)

Clinical validation of the Tempus xO assay

Science.gov (United States)

Beaubier, Nike; Tell, Robert; Huether, Robert; Bontrager, Martin; Bush, Stephen; Parsons, Jerod; Shah, Kaanan; Baker, Tim; Selkov, Gene; Taxter, Tim; Thomas, Amber; Bettis, Sam; Khan, Aly; Lau, Denise; Lee, Christina; Barber, Matthew; Cieslik, Marcin; Frankenberger, Casey; Franzen, Amy; Weiner, Ali; Palmer, Gary; Lonigro, Robert; Robinson, Dan; Wu, Yi-Mi; Cao, Xuhong; Lefkofsky, Eric; Chinnaiyan, Arul; White, Kevin P.

2018-01-01

We have developed a clinically validated NGS assay that includes tumor, germline and RNA sequencing. We apply this assay to clinical specimens and cell lines, and we demonstrate a clinical sensitivity of 98.4% and positive predictive value of 100% for the clinically actionable variants measured by the assay. We also demonstrate highly accurate copy number measurements and gene rearrangement identification. PMID:29899824

Comparative analysis of gene expression by microarray analysis of male and female flowers of Asparagus officinalis.

Science.gov (United States)

Gao, Wu-Jun; Li, Shu-Fen; Zhang, Guo-Jun; Wang, Ning-Na; Deng, Chuan-Liang; Lu, Long-Dou

2013-01-01

To identify rapidly a number of genes probably involved in sex determination and differentiation of the dioecious plant Asparagus officinalis, gene expression profiles in early flower development for male and female plants were investigated by microarray assay with 8,665 probes. In total, 638 male-biased and 543 female-biased genes were identified. These genes with biased-expression for male and female were involved in a variety of processes associated with molecular functions, cellular components, and biological processes, suggesting that a complex mechanism underlies the sex development of asparagus. Among the differentially expressed genes involved in the reproductive process, a number of genes associated with floral development were identified. Reverse transcription-PCR was performed for validation, and the results were largely consistent with those obtained by microarray analysis. The findings of this study might contribute to understanding of the molecular mechanisms of sex determination and differentiation in dioecious asparagus and provide a foundation for further studies of this plant.

Specificity of B-type natriuretic peptide assays

DEFF Research Database (Denmark)

Saenger, Amy K.; Rodriguez-Fraga, Olaia; Ler, Ranka

2017-01-01

BACKGROUND: B-type natriuretic peptides (BNPs) are used clinically to diagnose and monitor heart failure and are present in the circulation as multiple proBNP-derived fragments. We investigated the specificity of BNP immunoassays with glycosylated and nonglycosylated BNP, N-terminal proBNP (NT......-proBNP), and proBNP peptides to probe the cross-reactivity of each assay. METHODS: Nine B-type natriuretic peptides were studied, including synthetic and recombinant BNP (Shionogi, Scios, Mayo), human and synthetic glycosylated and nonglycosylated NT-proBNP (HyTest, Roche Diagnostics), and human glycosylated......-Rad, Goetze] were evaluated. Specificity was assessed by calculating the recovery between baseline and peptide-spiked human plasma pools at target concentrations of 100 ng/L BNP, 300 ng/L proBNP, or 450 ng/L NT-proBNP. All assays were performed in duplicate. RESULTS: BNP and NT-proBNP assays demonstrated...

Specificity tests of an oligonucleotide probe against food-outbreak salmonella for biosensor detection

Science.gov (United States)

Chen, I.-H.; Horikawa, S.; Xi, J.; Wikle, H. C.; Barbaree, J. M.; Chin, B. A.

2017-05-01

Phage based magneto-elastic (ME) biosensors have been shown to be able to rapidly detect Salmonella in various food systems to serve food pathogen monitoring purposes. In this ME biosensor platform, the free-standing strip-shaped magneto-elastic sensor is the transducer and the phage probe that recognizes Salmonella in food serves as the bio-recognition element. According to Sorokulova et al. at 2005, a developed oligonucleotide probe E2 was reported to have high specificity to Salmonella enterica Typhimurium. In the report, the specificity tests were focused in most of Enterobacterace groups outside of Salmonella family. Here, to understand the specificity of phage E2 to different Salmonella enterica serotypes within Salmonella Family, we further tested the specificity of the phage probe to thirty-two Salmonella serotypes that were present in the major foodborne outbreaks during the past ten years (according to Centers for Disease Control and Prevention). The tests were conducted through an Enzyme linked Immunosorbent Assay (ELISA) format. This assay can mimic probe immobilized conditions on the magnetoelastic biosensor platform and also enable to study the binding specificity of oligonucleotide probes toward different Salmonella while avoiding phage/ sensor lot variations. Test results confirmed that this oligonucleotide probe E2 was high specific to Salmonella Typhimurium cells but showed cross reactivity to Salmonella Tennessee and four other serotypes among the thirty-two tested Salmonella serotypes.

Automated brightfield dual-color in situ hybridization for detection of mouse double minute 2 gene amplification in sarcomas.

Science.gov (United States)

Zhang, Wenjun; McElhinny, Abigail; Nielsen, Alma; Wang, Maria; Miller, Melanie; Singh, Shalini; Rueger, Ruediger; Rubin, Brian P; Wang, Zhen; Tubbs, Raymond R; Nagle, Raymond B; Roche, Pat; Wu, Ping; Pestic-Dragovich, Lidija

2011-01-01

The human homolog of the mouse double minute 2 (MDM2) oncogene is amplified in about 20% of sarcomas. The measurement of the MDM2 amplification can aid in classification and may provide a predictive value for recently formulated therapies targeting MDM2. We have developed and validated an automated bright field dual-color in situ hybridization application to detect MDM2 gene amplification. A repeat-depleted MDM2 probe was constructed to target the MDM2 gene region at 12q15. A chromosome 12-specific probe (CHR12) was generated from a pα12H8 plasmid. The in situ hybridization assay was developed by using a dinitrophenyl-labeled MDM2 probe and a digoxigenin-labeled CHR12 probe on the Ventana Medical Systems' automated slide-staining platforms. The specificity of the MDM2 and CHR12 probes was shown on metaphase spreads and further validated against controls, including normal human tonsil and known MDM2-amplified samples. The assay performance was evaluated on a cohort of 100 formalin-fixed, paraffin-embedded specimens by using a conventional bright field microscope. Simultaneous hybridization and signal detection for MDM2 and CHR12 showed that both DNA targets were present in the same cells. One hundred soft tissue specimens were stained for MDM2 and CHR12. Although 26 of 29 lipomas were nonamplified and eusomic, MDM2 amplification was noted in 78% of atypical lipomatous tumors or well-differentiated liposarcomas. Five of 6 dedifferentiated liposarcoma cases were amplified for MDM2. MDM2 amplification was observed in 1 of 8 osteosarcomas; 3 showed CHR12 aneusomy. MDM2 amplification was present in 1 of 4 chondrosarcomas. Nine of 10 synovial sarcomas displayed no evidence of MDM2 amplification in most tumor cells. In pleomorphic sarcoma, not otherwise specified (pleomorphic malignant fibrous histiocytoma), MDM2 was amplified in 38% of cases, whereas 92% were aneusomic for CHR12. One alveolar rhabdomyosarcoma and 2 embryonal rhabdomyosarcomas displayed low-level aneusomy

Study of hepatitis B virus gene mutations with enzymatic colorimetry-based DNA microarray.

Science.gov (United States)

Mao, Hailei; Wang, Huimin; Zhang, Donglei; Mao, Hongju; Zhao, Jianlong; Shi, Jian; Cui, Zhichu

2006-01-01

To establish a modified microarray method for detecting HBV gene mutations in the clinic. Site-specific oligonucleotide probes were immobilized to microarray slides and hybridized to biotin-labeled HBV gene fragments amplified from two-step PCR. Hybridized targets were transferred to nitrocellulose membranes, followed by intensity measurement using BCIP/NBT colorimetry. HBV genes from 99 Hepatitis B patients and 40 healthy blood donors were analyzed. Mutation frequencies of HBV pre-core/core and basic core promoter (BCP) regions were found to be significantly higher in the patient group (42%, 40% versus 2.5%, 5%, P colorimetry method exhibited the same level of sensitivity and reproducibility. An enzymatic colorimetry-based DNA microarray assay was successfully established to monitor HBV mutations. Pre-core/core and BCP mutations of HBV genes could be major causes of HBV infection in HBeAg-negative patients and could also be relevant to chronicity and aggravation of hepatitis B.

Identification and validation of a gene causing cross-resistance between insecticide classes in Anopheles gambiae from Ghana.

Science.gov (United States)

Mitchell, Sara N; Stevenson, Bradley J; Müller, Pie; Wilding, Craig S; Egyir-Yawson, Alexander; Field, Stuart G; Hemingway, Janet; Paine, Mark J I; Ranson, Hilary; Donnelly, Martin James

2012-04-17

In the last decade there have been marked reductions in malaria incidence in sub-Saharan Africa. Sustaining these reductions will rely upon insecticides to control the mosquito malaria vectors. We report that in the primary African malaria vector, Anopheles gambiae sensu stricto, a single enzyme, CYP6M2, confers resistance to two classes of insecticide. This is unique evidence in a disease vector of cross-resistance associated with a single metabolic gene that simultaneously reduces the efficacy of two of the four classes of insecticide routinely used for malaria control. The gene-expression profile of a highly DDT-resistant population of A. gambiae s.s. from Ghana was characterized using a unique whole-genome microarray. A number of genes were significantly overexpressed compared with two susceptible West African colonies, including genes from metabolic families previously linked to insecticide resistance. One of the most significantly overexpressed probe groups (false-discovery rate-adjusted P P450 gene CYP6M2. This gene is associated with pyrethroid resistance in wild A. gambiae s.s. populations) and can metabolize both type I and type II pyrethroids in recombinant protein assays. Using in vitro assays we show that recombinant CYP6M2 is also capable of metabolizing the organochlorine insecticide DDT in the presence of solubilizing factor sodium cholate.

A substrate-optimized electrophoretic mobility shift assay for ADAM12

DEFF Research Database (Denmark)

Kotzsch, Alexander; Skovgaard, Tine; Buus, Uwe

2014-01-01

long been investigated as pharmaceutical drug targets; however, due to lack of potency and in vivo side effects, none of the small-molecule inhibitors discovered so far has made it beyond clinical testing. Ongoing research on novel selective inhibitors of ADAMs requires reliable biochemical assays...... to validate molecular probes from large-scale screening efforts. Here we describe an electrophoretic mobility shift assay for ADAM12 based on the identification of an optimized peptide substrate that is characterized by excellent performance and reproducibility....

Serodiagnosis of Leishmania donovani infections: assessment of enzyme-linked immunosorbent assays using recombinant L. donovani gene B protein (GBP) and a peptide sequence of L. donovani GBP

DEFF Research Database (Denmark)

Jensen, A T; Gasim, S; Moller, T

1999-01-01

The repetitive sequence of Leishmania major gene B protein (GBP) has previously been shown to be a useful tool in the diagnosis of cutaneous leishmaniasis (CL). Here, we have assessed enzyme-linked immunosorbent assays (ELISAs) using recombinant L. donovani GBP (rGBP) and a peptide sequence of L...... for malaria but free of leishmaniasis was negative in both assays....

Analysis of the Kinetics and Regulation of Cytokine Gene Expression During the Primary In Vivo Immune Response to Killed Brucella Abortus

Science.gov (United States)

1992-08-10

Purified protein derivative of Mycobacterium tuberculosis and excretory-secretory antigen(s) of Toxocara canis expand in vitro human T cells with...day after immunization viii 47 49 LIST OF TABLES I. PCR primers and Southern blot probes of Th 11Th2 cytokines II. Cytokine mRNA levels in Thyl...sensitive quantitative reverse transcriptase-polymerase chain reaction (RT- PCR ) assay to measure changes in Thl and Th2 cytokine gene expression during

Oestrogenic activity of a textile industrial wastewater treatment plant effluent evaluated by the E-screen test and MELN gene-reporter luciferase assay

Energy Technology Data Exchange (ETDEWEB)

Schiliro, Tiziana, E-mail: tiziana.schiliro@unito.it [Department of Public Health and Microbiology, University of Torino, Via Santena 5bis, 10126 Torino (Italy); Porfido, Arianna [Department of Public Health and Microbiology, University of Torino, Via Santena 5bis, 10126 Torino (Italy); Spina, Federica; Varese, Giovanna Cristina [Department of Life Sciences and Systems Biology, University of Torino, Viale Mattioli 25, 10125 Torino (Italy); Gilli, Giorgio [Department of Public Health and Microbiology, University of Torino, Via Santena 5bis, 10126 Torino (Italy)

2012-08-15

This study quantified the biological oestrogenic activity in the effluent of a textile industrial wastewater treatment plant (IWWTP) in northwestern Italy. Samples of the IWWTP effluent were collected monthly, both before and after tertiary treatment (ozonation). After solid phase extraction, all samples were subjected to two in vitro tests of total estrogenic activity, the human breast cancer cell line (MCF-7 BUS) proliferation assay, or E-screen test, and the luciferase-transfected human breast cancer cell line (MELN) gene-reporter assay, to measure the 17{beta}-oestradiol equivalent quantity (EEQ). In the E-screen test, the mean EEQ values were 2.35 {+-} 1.68 ng/L pre-ozonation and 0.72 {+-} 0.58 ng/L post-ozonation; in the MELN gene-reporter luciferase assay, the mean EEQ values were 4.18 {+-} 3.54 ng/L pre-ozonation and 2.53 {+-} 2.48 ng/L post-ozonation. These results suggest that the post-ozonation IWWTP effluent had a lower oestrogenic activity (simple paired t-tests, p < 0.05). The average reduction of estrogenic activity of IWWTP effluent after ozonation was 67 {+-} 26% and 52 {+-} 27% as measured by E-screen test and MELN gene-reporter luciferase assay, respectively. There was a positive and significant correlation between the two tests (Rho S = 0.650, p = 0.022). This study indicates that the environmental risk is low because oestrogenic substances are deposited into the river via IWWTP at concentrations lower than those at which chronic exposure has been reported to affect the endocrine system of living organisms. -- Highlights: Black-Right-Pointing-Pointer The two in vitro tests are suited for oestrogenic activity assessment in textile WWTP. Black-Right-Pointing-Pointer There is a significant correlation between the results of the two in vitro tests. Black-Right-Pointing-Pointer The oestrogenic activity of the effluent is reduced by ozonation. Black-Right-Pointing-Pointer The input of estrogenic substances into the river via textile WWTP is low.

Multiplex Ligation-Dependent Probe Amplification Analysis of GATA4 Gene Copy Number Variations in Patients with Isolated Congenital Heart Disease

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Valentina Guida

2010-01-01

Full Text Available GATA4 mutations are found in patients with different isolated congenital heart defects (CHDs, mostly cardiac septal defects and tetralogy of Fallot. In addition, GATA4 is supposed to be the responsible gene for the CHDs in the chromosomal 8p23 deletion syndrome, which is recognized as a malformation syndrome with clinical symptoms of facial anomalies, microcephaly, mental retardation, and congenital heart defects. Thus far, no study has been carried out to investigate the role of GATA4 copy number variations (CNVs in non-syndromic CHDs. To explore the possible occurrence of GATA4 gene CNVs in isolated CHDs, we analyzed by multiplex ligation-dependent probe amplification (MLPA a cohort of 161 non-syndromic patients with cardiac anomalies previously associated with GATA4 gene mutations. The patients were mutation-negative for GATA4, NKX2.5, and FOG2 genes after screening with denaturing high performance liquid chromatography. MLPA analysis revealed that normalized MLPA signals were all found within the normal range values for all exons in all patients, excluding a major contribution of GATA4 gene CNVs in CHD pathogenesis.

Development of a real-time RT-PCR assay for the simultaneous identification, quantitation and differentiation of avian metapneumovirus subtypes A and B.

Science.gov (United States)

Cecchinato, Mattia; Lupini, Caterina; Munoz Pogoreltseva, Olga Svetlana; Listorti, Valeria; Mondin, Alessandra; Drigo, Michele; Catelli, Elena

2013-01-01

In recent years, special attention has been paid to real-time polymerase chain reaction (PCR) for avian metapneumovirus (AMPV) diagnosis, due to its numerous advantages over classical PCR. A new multiplex quantitative real-time reverse transcription-PCR (qRT-PCR) with molecular beacon probe assay, designed to target the SH gene, was developed. The test was evaluated in terms of specificity, sensitivity and repeatability, and compared with conventional RT nested-PCR based on the G gene. All of the AMPV subtype A and B strains tested were amplified and specifically detected while no amplification occurred with other non-target bird respiratory pathogens. The detection limit of the assay was 10(-0.41) median infectious dose/ml and 10(1.15) median infectious dose/ml when the AMPV-B strain IT/Ty/B/Vr240/87 and the AMPV-A strain IT/Ty/A/259-01/03 were used, respectively, as templates. In all cases, the amplification efficiency was approximately 2 and the error values were 0.9375) between crossing point values and virus quantities, making the assay herein designed reliable for quantification. When the newly developed qRT-PCR was compared with a conventional RT nested-PCR, it showed greater sensitivity with RNA extracted from both positive controls and from experimentally infected birds. This assay can be effectively used for the detection, identification, differentiation and quantitation of AMPV subtype A or subtype B to assist in disease diagnosis and to carry out rapid surveillance with high levels of sensitivity and specificity.

Rapid and sensitive reporter gene assays for detection of antiandrogenic and estrogenic effects of environmental chemicals

DEFF Research Database (Denmark)

Vinggaard, Anne; Jørgensen, E.C.B.; Larsen, John Christian

1999-01-01

Reports on increasing incidences in developmental abnormalities of the human male reproductive tract and the recent identifications of environmental chemicals with antiandrogenic activity necessitate the screening of a larger number of compounds in order to get an overview of potential antiandrog......Reports on increasing incidences in developmental abnormalities of the human male reproductive tract and the recent identifications of environmental chemicals with antiandrogenic activity necessitate the screening of a larger number of compounds in order to get an overview of potential...... antiandrogenic chemicals present in our environment. Thus, there is a great need for an effective in vitro screening method for (anti)androgenic chemicals. We have developed a rapid, sensitive, and reproducible reporter gene assay for detection of antiandrogenic chemicals. Chinese Hamster Ovary cells were...... calcium phosphate transfection method, this method has the advantage of being more feasible, as the assay can be scaled down to the microtiter plate format. Furthermore, the transfection reagent is noncytotoxic, allowing its addition together with the test compounds thereby reducing the hands...

Mechano-genetic DNA hydrogels as a simple, reconstituted model to probe the effect of active fluctuations on gene transcription

Science.gov (United States)

Nguyen, Dan; Saleh, Omar

Active fluctuations - non-directed fluctuations attributable, not to thermal energy, but to non-equilibrium processes - are thought to influence biology by increasing the diffusive motion of biomolecules. Dense DNA regions within cells (i.e. chromatin) are expected to exhibit such phenomena, as they are cross-linked networks that continually experience propagating forces arising from dynamic cellular activity. Additional agitation within these gene-encoding DNA networks could have potential genetic consequences. By changing the local mobility of transcriptional machinery and regulatory proteins towards/from their binding sites, and thereby influencing transcription rates, active fluctuations could prove to be a physical means of modulating gene expression. To begin probing this effect, we construct genetic DNA hydrogels, as a simple, reconstituted model of chromatin, and quantify transcriptional output from these hydrogels in the presence/absence of active fluctuations.

A novel TaqMan® assay for Nosema ceranae quantification in honey bee, based on the protein coding gene Hsp70.

Science.gov (United States)

Cilia, Giovanni; Cabbri, Riccardo; Maiorana, Giacomo; Cardaio, Ilaria; Dall'Olio, Raffaele; Nanetti, Antonio

2018-04-01

Nosema ceranae is now a widespread honey bee pathogen with high incidence in apiculture. Rapid and reliable detection and quantification methods are a matter of concern for research community, nowadays mainly relying on the use of biomolecular techniques such as PCR, RT-PCR or HRMA. The aim of this technical paper is to provide a new qPCR assay, based on the highly-conserved protein coding gene Hsp70, to detect and quantify the microsporidian Nosema ceranae affecting the western honey bee Apis mellifera. The validation steps to assess efficiency, sensitivity, specificity and robustness of the assay are described also. Copyright © 2018 Elsevier GmbH. All rights reserved.

A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses.

Science.gov (United States)

Wadhwa, Ashutosh; Wilkins, Kimberly; Gao, Jinxin; Condori Condori, Rene Edgar; Gigante, Crystal M; Zhao, Hui; Ma, Xiaoyue; Ellison, James A; Greenberg, Lauren; Velasco-Villa, Andres; Orciari, Lillian; Li, Yu

2017-01-01

Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high

A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses.

Directory of Open Access Journals (Sweden)

Ashutosh Wadhwa

2017-01-01

Full Text Available Rabies, resulting from infection by Rabies virus (RABV and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses

Automated 5 ' nuclease assay for detection of virulence factors in porcine Escherichia coli

DEFF Research Database (Denmark)

Frydendahl, K.; Imberechts, H.; Lehmann, S.

2001-01-01

(STa, STb, EAST1) and heat labile LT) enterotoxins and the verocytotoxin variant 2e (VT2e). To correctly identify false negative results, an endogenous internal control targeting the E. coil 16S rRNA gene was incorporated in each test tube. The assay was evaluated using a collection of E. coil...... reference strains which have previously been examined with phenotypical assays or DNA hybridization. Furthermore, the assay was evaluated by testing porcine E. coil field strains, previously characterized. The 5' nuclease assay correctly detected the presence of virulence genes in all reference strains....... When testing field strains there was generally excellent agreement with results obtained by laboratories in Belgium and Germany. In conclusion, the 5' nuclease assay developed is a fast and specific tool for detection of E. coli virulence genes in the veterinary diagnostic laboratory....

Detection and quantification of alternative splice sites in Arabidopsis genes AtDCL2 and AtPTB2 with highly sensitive surface enhanced Raman spectroscopy (SERS) and gold nanoprobes.

Science.gov (United States)

Kadam, Ulhas S; Schulz, Burkhard; Irudayaraj, Joseph

2014-05-02

Alternative splicing (AS) increases the size of the transcriptome and proteome to enhance the physiological capacity of cells. We demonstrate surface enhanced Raman spectroscopy (SERS) in combination with a DNA hybridization analytical platform to identify and quantify AS genes in plants. AS in AtDCL2 and AtPTB2 were investigated using non-fluorescent Raman probes using a 'sandwich assay'. Utilizing Raman probes conjugated to gold nanoparticles we demonstrate the recognition of RNA sequences specific to AtDCL2 and AtPTB2 splice junction variants with detection sensitivity of up to 0.1 fM. Published by Elsevier B.V.

Development of an ultrasensitive PCR assay for polycyclic musk determination in fish.

Science.gov (United States)

Zhang, Xiaohan; Zhuang, Huisheng

2018-05-01

Polycyclic musks (PCMs) in the aquatic environment and organisms have become an emerging environmental issue because of their potential risk. The most used method for polycyclic musk determination is gas chromatography-mass spectrometry (GC-MS) with different sample extractions, which are somewhat expensive to operate, complex and laborious. In this study, a novel and ultrasensitive real-time polymerase chain reaction (PCR) assay with multiple signal amplification of carboxylic-DNA by gold nanoparticle-polyamidoamine conjugation (Au-PAMAM) was developed for determining polycyclic musks in fish. Hapten and immunogen were specially prepared. Polyclonal antibodies were produced based on the optimal immunisation, and the antibodies were characterised. Due to PAMAM's unique nanostructure of numerous functional amino groups, polyclonal antibody and carboxylic-DNA were immobilised by Au-PAMAM conjugation to develop the antibody-Au-PAMAM-DNA probes, which were used as a signal DNA amplifier in the PCR system. Compared with real-time immuno-PCR, this biological probe-amplified immuno-PCR (BPAI-PCR) assay had higher sensitivity due to the probes' higher ratio of signal DNA. Finally, the BPAI-PCR assay was applied to analyse AHTN (7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene,Tonalide) concentrations in fish samples in the range from 1 pg/L to 10 ng/L, giving an of LOD 0.61 pg/L. In general, due to the specificity of the antibody and novel nanoprobe design, this BPAI-PCR assay provided a potential way for trace analysis of AHTN in the aquatic organisms. The high concentrations of AHTN found in cultivated fish should encourage further toxicological studies.

Development and validation of a 36-gene sequencing assay for hereditary cancer risk assessment

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Valentina S. Vysotskaia

2017-02-01

Full Text Available The past two decades have brought many important advances in our understanding of the hereditary susceptibility to cancer. Numerous studies have provided convincing evidence that identification of germline mutations associated with hereditary cancer syndromes can lead to reductions in morbidity and mortality through targeted risk management options. Additionally, advances in gene sequencing technology now permit the development of multigene hereditary cancer testing panels. Here, we describe the 2016 revision of the Counsyl Inherited Cancer Screen for detecting single-nucleotide variants (SNVs, short insertions and deletions (indels, and copy number variants (CNVs in 36 genes associated with an elevated risk for breast, ovarian, colorectal, gastric, endometrial, pancreatic, thyroid, prostate, melanoma, and neuroendocrine cancers. To determine test accuracy and reproducibility, we performed a rigorous analytical validation across 341 samples, including 118 cell lines and 223 patient samples. The screen achieved 100% test sensitivity across different mutation types, with high specificity and 100% concordance with conventional Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA. We also demonstrated the screen’s high intra-run and inter-run reproducibility and robust performance on blood and saliva specimens. Furthermore, we showed that pathogenic Alu element insertions can be accurately detected by our test. Overall, the validation in our clinical laboratory demonstrated the analytical performance required for collecting and reporting genetic information related to risk of developing hereditary cancers.

Use of Ti plasmid DNA probes for determining tumorigenicity of agrobacterium strains

International Nuclear Information System (INIS)

Burr, T.J.; Norelli, J.L.; Katz, B.H.; Bishop, A.L.

1990-01-01

Probes consisting of T-DNA genes from the Ti plasmid of Agrobacterium tumefaciens were used for determining tumorigenicity of strains. Two 32 P-labeled probes hybridized with 28 of 28 tumorigenic strains of the pathogen but not with 20 of 22 nontumorigenic strains. One probe, pTHE17, consists of all but the far left portion of the T-DNA of strain C58. Probe SmaI7 consists of SmaI fragment 7 of pTiC58, including onc genes 1, 4, and 6a and most of 2. Another probe, pAL4044, consisting of the vir region of strain Ach-5, hybridized with several nontumorigenic as well as tumorigenic strains. Colony hybridizations were done with 28 tumorigenic and 22 nontumorigenic Agrobacterium strains. About 10 6 CFU of the different tumorigenic strains were detectable with this method. Southern analyses confirmed the presence or absence of Ti plasmids in strains for which tumorigenicity was questioned. Colony hybridization with the T-DNA probes provides a rapid and sensitive means for determining the tumorigenic nature of Agrobacterium strains

Novel Multiplex PCR Assay for Detection of Chlorhexidine-Quaternary Ammonium, Mupirocin, and Methicillin Resistance Genes, with Simultaneous Discrimination of Staphylococcus aureus from Coagulase-Negative Staphylococci.

Science.gov (United States)

McClure, Jo-Ann; Zaal DeLongchamp, Johanna; Conly, John M; Zhang, Kunyan

2017-06-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a clinically significant pathogen that is resistant to a wide variety of antibiotics and responsible for a large number of nosocomial infections worldwide. The Agency for Healthcare Research and Quality and the Centers for Disease Control and Prevention recently recommended the adoption of universal mupirocin-chlorhexidine decolonization of all admitted intensive care unit patients rather than MRSA screening with targeted treatments, which raises a serious concern about the selection of resistance to mupirocin and chlorhexidine in strains of staphylococci. Thus, a simple, rapid, and reliable approach is paramount in monitoring the prevalence of resistance to these agents. We developed a simple multiplex PCR assay capable of screening Staphylococcus isolates for the presence of antiseptic resistance genes for chlorhexidine and quaternary ammonium compounds, as well as mupirocin and methicillin resistance genes, while simultaneously discriminating S. aureus from coagulase-negative staphylococci (CoNS). The assay incorporates 7 PCR targets, including the Staphylococcus 16S rRNA gene (specifically detecting Staphylococcus spp.), nuc (distinguishing S. aureus from CoNS), mecA (distinguishing MRSA from methicillin-susceptible S. aureus ), mupA and mupB (identifying high-level mupirocin resistance), and qac and smr (identifying chlorhexidine and quaternary ammonium resistance). Our assay demonstrated 100% sensitivity, specificity, and accuracy in a total of 23 variant antiseptic- and/or antibiotic-resistant control strains. Further validation of our assay using 378 randomly selected and previously well-characterized local clinical isolates confirmed its feasibility and practicality. This may prove to be a useful tool for multidrug-resistant Staphylococcus monitoring in clinical laboratories, particularly in the wake of increased chlorhexidine and mupirocin treatments. Copyright © 2017 American Society for Microbiology.

Using genomic DNA-based probe-selection to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species

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Townsend Henrik J

2005-11-01

Full Text Available Abstract High-density oligonucleotide (oligo arrays are a powerful tool for transcript profiling. Arrays based on GeneChip® technology are amongst the most widely used, although GeneChip® arrays are currently available for only a small number of plant and animal species. Thus, we have developed a method to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species and tested the method by analysing the transcriptome of Brassica oleracea L., a species for which no GeneChip® array is available, using a GeneChip® array designed for Arabidopsis thaliana (L. Heynh. Genomic DNA from B. oleracea was labelled and hybridised to the ATH1-121501 GeneChip® array. Arabidopsis thaliana probe-pairs that hybridised to the B. oleracea genomic DNA on the basis of the perfect-match (PM probe signal were then selected for subsequent B. oleracea transcriptome analysis using a .cel file parser script to generate probe mask files. The transcriptional response of B. oleracea to a mineral nutrient (phosphorus; P stress was quantified using probe mask files generated for a wide range of gDNA hybridisation intensity thresholds. An example probe mask file generated with a gDNA hybridisation intensity threshold of 400 removed > 68 % of the available PM probes from the analysis but retained >96 % of available A. thaliana probe-sets. Ninety-nine of these genes were then identified as significantly regulated under P stress in B. oleracea, including the homologues of P stress responsive genes in A. thaliana. Increasing the gDNA hybridisation intensity thresholds up to 500 for probe-selection increased the sensitivity of the GeneChip® array to detect regulation of gene expression in B. oleracea under P stress by up to 13-fold. Our open-source software to create probe mask files is freely available http://affymetrix.arabidopsis.info/xspecies/ and may be used to facilitate transcriptomic analyses of a wide range of plant and animal

Detection and removal of spatial bias in multiwell assays.

Science.gov (United States)

Lachmann, Alexander; Giorgi, Federico M; Alvarez, Mariano J; Califano, Andrea

2016-07-01

Multiplex readout assays are now increasingly being performed using microfluidic automation in multiwell format. For instance, the Library of Integrated Network-based Cellular Signatures (LINCS) has produced gene expression measurements for tens of thousands of distinct cell perturbations using a 384-well plate format. This dataset is by far the largest 384-well gene expression measurement assay ever performed. We investigated the gene expression profiles of a million samples from the LINCS dataset and found that the vast majority (96%) of the tested plates were affected by a significant 2D spatial bias. Using a novel algorithm combining spatial autocorrelation detection and principal component analysis, we could remove most of the spatial bias from the LINCS dataset and show in parallel a dramatic improvement of similarity between biological replicates assayed in different plates. The proposed methodology is fully general and can be applied to any highly multiplexed assay performed in multiwell format. ac2248@columbia.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Genotypic characterization of Rickettsiae by DNA probes generated from Rickettsia Prowazekii DNA

International Nuclear Information System (INIS)

Demkin, V.V.; Rydkina, E.B.; Likhoded, L.Ya.; Ignatovich, V.F.; Genig, V.A.; Balayeva, N.M.

1994-01-01

Southern blot analysis of HindIII-cleaved rickettsial DNA was used for genotypic characterization of the typhus group (TG) species (R. prowazekii, R. typhi, R. canada) and a few species were of the spotted fever group (SFG)rickettsiae (R. sibirica, R. conorii, R. akari). Four different DNA probes were employed. PBH11 and PBH13 probes were morphospecific HindIII fragment of R prowazekii DNA. MW218 probe contained the gene for 51 K antigen and MW264 probe contained the citrate synthase gene of R. prowazekii. All the probes hybridized with the tested TG and SFG rickettsial DNAs, forming from 1 to 5 bands, but they did not with R. tsutsudamushi or C. burnetii DNAs. All the probes demonstrated specific hybridization pattern with TG species and R. akari. PBH11. PBH13 and MW264 probes clearly distinguished R. sibirica and R. conorii from the other tested rickettsiae, but not from each other. However, these two species differed slightly with MW218 probe. Several strains of each species were analyzed in this way and except for strains of R. conorii identical intra-species pattern were obtained. These data lead us to consider the obtained hybridization patterns as criteria for genotypic identification. (author)

Detection of 22 common leukemic fusion genes using a single-step multiplex qRT-PCR-based assay.

Science.gov (United States)

Lyu, Xiaodong; Wang, Xianwei; Zhang, Lina; Chen, Zhenzhu; Zhao, Yu; Hu, Jieying; Fan, Ruihua; Song, Yongping

2017-07-25

Fusion genes generated from chromosomal translocation play an important role in hematological malignancies. Detection of fusion genes currently employ use of either conventional RT-PCR methods or fluorescent in situ hybridization (FISH), where both methods involve tedious methodologies and require prior characterization of chromosomal translocation events as determined by cytogenetic analysis. In this study, we describe a real-time quantitative reverse transcription PCR (qRT-PCR)-based multi-fusion gene screening method with the capacity to detect 22 fusion genes commonly found in leukemia. This method does not require pre-characterization of gene translocation events, thereby facilitating immediate diagnosis and therapeutic management. We performed fluorescent qRT-PCR (F-qRT-PCR) using a commercially-available multi-fusion gene detection kit on a patient cohort of 345 individuals comprising 108 cases diagnosed with acute myeloid leukemia (AML) for initial evaluation; remaining patients within the cohort were assayed for confirmatory diagnosis. Results obtained by F-qRT-PCR were compared alongside patient analysis by cytogenetic characterization. Gene translocations detected by F-qRT-PCR in AML cases were diagnosed in 69.4% of the patient cohort, which was comparatively similar to 68.5% as diagnosed by cytogenetic analysis, thereby demonstrating 99.1% concordance. Overall gene fusion was detected in 53.7% of the overall patient population by F-qRT-PCR, 52.9% by cytogenetic prediction in leukemia, and 9.1% in non-leukemia patients by both methods. The overall concordance rate was calculated to be 99.0%. Fusion genes were detected by F-qRT-PCR in 97.3% of patients with CML, followed by 69.4% with AML, 33.3% with acute lymphoblastic leukemia (ALL), 9.1% with myelodysplastic syndromes (MDS), and 0% with chronic lymphocytic leukemia (CLL). We describe the use of a F-qRT-PCR-based multi-fusion gene screening method as an efficient one-step diagnostic procedure as an

Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

Energy Technology Data Exchange (ETDEWEB)

Greulich-Bode, Karin; Wang, Mei; Rhein, Andreas; Weier, Jingly; Weier, Heinz-Ulli

2008-12-16

Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100kb, careful probe selection and characterization are of paramount importance. We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific {approx}6kb plasmid onto an unusually small, {approx}55kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-?B2 locus. The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

Energy Technology Data Exchange (ETDEWEB)

Greulich-Bode, Karin M.; Wang, Mei; Rhein, Andreas P.; Weier, Jingly F.; Weier, Heinz-Ulli G.

2008-12-04

Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100kb, careful probe selection and characterization are of paramount importance. We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific {approx}6kb plasmid onto an unusually small, {approx}55kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-{kappa}B2 locus. The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis

DEFF Research Database (Denmark)

Thierry, Simon; Hamidjaja, Raditijo A.; Girault, Guillaume

2013-01-01

been modified and adapted for simultaneous interrogation of 13 biallelic canonical SNPs in a 13-plex assay. Changes made to the originally published method include the design of allele-specific dual-priming-oligonucleotides (DPOs) as competing detection probes (MOLigo probes) and use of asymmetric PCR...

[A cell-based detection of ciguatoxin using sodium fluorescence probe].

Science.gov (United States)

Yuan, Jian-hui; Yang, Hui; Tang, Huan-wen; Huang, Wei; Xu, Xin-yun; Liu, Jian-jun; Ke, Yue-bin; Cheng, Jin-quan; Zhuang, Zhi-xiong

2011-04-01

To establish a cell-based detection method of ciguatoxin using fluorescence assay. Mouse neuroblastoma N-2A cells were exposed to ouabain and veratridine and different concentrations of standard ciguatoxin samples (P-CTX-1) to establish the curvilinear relationship between the toxin dosage and fluorescence intensity using the sodium fluorescence probe CoroNaTM Green. The toxicity curvilinear relationship was also generated between the toxin dosage and cell survival using CCK-8 method. Based on these standard curves, the presence of ciguatoxin was detected in 33 samples of deep-sea coral fish. A correlation was found between the detection results of cell-based fluorescence assay and cytotoxicity assay, whose detection limit reached 103 g/ml and 1012 g/ml, respectively. The cell-based fluorescent assay sensitivity showed a higher sensitivity than cytotoxicity assay with a 2-4 h reduction of the detection time. The cell-based fluorescent assay can quickly and sensitively detect ciguatoxin and may serve as a good option for preliminary screening of the toxin.

SYTO probes: markers of apoptotic cell demise.

Science.gov (United States)

Wlodkowic, Donald; Skommer, Joanna

2007-10-01

As mechanistic studies on tumor cell death advance towards their ultimate translational goal, there is a need for specific, rapid, and high-throughput analytical tools to detect diverse cell demise modes. Patented DNA-binding SYTO probes, for example, are gaining increasing interest as easy-to-use markers of caspase-dependent apoptotic cell death. They are proving convenient for tracking apoptosis in diverse hematopoietic cell lines and primary tumor samples, and, due to their spectral characteristics, appear to be useful for the development of multiparameter flow cytometry assays. Herein, several protocols for multiparametric assessment of apoptotic events using SYTO probes are provided. There are protocols describing the use of green fluorescent SYTO 16 and red fluorescent SYTO 17 dyes in combination with plasma membrane permeability markers. Another protocol highlights the multiparametric use of SYTO 16 dye in conjunction with the mitochondrial membrane potential sensitive probe, tetramethylrhodamine methyl ester (TMRM), and the plasma membrane permeability marker, 7-aminoactinomycin D (7-AAD).

The development and application of the two real-time RT-PCR assays to detect the pathogen of HFMD.

Directory of Open Access Journals (Sweden)

Aili Cui

Full Text Available Large-scale Hand, Foot, and Mouth Disease (HFMD outbreaks have frequently occurred in China since 2008, affecting more than one million children and causing several hundred children deaths every year. The pathogens of HFMD are mainly human enteroviruses (HEVs. Among them, human enterovirus 71 (HEV71 and coxsackievirus A16 (CVA16 are the most common pathogens of HFMD. However, other HEVs could also cause HFMD. To rapidly detect HEV71 and CVA16, and ensure detection of all HEVs causing HFMD, two real-time hybridization probe-based RT-PCR assays were developed in this study. One is a multiplex real-time RT-PCR assay, which was developed to detect and differentiate HEV71 specifically from CVA16 directly from clinical specimens within 1-2 h, and the other is a broad-spectrum real-time RT-PCR assay, which targeted almost all HEVs. The experiments confirmed that the two assays have high sensitivity and specificity, and the sensitivity was up to 0.1 TCID50/ml for detection of HEVs, HEV71, and CVA16, respectively. A total of 213 clinical specimens were simultaneously detected by three kinds of assays, including the two real-time RT-PCR assays, direct conventional RT-PCR assay, and virus isolation assay on human rhabdomyosarcoma cells (RD cells. The total positive rate of both HEV71 and CVA16 was 69.48% with real-time RT-PCR assay, 47.42% with RT-PCR assay, and 34.58% with virus isolation assay. One HFMD clinical specimen was positive for HEV, but negative for HEV71 or CVA16, which was identified as Echovirus 11 (Echo11 by virus isolation, RT-PCR, and sequencing for the VP1 gene. The two real-time RT-PCR assays had been applied in 31 provincial HFMD labs to detect the pathogens of HFMD, which has contributed to the rapid identification of the pathogens in the early stages of HFMD outbreaks, and helped to clarify the etiologic agents of HFMD in China.

Homology of yeast photoreactivating gene fragment with human genomic digests

International Nuclear Information System (INIS)

Meechan, P.J.; Milam, K.M.; Cleaver, J.E.

1984-01-01

Enzymatic photoreactivation of UV-induced DNA lesions has been demonstrated for a variety of prokaryotic and eukaryotic organisms. Its presence in placental mammals, however, has not been clearly established. The authors attempted to resolve this question by assaying for the presence (or absence) of sequences in human DNA complimentary to a fragment of the photoreactivating gene from S. cerevisiae that has recently been cloned. In another study, DNA from human, chick E. coli and yeast cells was digested with either HindIII of BglII, electrophoresed on a 0.5% agarose gel, transferred (Southern blot) to a nylon membrane and probed for homology against a Sau3A restriction fragment from S. cerevisiae that compliments phr/sup -/ cells. Hybridization to human DNA digests was observed only under relatively non-stringent conditions indicating the gene is not conserved in placental mammals. These results are correlated with current literature data concerning photoreactivating enzymes

Luminescence resonance energy transfer-based nucleic acid hybridization assay on cellulose paper with upconverting phosphor as donors.

Science.gov (United States)

Zhou, Feng; Noor, M Omair; Krull, Ulrich J

2014-03-04

A bioassay based on DNA hybridization on cellulose paper is a promising format for gene fragment detection that may be suited for in-field and rapid diagnostic applications. We demonstrate for the first time that luminescence resonance energy transfer (LRET) associated with upconverting phosphors (UCPs) can be used to develop a paper-based DNA hybridization assay with high sensitivity, selectivity and fast response. UCPs with strong green emission were synthesized and subsequently functionalized with streptavidin (UCP-strep). UCP-strep particles were immobilized on cellulose paper, and then biotinylated single-stranded oligonucleotide probes were conjugated onto the UCPs via streptavidin-biotin linkage. The UCPs served as donors that were LRET-paired with Cy3-labeled target DNA. Selective DNA hybridization enabled the proximity required for LRET-sensitized emission from Cy3, which was used as the detection signal. Hybridization was complete within 2 min, and the limit of detection of the method was 34 fmol, which is a significant improvement in comparison to an analogous fluorescence resonance energy transfer (FRET) assay based on quantum dots. The assay exhibited excellent resistance to nonspecific adsorption of noncomplementary short/long DNA and protein. The selectivity of the assay was further evaluated by one base pair mismatched (1BPM) DNA detection, where a maximum signal ratio of 3.1:1 was achieved between fully complementary and 1BPM samples. This work represents a preliminary but significant step for the development of paper-based UCP-LRET nucleic acid hybridization assays, which offer potential for lowering the limit of detection of luminescent hybridization assays due to the negligible background signal associated with optical excitation by near-infrared (NIR) light.

Evaluation of the Branched-Chain DNA Assay for Measurement of RNA in Formalin-Fixed Tissues

Science.gov (United States)

Knudsen, Beatrice S.; Allen, April N.; McLerran, Dale F.; Vessella, Robert L.; Karademos, Jonathan; Davies, Joan E.; Maqsodi, Botoul; McMaster, Gary K.; Kristal, Alan R.

2008-01-01

We evaluated the branched-chain DNA (bDNA) assay QuantiGene Reagent System to measure RNA in formalin-fixed, paraffin-embedded (FFPE) tissues. The QuantiGene Reagent System does not require RNA isolation, avoids enzymatic preamplification, and has a simple workflow. Five selected genes were measured by bDNA assay; quantitative polymerase chain reaction (qPCR) was used as a reference method. Mixed-effect statistical models were used to partition the overall variance into components attributable to xenograft, sample, and assay. For FFPE tissues, the coefficients of reliability were significantly higher for the bDNA assay (93–100%) than for qPCR (82.4–95%). Correlations between qPCRFROZEN, the gold standard, and bDNAFFPE ranged from 0.60 to 0.94, similar to those from qPCRFROZEN and qPCRFFPE. Additionally, the sensitivity of the bDNA assay in tissue homogenates was 10-fold higher than in purified RNA. In 9- to 13-year-old blocks with poor RNA quality, the bDNA assay allowed the correct identification of the overexpression of known cancer genes. In conclusion, the QuantiGene Reagent System is considerably more reliable, reproducible, and sensitive than qPCR, providing an alternative method for the measurement of gene expression in FFPE tissues. It also appears to be well suited for the clinical analysis of FFPE tissues with diagnostic or prognostic gene expression biomarker panels for use in patient treatment and management. PMID:18276773

Transforming thymidine into a magnetic resonance imaging probe for monitoring gene expression.

Science.gov (United States)

Bar-Shir, Amnon; Liu, Guanshu; Liang, Yajie; Yadav, Nirbhay N; McMahon, Michael T; Walczak, Piotr; Nimmagadda, Sridhar; Pomper, Martin G; Tallman, Keri A; Greenberg, Marc M; van Zijl, Peter C M; Bulte, Jeff W M; Gilad, Assaf A

2013-01-30

Synthetic chemistry has revolutionized the understanding of many biological systems. Small compounds that act as agonists and antagonists of proteins, and occasionally as imaging probes, have contributed tremendously to the elucidation of many biological pathways. Nevertheless, the function of thousands of proteins is still elusive, and designing new imaging probes remains a challenge. Through screening and characterization, we identified a thymidine analogue as a probe for imaging the expression of herpes simplex virus type-1 thymidine kinase (HSV1-TK). To detect the probe, we used chemical exchange saturation transfer magnetic resonance imaging (CEST-MRI), in which a dynamic exchange process between an exchangeable proton and the surrounding water protons is used to amplify the desired contrast. Initially, five pyrimidine-based molecules were recognized as putative imaging agents, since their exchangeable imino protons resonate at 5-6 ppm from the water proton frequency and their detection is therefore less affected by endogenous CEST contrast or confounded by direct water saturation. Increasing the pK(a) value of the imino proton by reduction of its 5,6-double bond results in a significant reduction of the exchange rate (k(ex)) between this proton and the water protons. This reduced k(ex) of the dihydropyrimidine nucleosides fulfills the "slow to intermediate regime" condition for generating high CEST-MRI contrast. Consequently, we identified 5-methyl-5,6-dihydrothymidine as the optimal probe and demonstrated its feasibility for in vivo imaging of HSV1-TK. In light of these findings, this new approach can be generalized for designing specific probes for the in vivo imaging of a variety of proteins and enzymes.

Absolute and direct microRNA quantification using DNA-gold nanoparticle probes.

Science.gov (United States)

Degliangeli, Federica; Kshirsagar, Prakash; Brunetti, Virgilio; Pompa, Pier Paolo; Fiammengo, Roberto

2014-02-12

DNA-gold nanoparticle probes are implemented in a simple strategy for direct microRNA (miRNA) quantification. Fluorescently labeled DNA-probe strands are immobilized on PEGylated gold nanoparticles (AuNPs). In the presence of target miRNA, DNA-RNA heteroduplexes are formed and become substrate for the endonuclease DSN (duplex-specific nuclease). Enzymatic hydrolysis of the DNA strands yields a fluorescence signal due to diffusion of the fluorophores away from the gold surface. We show that the molecular design of our DNA-AuNP probes, with the DNA strands immobilized on top of the PEG-based passivation layer, results in nearly unaltered enzymatic activity toward immobilized heteroduplexes compared to substrates free in solution. The assay, developed in a real-time format, allows absolute quantification of as little as 0.2 fmol of miR-203. We also show the application of the assay for direct quantification of cancer-related miR-203 and miR-21 in samples of extracted total RNA from cell cultures. The possibility of direct and absolute quantification may significantly advance the use of microRNAs as biomarkers in the clinical praxis.

Gain-of-function analysis of poplar CLE genes in Arabidopsis by exogenous application and over-expression assays.

Science.gov (United States)

Liu, Yisen; Yang, Shaohui; Song, Yingjin; Men, Shuzhen; Wang, Jiehua

2016-04-01

Among 50 CLE gene family members in the Populus trichocarpa genome, three and six PtCLE genes encode a CLE motif sequence highly homologous to Arabidopsis CLV3 and TDIF peptides, respectively, which potentially make them functional equivalents. To test and compare their biological activity, we first chemically synthesized each dodecapeptide and analysed itsi n vitro bioactivity on Arabidopsis seedlings. Similarly, but to a different extent, three types of poplar CLV3-related peptides caused root meristem consumption, phyllotaxis disorder, anthocyanin accumulation and failure to enter the bolting stage. In comparison, application of two poplar TDIF-related peptides led to root length promotion in a dose-dependent manner with an even stronger effect observed for poplar TDIF-like peptide than TDIF. Next, we constructed CaMV35S:PtCLE transgenic plants for each of the nine PtCLE genes. Phenotypic abnormalities exemplified by arrested shoot apical meristem and abnormal flower structure were found to be more dominant and severe in 35S:PtCLV3 and 35S:PtCLV3-like2 lines than in the 35S:PtCLV3-like line. Disordered vasculature was detected in both stem and hypocotyl cross-sections in Arabidopsis plants over-expressing poplar TDIF-related genes with the most defective vascular patterning observed for TDIF2 and two TDIF-like genes. Phenotypic difference consistently observed in peptide application assay and transgenic analysis indicated the functional diversity of nine poplar PtCLE genes under investigation. This work represents the first report on the functional analysis of CLE genes in a tree species and constitutes a basis for further study of the CLE peptide signalling pathway in tree development. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Screening for common copy-number variants in cancer genes.

Science.gov (United States)

Tyson, Jess; Majerus, Tamsin M O; Walker, Susan; Armour, John A L

2010-12-01

For most cases of colorectal cancer that arise without a family history of the disease, it is proposed that an appreciable heritable component of predisposition is the result of contributions from many loci. Although progress has been made in identifying single nucleotide variants associated with colorectal cancer risk, the involvement of low-penetrance copy number variants is relatively unexplored. We have used multiplex amplifiable probe hybridization (MAPH) in a fourfold multiplex (QuadMAPH), positioned at an average resolution of one probe per 2 kb, to screen a total of 1.56 Mb of genomic DNA for copy number variants around the genes APC, AXIN1, BRCA1, BRCA2, CTNNB1, HRAS, MLH1, MSH2, and TP53. Two deletion events were detected, one upstream of MLH1 in a control individual and the other in APC in a colorectal cancer patient, but these do not seem to correspond to copy number polymorphisms with measurably high population frequencies. In summary, by means of our QuadMAPH assay, copy number measurement data were of sufficient resolution and accuracy to detect any copy number variants with high probability. However, this study has demonstrated a very low incidence of deletion and duplication variants within intronic and flanking regions of these nine genes, in both control individuals and colorectal cancer patients. Copyright © 2010 Elsevier Inc. All rights reserved.

Chromatin immunoprecipitation assays revealed CREB and serine 133 phospho-CREB binding to the CART gene proximal promoter.

Science.gov (United States)

Rogge, George A; Shen, Li-Ling; Kuhar, Michael J

2010-07-16

Both over expression of cyclic AMP response element binding protein (CREB) in the nucleus accumbens (NAc), and intra-accumbal injection of cocaine- and amphetamine-regulated transcript (CART) peptides, have been shown to decrease cocaine reward. Also, over expression of CREB in the rat NAc increased CART mRNA and peptide levels, but it is not known if this was due to a direct action of P-CREB on the CART gene promoter. The goal of this study was to test if CREB and P-CREB bound directly to the CRE site in the CART promoter, using chromatin immunoprecipitation (ChIP) assays. ChIP assay with anti-CREB antibodies showed an enrichment of the CART promoter fragment containing the CRE region over IgG precipitated material, a non-specific control. Forskolin, which was known to increase CART mRNA levels in GH3 cells, was utilized to show that the drug increased levels of P-CREB protein and P-CREB binding to the CART promoter CRE-containing region. A region of the c-Fos promoter containing a CRE cis-regulatory element was previously shown to bind P-CREB, and it was used here as a positive control. These data suggest that the effects of CREB over expression on blunting cocaine reward could be, at least in part, attributed to the increased expression of the CART gene by direct interaction of P-CREB with the CART promoter CRE site, rather than by some indirect action. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Improving comparability between microarray probe signals by thermodynamic intensity correction

DEFF Research Database (Denmark)

Bruun, G. M.; Wernersson, Rasmus; Juncker, Agnieszka

2007-01-01

different probes. It is therefore of great interest to correct for the variation between probes. Much of this variation is sequence dependent. We demonstrate that a thermodynamic model for hybridization of either DNA or RNA to a DNA microarray, which takes the sequence-dependent probe affinities...... determination of transcription start sites for a subset of yeast genes. In another application, we identify present/absent calls for probes hybridized to the sequenced Escherichia coli strain O157:H7 EDL933. The model improves the correct calls from 85 to 95% relative to raw intensity measures. The model thus...... makes applications which depend on comparisons between probes aimed at different sections of the same target more reliable....

Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli

Directory of Open Access Journals (Sweden)

Bechard Matthew E.

2003-01-01

Full Text Available Tetrahydromethanopterin (H4MPT is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase. Given the importance of RFAP synthase in H4MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H4MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli.

Science.gov (United States)

Bechard, Matthew E.; Chhatwal, Sonya; Garcia, Rosemarie E.; Rasche, Madeline E.

2003-01-01

Tetrahydromethanopterin (H(4)MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H(4)MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H(4)MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase). Given the importance of RFAP synthase in H(4)MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H(4)MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

Terbium fluorescence as a sensitive, inexpensive probe for UV-induced damage in nucleic acids

International Nuclear Information System (INIS)

El-Yazbi, Amira F.; Loppnow, Glen R.

2013-01-01

Graphical abstract: -- Highlights: •Simple, inexpensive, mix-and-read assay for positive detection of DNA damage. •Recognition of undamaged DNA via hybridization to a hairpin probe. •Terbium(III) fluorescence reports the amount of damage by binding to ssDNA. •Tb/hairpin is a highly selective and sensitive fluorescent probe for DNA damage. -- Abstract: Much effort has been focused on developing methods for detecting damaged nucleic acids. However, almost all of the proposed methods consist of multi-step procedures, are limited, require expensive instruments, or suffer from a high level of interferences. In this paper, we present a novel simple, inexpensive, mix-and-read assay that is generally applicable to nucleic acid damage and uses the enhanced luminescence due to energy transfer from nucleic acids to terbium(III) (Tb 3+ ). Single-stranded oligonucleotides greatly enhance the Tb 3+ emission, but duplex DNA does not. With the use of a DNA hairpin probe complementary to the oligonucleotide of interest, the Tb 3+ /hairpin probe is applied to detect ultraviolet (UV)-induced DNA damage. The hairpin probe hybridizes only with the undamaged DNA. However, the damaged DNA remains single-stranded and enhances the intrinsic fluorescence of Tb 3+ , producing a detectable signal directly proportional to the amount of DNA damage. This allows the Tb 3+ /hairpin probe to be used for sensitive quantification of UV-induced DNA damage. The Tb 3+ /hairpin probe showed superior selectivity to DNA damage compared to conventional molecular beacons probes (MBs) and its sensitivity is more than 2.5 times higher than MBs with a limit of detection of 4.36 ± 1.2 nM. In addition, this probe is easier to synthesize and more than eight times cheaper than MBs, which makes its use recommended for high-throughput, quantitative analysis of DNA damage

A cost-effective melting temperature assay for the detection of single-nucleotide polymorphism in the MBL2 gene of HIV-1-infected children

Directory of Open Access Journals (Sweden)

Arraes L.C.

2006-01-01

Full Text Available We report a fast (less than 3 h and cost-effective melting temperature assay method for the detection of single-nucleotide polymorphisms in the MBL2 gene. The protocol, which is based on the Corbett Rotor Gene real time PCR platform and SYBR Green I chemistry, yielded, in the cohorts studied, sensitive (100% and specific (100% PCR amplification without the use of costly fluorophore-labeled probes or post-PCR manipulation. At the end of the PCR, the dissociation protocol included a slow heating from 60º to 95ºC in 0.2ºC steps, with an 8-s interval between steps. Melting curve profiles were obtained using the dissociation software of the Rotor Gene-3000 apparatus. Samples were analyzed in duplicate and in different PCR runs to test the reproducibility of this technique. No supplementary data handling is required to determine the MBL2 genotype. MBL2 genotyping performed on a cohort of 164 HIV-1-positive Brazilian children and 150 healthy controls, matched for age and sex and ethnic origin, yielded reproducible results confirmed by direct sequencing of the amplicon performed in blind. The three MBL2 variants (Arg52Cys, Gly54Asp, Gly57Glu were grouped together and called allele 0, while the combination of three wild-type alleles was called allele A. The frequency of the A/A homozygotes was significantly higher among healthy controls (0.68 than in HIV-infected children (0.55; P = 0.0234 and the frequency of MBL2 0/0 homozygotes was higher among HIV-1-infected children than healthy controls (P = 0.0296. The 0 allele was significantly more frequent among the 164 HIV-1-infected children (0.29 than among the 150 healthy controls (0.18; P = 0.0032. Our data confirm the association between the presence of the mutated MBL2 allele (allele 0 and HIV-1 infection in perinatally exposed children. Our results are in agreement with the literature data which indicate that the presence of the allele 0 confers a relative risk of 1.37 for HIV-1 infection through

Sensitive detection of point mutation by electrochemiluminescence and DNA ligase-based assay

Science.gov (United States)

Zhou, Huijuan; Wu, Baoyan

2008-12-01

The technology of single-base mutation detection plays an increasingly important role in diagnosis and prognosis of genetic-based diseases. Here we reported a new method for the analysis of point mutations in genomic DNA through the integration of allele-specific oligonucleotide ligation assay (OLA) with magnetic beads-based electrochemiluminescence (ECL) detection scheme. In this assay the tris(bipyridine) ruthenium (TBR) labeled probe and the biotinylated probe are designed to perfectly complementary to the mutant target, thus a ligation can be generated between those two probes by Taq DNA Ligase in the presence of mutant target. If there is an allele mismatch, the ligation does not take place. The ligation products are then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. Results showed that the new method held a low detection limit down to 10 fmol and was successfully applied in the identification of point mutations from ASTC-α-1, PANC-1 and normal cell lines in codon 273 of TP53 oncogene. In summary, this method provides a sensitive, cost-effective and easy operation approach for point mutation detection.

A TaqMan Real-Time PCR Assay for Detection and Quantification of Sporisorium scitamineum in Sugarcane

Directory of Open Access Journals (Sweden)

Yachun Su

2013-01-01

Full Text Available Sporisorium scitamineum is a fungal smut pathogen epidemic in sugarcane producing areas. Early detection and proper identification of the smut are an essential requirement in its management practice. In this study, we developed a TaqMan real-time PCR assay using specific primers (bEQ-F/bEQ-R and a TaqMan probe (bEQ-P which were designed based on the bE (b East mating type gene (Genbank Accession no. U61290.1. This method was more sensitive (a detection limit of 10 ag pbE DNA and 0.8 ng sugarcane genomic DNA than that of conventional PCR (10 fg and 100 ng, resp.. Reliability was demonstrated through the positive detection of samples collected from artificially inoculated sugarcane plantlets (FN40. This assay was capable of detecting the smut pathogen at the initial stage (12 h of infection and suitable for inspection of sugarcane pathogen-free seed cane and seedlings. Furthermore, quantification of pathogen was verified in pathogen-challenged buds in different sugarcane genotypes, which suggested its feasibility for evaluation of smut resistance in different sugarcane genotypes. Taken together, this novel assay can be used as a diagnostic tool for sensitive, accurate, fast, and quantitative detection of the smut pathogen especially for asymptomatic seed cane or plants and evaluation of smut resistance of sugarcane genotypes.

Cytotoxicity and genotoxicity of Agaricus blazei methanolic extract fractions assessed using gene and chromosomal mutation assays

Directory of Open Access Journals (Sweden)

Marilanda Ferreira Bellini

2008-01-01

Full Text Available Functional food investigations have demonstrated the presence of substances that could be beneficial to human health when consumed. However, the toxic effects of some substances contained in foods have been determined. Reported medicinal and nutritive properties have led to the extensive commercialization of the basidiomycete fungi Agaricus blazei Murrill (sensu Heinemann, also known as Agaricus brasiliensis Wasser et al., Agaricus subrufescens Peck or the Brazilian medical mushroom (BMM. Different methanolic extract fractions (ME of this mushroom were submitted to the cytokinesis-block micronucleus (CBMN clastogenic assay and the hypoxanthine-guanine phosphoribosyl transferase locus (HGPRT assay for gene mutation, both using Chinese hamster ovary cells clone K1 (CHO-K1. The results suggest that all the fractions tested possess cytotoxic and mutagenic potential but no clastogenic effects. Further information is needed on the biochemical components of the A. blazei methanol fractions to identify any substances with cytotoxic and/or mutagenicity potential. These findings indicate that A. blazei methanolic extract should not be used due to their genotoxicity and care should be taken in the use of A. blazei by the general population until further biochemical characterization of this fungi is completed.

Assaying locomotor, learning, and memory deficits in Drosophila models of neurodegeneration.

Science.gov (United States)

Ali, Yousuf O; Escala, Wilfredo; Ruan, Kai; Zhai, R Grace

2011-03-11

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination. Here we use behavioral assays, including the negative geotaxis assay and the aversive phototaxic suppression assay (APS assay), to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

G-stack modulated probe intensities on expression arrays - sequence corrections and signal calibration

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Fasold Mario

2010-04-01

Full Text Available Abstract Background The brightness of the probe spots on expression microarrays intends to measure the abundance of specific mRNA targets. Probes with runs of at least three guanines (G in their sequence show abnormal high intensities which reflect rather probe effects than target concentrations. This G-bias requires correction prior to downstream expression analysis. Results Longer runs of three or more consecutive G along the probe sequence and in particular triple degenerated G at its solution end ((GGG1-effect are associated with exceptionally large probe intensities on GeneChip expression arrays. This intensity bias is related to non-specific hybridization and affects both perfect match and mismatch probes. The (GGG1-effect tends to increase gradually for microarrays of later GeneChip generations. It was found for DNA/RNA as well as for DNA/DNA probe/target-hybridization chemistries. Amplification of sample RNA using T7-primers is associated with strong positive amplitudes of the G-bias whereas alternative amplification protocols using random primers give rise to much smaller and partly even negative amplitudes. We applied positional dependent sensitivity models to analyze the specifics of probe intensities in the context of all possible short sequence motifs of one to four adjacent nucleotides along the 25meric probe sequence. Most of the longer motifs are adequately described using a nearest-neighbor (NN model. In contrast, runs of degenerated guanines require explicit consideration of next nearest neighbors (GGG terms. Preprocessing methods such as vsn, RMA, dChip, MAS5 and gcRMA only insufficiently remove the G-bias from data. Conclusions Positional and motif dependent sensitivity models accounts for sequence effects of oligonucleotide probe intensities. We propose a positional dependent NN+GGG hybrid model to correct the intensity bias associated with probes containing poly-G motifs. It is implemented as a single-chip based calibration

Developing a yeast-based assay protocol to monitor total ...

African Journals Online (AJOL)

A yeast-based assay protocol developed for detecting oestrogenic activity in activated sludge (AS) supernatant is described. The protocol used Saccharomyces cerevisiae construct RMY/ER-ERE with human oestrogen receptor (ERα) and lacZ reporter genes, and was developed by modifying existing assays for use with AS ...

Development of a non invasion real-time PCR assay for the quantitation of chicken parvovirus in fecal swabs

Science.gov (United States)

The present study describes the development of a real time Taqman polymerase chain reaction (PCR) assay using a fluorescent labeled probe for the detection and quantitation of chicken parvovirus (ChPV) in feces. The primers and probes were designed based on the nucleotide sequence of the non struct...

Comparison of kDNA PCR-hybridization assay with three PCR methods for canines visceral Leishmaniasis diagnosis

Energy Technology Data Exchange (ETDEWEB)

Pilatti, Marcia M.; Andrade, Antero S.R. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)], e-mail: marciapilatti@yahoo.com.br, e-mail: antero@cdtn.br; Ferreira, Sidney A. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Parasitologia], e-mail: saninoalmeida@gmail.com

2009-07-01

The sensitivity of the kDNA PCR-Hybridization assay, which uses radioactive DNA probes (labeled with {sup 32}P), was compared with three conventional PCR methods used for canine visceral leishmaniasis diagnosis. All PCR methods had two steps: a first amplification followed by hybridization or by a new amplification (nested or semi nested). Two methods (kDNA PCR-Hybridization and kDNA snPCR) used primers addressed to kinetoplast minicircles and the other two methods to the coding (LnPCR) and intergenic noncoding regions (ITS-1 nPCR) of the ribosomal rRNA genes. The comparison was accomplished in two groups of 23 infected dogs using samples collected by the conjunctival swab procedure. In the Group 1 the DNA was extracted from cotton swabs by phenol-chloroform and in Group 2 by boiling. The most efficient PCR methods in the Group 1 were those based on kDNA targets. The kDNA PCR-Hybridization was able to detect parasites in 22/23 dogs (95.6%) and in 40/46 samples (86.9%). The kDNA snPCR was positive for 21/23 dogs (91.3%) and for 40/46 samples (86.9%). The positivities of the kDNA based methods were significantly higher than the positivities verified for the methods based on ribosomal rRNA genes (p<0.05). In the Group 2 the kDNA PCR- Hybridization showed a better performance detecting parasites in 18/23 dogs (78.3%) and in 31/46 samples (67.4%), significantly higher than the other three methods (p<0.05). The higher sensitivity of the minicircle kDNA based assays reported by others was confirmed in this study and kDNA PCR-Hybridization showed the best sensitivity among the assays evaluated. (author)

Comparison of kDNA PCR-hybridization assay with three PCR methods for canines visceral Leishmaniasis diagnosis